CA2395787A1 - Nucleic acids, proteins, and antibodies - Google Patents
Nucleic acids, proteins, and antibodies Download PDFInfo
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- CA2395787A1 CA2395787A1 CA002395787A CA2395787A CA2395787A1 CA 2395787 A1 CA2395787 A1 CA 2395787A1 CA 002395787 A CA002395787 A CA 002395787A CA 2395787 A CA2395787 A CA 2395787A CA 2395787 A1 CA2395787 A1 CA 2395787A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.
Description
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
~~ TTENANT LES PAGES 1 A 285 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
Nucleic Acids, Proteins, and Antibodies [1] This application refers to a "Sequence Listing" that is provided only on electronic media in computer readable form pursuant to Administrative Instructions Section 801 (a)(i).
The Sequence Listing forms a part of this description pursuant to Rule 5.2 and Administrative Instructions Sections 801 to 806, and is hereby incorporated in its entirety.
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
~~ TTENANT LES PAGES 1 A 285 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
Nucleic Acids, Proteins, and Antibodies [1] This application refers to a "Sequence Listing" that is provided only on electronic media in computer readable form pursuant to Administrative Instructions Section 801 (a)(i).
The Sequence Listing forms a part of this description pursuant to Rule 5.2 and Administrative Instructions Sections 801 to 806, and is hereby incorporated in its entirety.
(2] The Sequence Listing is provided as an electronic file (PTZ07PCT
seqList.txt, 370,456 bytes in size, created on January 13, 2001) on four identical compact discs (CD-R), labeled "COPY 1," "COPY 2," "COPY 3," and "CRF." The Sequence Listing complies with Annex C of the Administrative Instructions, and may be viewed, for example, on an 1BM-PC machine running the MS-Windows operating system by using the V viewer soilware, version 2000 (see World Wide Web URL: http://www.fileviewer.com).
Field of the Invention [3] The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing andlor prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention.
The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.
Background of the Invention [4] Calcium ions are major players in an intracellular signaling system that translates extracellular stimuli into the regulation~of a bewildering number of phenomena such as muscle contraction, neurotransmitter release and other secretion processes, cell proliferation, gene expression, and cell death. In order to accomplish such as wide array of functions, changes in calcium concentrations must be tightly regulated.
seqList.txt, 370,456 bytes in size, created on January 13, 2001) on four identical compact discs (CD-R), labeled "COPY 1," "COPY 2," "COPY 3," and "CRF." The Sequence Listing complies with Annex C of the Administrative Instructions, and may be viewed, for example, on an 1BM-PC machine running the MS-Windows operating system by using the V viewer soilware, version 2000 (see World Wide Web URL: http://www.fileviewer.com).
Field of the Invention [3] The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing andlor prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention.
The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.
Background of the Invention [4] Calcium ions are major players in an intracellular signaling system that translates extracellular stimuli into the regulation~of a bewildering number of phenomena such as muscle contraction, neurotransmitter release and other secretion processes, cell proliferation, gene expression, and cell death. In order to accomplish such as wide array of functions, changes in calcium concentrations must be tightly regulated.
[5] The calcium-binding domain of many proteins contains the high-affinity motif known as the EF-hand. The EF-hand appears in many proteins that exert calcium dependent actions in the nucleus or the cytoplasm, as well as a fev that function extracellularly (Maurer et al., Current Opinion in Cell Biology 8: 609-617 (1996)). This motif exhibits helix-loop-helix architecture and is normally paired with a second EF-hand motif - 'the two calcium-binding loops coupled through a short antiparallel beta sheet (Kretsinger, Nature Structural Biology 3: 12-15 (1996)). Prominent calcium binding proteins exhibiting the EF-hand include calmodulin and the S 100 family.
[6] Calmodulin (CaM) is the most widely distributed and most common mediator of calcium effects (Celio et al., Guidebook to Calcium-Binding Proteins, Oxford University Press, Oxford, UK, pp. 34-40 (1996)), as well as the primary sensor of intracellular calcium changes in eukaryotic cells. The binding of calcium to CaM induces marked conformational changes in the protein, permitting its interaction with and regulation of over one~hvndred different proteins. In particular, CaM interactions are involved in a multitude of cellular processes including, for example, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation.
[7] The 5100 proteins are a group of acidic calcium binding proteins. Each of these proteins contains two calcium-binding domains, one that binds with high affinity and the other with low affinity. The distribution~of particular 5100 proteins is dependent on the specific cell type, suggesting that these proteins may be involved in transducing intracellular calcium changes in a cell-specific manner. For example, S100A13 protein is present in human and murine heart and skeletal muscle, S100A2 is localized to lung and kidney cells, and S 100B is abundant in the glial cells of the central and peripheral nervous system (Donato, Cell Calcium 12: 713-726 (1991); Zimmer et al., Brain Research Bulletin 37: 417-429 (1995); Schafer and Heizmann, Trends in Biochemical Sciences 21:
(1996)).
[8J Several families of calcium-binding proteins bind calcium at EGF-like domains rather than EF-hands. The epidermal growth factor-like (EGF) domain is a widely distributed, independently folding protein module that is thought to play a general role in.
extracellular events such as adhesion, coagulation, and receptor-ligand interactions (Campbell and Bork, Current Opinions in Structural Biology 3: 38S-392 (1993)).
A distinct class of these domains has been identified containing a consensus sequence associated with calcium binding: D/N-x-D/N-E/Q-xm-D/N*-xn-Y/F (where m and n are variable and indicates possible -hydroxylation) (Reel et al., EMBO Journal 7:2053-2067 (1988);
Handford et al., Nature 351:.164-167 (1991); Mayhew et al., Protein Engineering S:_489-494 (1992)). The importance of the calcium-binding EGF domain is emphasized by its occurrence in functionally diverse proteins, including those involved iri extracellular matrix architecture (fibrillin-1,2, fibulin-1,2, nidogen), control of blood coagulation (factors IX and X, proteins C and S, thrombomodulin), cholesterol uptake (low density lipoprotein receptor [LDLR]), and specification of cell fate (Drosophila Notch, Delta, and Serrate). Genetic mutations causing amino acid changes in calcium-binding EGF domains from some of these proteins have been identified in patients with the Marfan syndrome,, familial hypercholesterolemia, hemophilia B (diseases caused by mutations in fibrillin-1, LDLR, and factor IX genes, respectively), and protein S deficiency (Dietz et al., Human Molecular Genetics 4: 1799-1809 (1995); Hobbs et al., Human Mutations 1: 45S-466 (I992);
Giannelli et al., Nucleic Acids Research 24: 103-118 (1996); Gandrille et al., Blood 8S:
(1995)).
[9] Further, annexins are a structurally conserved family of proteins characterized by reversible calcium-dependent membrane binding. High-resolution crystal structures of the soluble forms of several different annexins have been solved, and all structures share a common fold consisting of four domains, each of which contains five helices connected by short loops. The loop regions on the convex side of the protein are believed to account for the calcium-dependent binding of the protein to membranes by jointly coordinating calcium with phosphatidylserine. Membrane association is of critical importance for the proposed functions of annexins, which includes vesicular trafficking, membrane fusion, and ion-channel formation (Seaton, Annexins: Molecular Structure to Cellular Function, Landes, New York ( 1996)).
(10] Calcium is involved in a wide array of different biological events. New structural and biophysical studies of calcium-binding proteins have provided important information on the roles of calcium at the molecular level, however much remains to be done to establish links between structure, calcium levels, and biological function.
Accordingly, there is a clear need for identifying and exploiting novel calcium-binding proteins, such as those described above, that may contribute to diseases resulting from the aberrant calcium flux. Additionally, novel members of these protein families are useful as screening tools to identify antagonists and/or agonists that may enhance or block activities mediated by calcium-binding proteins. Further, blockers of these calcium-binding proteins may prove useful in the diagnosis, prevention, andlor treatment of disorders including, but not limited to, neurological diseases, immune dysfunction, digestive disorders, neoplastic diseases, blood disorders, and/or infectious disease.
Summary of the Invention [1l] The, present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention:
The present invention further relates to methods and/or compositions for inhibiting .or enhancing the production and function of the polypeptides of the pxesent invention.
Detailed Desct-iption Tables [12] Table 1A summarizes some of the polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) and contig nucleotide sequence identifier.
(SEQ ID NO:X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, "Clone ID NO:Z", for a cDNA clone related to each contig sequence disclosed in Table 1 A. The third column provides a unique contig identifier, "Contig ID:" for each of the contig sequences disclosed in Table 1A. The fourth column provides the sequence identifier, "SEQ ID
NO:X", for each of the contig sequences disclosed in Table 1A. The fifth column, "ORF
(From-To)", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineate the preferred open reading frame (ORF) that encodes the amino acid sequence shown in the sequence listing and referenced in Table IA
as SEQ ID NO:Y (column 6). Column 7 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID
NO:Y).
Identification of potential immunogenic regions was performed according to the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988)); specifically, the -Genetics Computer Group (GCG) implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE (Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wisc.). This method returns a measure of the probability that a given residue is found on the surface of the protein. Regions where the antigenic index score ~is greater than 0.9 over at least 6 amino acids are indicated in Table 1 A as "Predicted Epitopes". In particular embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table 1A. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly. Column 8, "Tissue Distribution" shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucIeotides of the invention. .The first number in column 8 (preceding the colon), represents the tissue/cell source identifier code corresponding to the key-provided in .
Table 4. Expression of these polynucleotides was not observed in.the other tissues and/or cell libraries .tested. For those identifier codes in which the first two letters are not "AR", the second number in column 8 (following the colon), represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID
NO:X) was identified in the tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are "AR" designate information generated using DNA array technology.
Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA
probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids, from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the Level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after "[array code]:"
represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissues) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression. Column 9 provides the chromosomal location of polynucleotides corresponding to SEQ ID NO:X. Chromosomal location was determined by finding exact matches. to EST and cDNA sequences contained in the NCBI
(National Center for Biotechnology Information) UniGene database. Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIMTM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD) 2000. World Wide Web URL:
http://www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM
identification number is disclosed in column 10 labeled "OMIM Disease References)". A key to the OMIM reference identification numbers is provided in Table 5.
[~3] Table 1B summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID
NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, "Clone ID NO:Z", for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, "SEQ ID NO:X", for each contig sequence. The third column provides a unique contig identifier, "Contig ID:" for each contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, "SEQ ID NO:B" for a fragment of the BAC clone identif ed in column four of the corresponding row of the table. The sixth column, "Exon From-To", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID
NO:B which delineate certain polynucleotides of the invention that are also exemplary .
members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
[14) ~ Table 2 summarizes homology and features of some of the polypeptides of the invention. The first column provides a unique clone identifier, "Clone ID
NO:Z", corresponding to a cDNA clone disclosed in Table IA. The second column provides the unique contig identifier, "Contig ID:" corresponding to contigs in Table 1A
and allowing for correlation with the information in Table 1A. The third column provides the sequence.
identifier, "SEQ ID NO:X", for the contig polynucleotide sequence. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. Comparisons were made between polypeptides encoded by the polynucleotides of the invention and either a non-redundant protein database (herein referred to as "NR"), or a database of protein families (herein referred to as "PFAM") as further described below.
The fifth column provides a description of the PFAM/NR hit having a significant match to a polypeptide of the invention. Column six provides the accession number of the PFAM/NR
hit disclosed in the fifth column. Column seven, "Score/Percent Identity", provides a quality score or the,percent identity, of the hit disclosed in columns five and six.
Columns 8 and 9, "NT From" and "NT To" respectively, delineate the polynucleotides in "SEQ ID
NO:X"
that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth and sixth columns. In specific embodiments polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence encoded by 'a polynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
(15] Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention. The first column provides a unique clone identifier, "Clone ID", for a cDNA clone related to contig sequences disclosed in Table l A. The second column provides the sequence identifier, "SEQ ID NO:X", for contig sequences disclosed in Table 1A. The third column provides the unique contig identifier, "Contig ID:", for contigs disclosed in Table 1A. The fourth column provides a unique integer 'a' where 'a' is any integer between 1 and the final nucleotide minus 15 of SEQ ID
NO:X, and the fifth column provides a unique integer 'b' where 'b' is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14.
For each of the polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be substituted into the general formula of a-b, and used to describe polynucleotides which may be preferably excluded , from the invention. In certain embodiments, preferably , excluded from the invention are at least one, two, three, four, five, ten, or more of the polynucleotide sequences) having the accession numbers) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC
clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequences) contained in the clones corresponding to at least one, two, three;
four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).
[16] Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1A, column 8. Column 1 provides the tissue/cell source identifier code disclosed in Table 1A, Column 8. Columns 2-5 provide a description of the tissue or cell source.
Codes corresponding to diseased tissues are indicated in column 6 with the word "disease". The use of the word "disease" in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the "disease"
designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissuelcell source is a library, column 7 identifies the vector used to generate the library.
[17] Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1A, column 10. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM.
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, MD) 2000. WorldWide Web URL: http://www.ncbi.nlm.nih.gov/omim/).
Column 2 provides diseases associated with the cytologic band disclosed in Table 1A, column 9, as determined using the Morbid Map database.
[18] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present application.
[19] ~ Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers and vector information relating to these cDNA libraries.
[20] Table 8 provides a physical characterization of clones encompassed by the invention. The first column provides the unique clone identifier, "Clone ID
NO:Z", for certain cDNA clones of the invention, as described in Table IA. The second column provides the size of the cDNA insert contained in the corresponding cDNA
clone. , Definitions [21] The following definitions are provided to facilitate understanding of certain terms used throughout this specification.
[22] ~ In the present invention, "isolated" refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the polyriucleotide. The term "isolated" does not refer to genomic or cDNA
libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
(23] As used herein, a "polynucleotide" refers to a molecule having a nucleic acid sequence encoding SEQ ID NO:Y or a fragment or variant thereof; a nucleic acid sequence contained in SEQ ID NO:X (as described in column 3 of-Table 1A) or the complement thereof; a cDNA sequence contained in Clone ID NO:Z (as described in column 2 of Table 1A and contained within a library deposited with the ATCC); a nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B or a fragment or variant thereof; or a nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1B or the complement thereof. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA
sequence, including the 5' and 3' untranslated sequences, the coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
Moreover, as used herein, a "polypeptide" refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-PhenyIalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).
(24] In the present invention, "SEQ ID NO:X" was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X is deposited at Hurnan Genome Sciences, Inc.
(HGS) in a catalogued and archived library. As shown, for example, in column 2 of Table 1A, each clone is identified by a cDNA Clone ID (identifier generally referred to herein as Clone ID NO:Z). Each Clone ID is unique to an individual clone and the Clone ID is all the information needed to retrieve a given clone from the HGS library.
Furthermore, certain clones disclosed in this application have been deposited with the ATCC on October 5, 2000, having the,ATCC designation numbers PTA 2574 and PTA 2575; and on January 5, 2001, having the depositor reference numbers TS-1', TS-2, AC-1, and AC-2. In addition to the individual cDNA clone deposits, most of the cDNA libraries from which the clones were derived were deposited at the Ariierican Type Culture Collection (hereinafter "ATCC").
Table 7 provides a list of the deposited cDNA libraries. One can use the Clone ID NO:Z to determine the library source by reference to Tables 6 and 7. Table 7 lists the deposited cDNA libraries by name and links each library to an ATCC Deposit. Library names contain four characters, for example, "HTWE." The name of a cDNA clone (Clone ID) isolated from that library begins with the same four characters, for example "HTWEP07".
As mentioned below, Table l A correlates the Clone ID names with SEQ ID NO:X..
Thus, .
starting with an SEQ ID NO:X, one can use Tables 1, 6 and 7 to determine the corresponding Clone ID, which library it came from and which ATCC deposit the library is contained in. Furthermore, it is possible to retrieve a given cDNA clone from the source library by techniques known in the art and described elsewhere herein. The ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC
deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
(25] In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.Skb, kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise alI or a portion of any intron. In another embodiment, the polynucleotides comprising~coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene ~of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
(26] A "polynucleotide" of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, or the complement, thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein),. the polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or cDNA sequences contained in Clone ID NO:Z (e.g.; the complement of any one, two, three, four, or more of the polynucleotide fragments, or the cDNA clone within the pool of cDNA
clones deposited with the ATCC, described herein), and/or the polynucleotide sequence delineated in column 6 of Table 1B or the complement thereof. "Stringent hybridization conditions" refers to an overnight incubation at 42 degree C in a solution comprising 50%
formamide, Sx SSC (750 mM NaCI, 75 mM trisodium citrate), SO mM sodium phosphate (pH 7.6), Sx Denhardt's solution, 10% dextran sulfate, and 20 ~g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C.
[27] Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions.
Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C in a solution comprising 6X SSPE (20X SSPE = 3M NaCI; 0.2M NaH2P04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e:g. 5X SSC).
(28j Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include .Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations: The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
[29] Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing}, or to a complementary stretch. of T {or U) residues, would not be included in the definition of "polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded. cDNA clone generated using oligo dT as a primer).
[30] The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid~molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA
or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified"
bases include, for example, tritylated bases and unusual bases such as inosine. A
variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
[31] The polypeptide of the present invention can be composed of amino acids joined to each., other by peptide bonds or' modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be.
modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic .texts and in more detailed monographs, as well as in a voluminous research literature. Modif canons can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that ,the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modif cations include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-Linking, cyclization, disulf de bond formation, demethylation, formation of covalent cross-Links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS -STRUCTURE AND' MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H.
Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT
MODIFICATION OF PROTEINS, B. C.. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646. (1990); Rattan et al., Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[32] "SEQ ID NO:X" refers to a polynucleotide sequence described, for example, in Tables lAor 2, while "SEQ ID NO:Y" refers to a polypeptide sequence described 'in column 6 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 4 of Table 1A.
The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. "Clone ID NO:Z" refers to a cDNA clone described in column 2 of Table 1 A.
[33] "A polypeptide having functional activity" refers to a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody which 'binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
(34], The polypeptides of the invention can be assayed for functional activity (e.g.
biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, one of skill in the art may routinely assay calcium binding polypeptides (including fragments and variants) of the invention for activity using assays as described in the examples section below.
[3S] "A polypeptide having biological activity" refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or .
without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and .most preferably, not more than about three-fold Iess activity relative to the polypeptide of the present invention).
(36] Table 1A summarizes some of the polynucleotides encompassed by the invention (including contig sequences (SEQ ID NO:X) and clones (Clone ID NO:Z) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby.
OAS
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j37] The first column in Table 1A provides the gene number in the application corresponding to the clone identifier. The second column in Table 1A provides a unique "Clone ID NO:Z" for a cDNA clone related to each contig sequence disclosed in Table 1A.
This clone ID references the cDNA clone which contains at least the 5' most sequence of the assembled contig and at least a portion of SEQ ID NO:X was determined by directly sequencing the referenced clone. The reference clone may have more sequence than described in the sequence listing or the clone may have less. In the vast majority of cases, however, the clone is believed to encode a full-length polypeptide. In the case where a clone is not full-length, a full-length cDNA can be obtained by methods described elsewhere herein.
[38] . The third column in Table 1A provides a unique "Contig ID"
identification for each contig sequence. The fourth column provides the "SEQ ID NO:" identifier for each of the contig polynucleotide sequences disclosed in Table 1 A. The fifth column, "ORF
(From-To)", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence "SEQ ID NO:X" that delineate the preferred open reading frame (ORF) shown in the sequence listing and referenced in Table 1A, column 6, as SEQ ID
NO:Y. Where the nucleotide position number "To" is lower than the nucleotide position number "From", the preferred ORF is the reverse complement of the referenced polynucleotide sequence.
[39] The sixth column in Table 1A provides the corresponding SEQ ID NO:Y for the polypeptide sequence encoded by the preferred ORF delineated in column 5. In one embodiment, the invention provides an amino acid sequence comprising, or alternatively consisting of, a polypeptide encoded by the portion of SEQ TD NO:X delineated by "ORF
(From-To)". Also provided are polynucleotides encoding such amino acid sequences and the complementary strand thereto. .
[40] Column 7 in Table 1A lists residues comprising epitopes contained in the polypeptides encoded by the preferred ORF (SEQ ID NO:Y), as predicted using the algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN
(Version 3.11 for the Power Macintosh, DNASTAR, Inc., 1228 South Park Street-Madison,-WI). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, at least one, two, three, four, five or more of the predicted epitopes as described in Table 1A.
58 ' It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly.
j41] Column 8 in Table 1A provides an expression profile and library code:
count for each of the contig sequences (SEQ ID NO:X) disclosed in Table 1A, which can routinely be combined with the information provided in Table 4 and used to determine the tissues, cells, and/or cell line libraries which predominantly express the polynucleotides of the invention.
The first number in column 8 (preceding the colon), represents the tissue/cell source identifier code corresponding to the code and description provided in Table 4.
For those identifier codes in which the first two letters axe not "AR", the second number in column 8 (following the colon) represents the number of times a sequence corresponding to the reference polynucleotide sequence was identified in the tissuelcell source.
Those tissue/cell source identifier codes in which the first two letters are "AR" designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA
probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of 33P ~dCTP, using oligo(dT) to prime reverse.
transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A
local background signal subtraction was performed. before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after "[array code]:" represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased. tissues) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.
j42] Column 9 in Table 1A provides a chromosomal map location for certain polynucleotides of the invention. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Tnformation) UniGene database. Each sequence in the UniGene database is assigned to a "cluster"; all of the ESTs, cDNAs, and STSs in a cluster are believed to be derived from a single gene. Chromosomal mapping data is often available for one or more sequences) in a UniGene cluster; this data (if consistent) is then applied to the cluster as a whole. Thus, .it is possible to infer the chromosomal location of a new polynucleotide sequence by determining its identity with a mapped UniGene cluster.
[43] A modified version of the computer program BLASTN (Altshul et al., J..MoI.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet. 3:266-272 (1993)) was used to search the UniGene database for EST or cDNA sequences that contain exact or near=exact matches to a polynucleotide sequence of the invention (the 'Query'). A
sequence from the UniGene database (the 'Subject') was said to be an exact match if it contained a segment of 50 nucleotides in length such that 4~ of those nucleotides were in the same order as found in the Query sequence. If all of the matches that met this criteria were in the same Uni.Gene cluster, and mapping data was available for this cluster, it is indicated in Table 1A under the heading "Cytologic Band". Where a cluster had been further localized to a distinct cytologic band, that band is disclosed; where no banding information was available, but the gene had been localized to a single chromosome, the chromosome is disclosed.
[44] Once a presumptive chromosomal location was determined for a polynucleotide of the invention, an associated disease locus was identified by comparison with a database of diseases which have been experimentally associated with genetic loci. The database used was the Morbid Map, derived from OMIMTM (supra). If the putative chromosomal location of a polynucleotide of the invention~(Query sequence) was associated with a disease in the Morbid Map 'database, an OMIM reference identification number was noted in column 10, Table 1A, labelled "OMIM Disease References)". Table 5 is a key to the OMIM
reference identification numbers (column 1), and provides a description of the associated disease in Column 2.
TABLE IB
Clone ID SEQ ID CONTIG BAC ID: SEQ ID EXON
A
NO:Z NO:X ID: NO:B From-To HWHQR25 31 947020 AC02066I. 173 1-342 ' 1234-1287 . 5504-5728 ' 606-683 HBGMG39 44 971414 AL390719 181 ~ 1-1344 ~
' 3205-3305 HE8PY29 51 887862 AC009948 183 _ _ _ ~
1249=1380 HSIA078 67, 889498 AC002302 189 1-303 _ 6990-7582 [45] Table 1B summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID
NO:X)), and genomic sequences (SEQ ,ID NO:B). The first column provides a unique clone identifier, "Clone ID NO:Z", for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, "SEQ ID NO:X", for each contig sequence. The third column provides a unique contig identifier, "Contig ID:" for each contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A" for the BAC,clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, "SEQ ID NO:B" for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, "Exon From-To", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID
NO:B which delineate certain polynucleotides. of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
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N O~ d' co 0o O O oo d' ,-, a1 M ~ 'O ~ I~
~ M
O
0 0 0 ~ o o ~ o 0 0 ~
N
d' ~ d' ~ ~ N
~ M
N in U U
d d bn on ~ 01 ~ M
_ O _ O ~
pMp '"' '-~
O N N O
O ~ N
N
M (. ~ ~ .,.-., i., vp r, O
.
O . ,~ ~ ,~ O
w M 's~.w w. ~ w o O ~ a O
d- ~., d' w ~ w r~N .~
M d1 ' -- N
w U
[46] Table 2 further characterizes certain encoded polypeptides of. the invention, by providing the results of comparisons to protein and protein family databases.
The f rst column provides a unique clone identifier, "Clone ID NO:", corresponding to a cDNA
clone disclosed in .Table 1 A. The second column provides the unique contig identifier, "Contig ID:" which allows correlation with the information in Table 1A. The third column provides the sequence identifier, "SEQ ID NO:", for the contig polynucleotide sequences.
The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. The fifth column provides a description of the PFAM/NR hit identified by each analysis. Column six provides the accession number of the PFAM/NR
hit disclosed in the fifth column. Column seven, score/percent identity, provides a quality score or the percent identity, of the hit disclosed in column five.
Comparisons were made between polypeptides encoded by polynucleotides of the invention and a non-redundant protein database (herein referred to as "NR"), or a database of protein families (herein referred to as "PFAM"), as described below.
[47] The NR database, which comprises the NBRF PIR database, the NCBI GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-redundant using the computer program nrdb2 (Warren Gish, Washington University in Saint Louis). Each of the polynucleotides shown in Table 1A, column 3 (e.g., SEQ TD NO:X or the 'Query' sequence) was used to search against the NR database. The computer program BLASTX
was used to compare a 6-frame translation of the Query sequence to the NR
database (for information about the BLASTX algorithm please see Altshul et al., J. Mol.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the sequence that is most similar to the Query sequence (the highest scoring 'Subject') is shown in column five of Table 2 and the database accession number for that sequence is provided in column six. The highest scoring 'Subject' is reported in Table 2 if (a) the estimated probability that the match occurred by chance alone is less than I.Oe-07, and (b) the match was not to a known repetitive element. BLASTX returns alignments of short polypeptide segments of the Query and Subject sequences which share a high degree of similarity; these segments are known as High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity between the Query and the Subject for each HSP as a percent identity in Column 7. The percent identity is determined by dividing the number of exact matches between the two aligned sequences in the HSP, dividing by. the number of Query amino acids in the HSP
and multiplying by 100. The polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that generates an HSP are delineated by columns 8 and 9 of Table 2.
[48] The PFAM database, PFAM version 2.1, (Sonnhammer et al., Nucl. Acids Res., 26:320-322, 1998)) consists of a series of multiple sequence alignments; one alignment for each protein family. Each multiple sequence alignment ~is converted into a probability model called a Hidden Markov Model, or HMM, that~ represents the position-specific variation among the sequences that make up the multiple sequence alignment (see, e.g., Durbin et al., Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998 for the theory of HMMs). The program HMMER
version 1.8 (Sean Eddy, Washington University in Saint Louis) was used to compare the predicted protein sequence for each Query sequence (SEQ ID NO:Y in Table 1A) to each of the HMMs derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to be a significant match to a polypeptide of the invention if the score returned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 score obtained with the most distantly related known member of that protein family. The description of the PFAM family which shares a significant match with a polypeptide of the invention is listed in column 5 of Table 2., and the database accession number of the PFAM hit is provided in column 6.
Column 7 provides the score returned by H1VIMER version 1.8 for the alignment.
Columns 8 and 9 delineate the polynucleotides of SEQ TD NO:X which encode the polypeptide sequence which show a significant match to a PFAM protein family.
[49] As mentioned, columns 8 and 9 in Table 2, "NT From" and "NT
To",.delineate the polynucleotides of "SEQ ID NO:X" that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth column. In one embodiment, the invention provides a protein comprising, or alternatively consisting of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2.
Also provided are polynucleotides encoding such proteins, and the complementary strand thereto.
[50] The nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, the nucleotide sequences of SEQ ID NO:X
are useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences, contained in SEQ ID NO:X or the cDNA contained in Clone ID NO:Z.
These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling immediate applications in chromosome mapping, linkage analysis, tissue identification and/or typing, and a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to these polypeptides, or fragments thereof, and/or to the polypeptides encoded by the cDNA clones identified in, for example, Table 1A.
[51] Nevertheless, DNA sequences generated by sequencing reactions can.contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9%
identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
[52] Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated.
nucleotide sequence identified as SEQ ID NO:X, and a predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA
containing cDNA
Clone ID NO:Z (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and having depositor reference numbers TS-1, TS-2, AC-1, and AC-2;
and/or as set forth, for example, in Table 1A, 6 and 7). The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. Further, techniques known in the art can be used to verify the nucleotide sequences of SEQ ID NO:X.
[53] The predicted amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence. .
R~4CE Protocol For Recovery of Full-Length Genes [54] Partial cDNA clones can be made full-length by utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M.A., et al., Proc. Nat'1.
Acad.
Sci. USA, 85:8998-9002 (1988). A cDNA clone, missing either the 5' or 3' end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start codon of translation, therefor. The following briefly describes a modification of this original 5' RACE
procedure. Poly A+ or total RNA is reverse transcribed with Superscript II
(Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA
is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL).
Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoI; SaII and CIaI) at the 5' end and a primer containing just these restriction sites. This double-stranded cDNA is PCR
amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA
is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or SaII, and ligated to a plasmid such as pBluescript SKII
(Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the' correct protein-coding inserts.
Correct 5' ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3' ends.
[55] Several quality-controlled kits are commercially available for purchase.
Similar reagents and methods to those above are supplied in kit form from Gibco/BRL
for both 5' and 3' RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure' are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
[56] An alternative to generating 5' or 3' cDNA from RNA is to use cDNA
library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
.RNA Ligase Protocol For Generating The S' or 3' End Sequences To Obtain Full Length Genes [57] Once a gene of interest is identified, several methods are available for the identification of the 5' or 3' portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5' and 3' RACE.
While the full length gene may be present in the library and can be identified by probing, a useful method for generating the 5' or 3' end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5' RACE
is available for generating the missing 5' end of a desired full-length gene.
(This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993)).
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA
presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specif c to a known sequence of the gene of interest, is used to PCR amplify the 5' portion of the desired full length gene which may then be sequenced and used to generate the full length gene. ~ This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used ~is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA
oligonucleotide using T4 RNA. ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis -reaction can then be used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide~ and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the relevant gene.
[58] The present invention also relates to vectors or plasmids which include such DNA sequences, as well as the use of the DNA sequences. The material deposited with the ATCC (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and receiving ATCC designation numbers TS-1, TS-2, AC-1, and AC-2; and/or as set forth, for example, in Table 1A, Table 6, or Table 7) is a mixture of cDNA clones derived from a variety of human tissue and cloned in either a plasmid vector or a phage vector, as described, for example, in Table 7. These deposits are referred to as "the deposits" herein.
The tissues from which some of the clones were derived are listed in Table 7, and the vector in which the corresponding cDNA is contained is also indicated in Table 7. The deposited material includes cDNA clones corresponding to SEQ ID NO:X described, for example, in Table 1A (Clone ID NO:Z). A clone which'is isolatable from the ATCC Deposits by use of a sequence listed as SEQ ID NO:X, may include the entire~coding region of a human gene or in other cases such clone may include a substantial portion of the coding region of a human gene. Furthermore, although the sequence listing may in some instances list only a portion of the DNA sequence in a clone included in the ATCC Deposits, it is well within the ability of one skilled in the art to sequence the DNA included in a clone contained in the ATCC Deposits by use of a sequence (or portion thereof) described in, for example Tables lAor 2 by procedures hereinafter further described, and others apparent to those skilled in the art.
[59] ~ Also provided in Table 7 is the name of the vector which contains the cDNA
clone. Each vector is routinely used in the art. The following additional information is provided for convenience.
[60] Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap XR
(U.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988);
Aping-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17: 9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies .5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037. pBS
contains an ampicillin resistance gene and pBK contains a neomycin resistance gene.
Phagemid pBS
may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK
may be excised from the Zap Express vector. Both phagemids may be transformed into E.
coli strain XL-1 Blue, also available from Stratagene.
[61] Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897.
AlfSport vectors contain an ampicillin resistance gene and may be transformed into E.
coli strain DHlOB, also available from Life Technologies. See, for instance, Gruber, C.
E., et al., Focus.15:59- (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR~'2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, GA 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, Clark, J:
M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., BiolTechnology 9: (I991).
[62] The present invention also relates to the genes corresponding to SEQ ID
NO:X, SEQ ID NO:Y, and/or the deposited clone (Clone ID NO:Z). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
[63] Also provided in the present invention are allelic variants, orthologs, and/or species homologs: Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, andlor species homologs of genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA contained in Clone ID NO:Z, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or.
primers from the sequences provided herein and screening a.suitable nucleic acid source for allelic variants and/or the desired homologue.
[64] The polypeptides of the ,invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
[65] The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
[66] The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art; such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.
[67] The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA
sequence contained in Clone ID NO:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ
ID NO:Y, a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or the polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1 B.
Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1 B are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of, the, complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA contained in Clone ID NO:Z.
[68] Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in Table 1 B column 6, or any combination thereof.
Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in Table 1 B column 6, or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column S). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1 B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC
ID NO:A (see Table 1 B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[69] Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten; or more of the complementary strands) of the sequences delineated in column 6 of Table 1 B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequence~~~lineated in~ column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is di~i rent from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1 B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1 B which correspond to the same Clone ID NO:Z (see Table 1 B, column 1) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[70J Further, representative examples of polynucleotides of the invention.
comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof.
Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in column 6 of Table 1B
which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X
(see Table 1B, column 2) and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2) and have a~
nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID
NO:X (see Table 1B, column 2) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (See Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[71] Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of Table 1B column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s). of the sequences delineated in the same row of Table 1B
column 6, or any combination thereof. In preferred embodiments, the polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in the same row of Table 1B column 6, wherein sequentially delineated sequences in the table (i.e.
corresponding to those exons located closest to each other) are directly contiguous in a 5' to 3' orientation. In further embodiments, above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the .
above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC TD
NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[72] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B, and the polynucleotide sequence of SEQ ID
NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[73J In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1 B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ ID NO:X
(e.g., as defined in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments, the delineated sequences) and polynucleotide sequence of SEQ ID NO:X correspond to the same Clone ID NO:Z. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[74] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, orie, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of column 6 of Table 1 B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments, the delineated sequences) and polynucleotide sequence of SEQ ID NO:X correspond to the same row of column 6 of Table 1B.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[75] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of the sequence of SEQ ID NO:X axe directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides andlor nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[76] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1 B and the 5' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies. that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[77) In specific embodiments, polynucleotides 'of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[78] In specific embodiments, ~olynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B axe directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides, are also encompassed by the invention.
[79] . In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 are directly contiguous.. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[80] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column,6 corresponding to the same Glone ID NO:Z (see Table 1B, column 1) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 lower stringency conditions, are also encompassed by the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[81] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one sequence in column 6 corresponding to the same contig sequence identifer SEQ ID
NO:X (see Table 1B, column 2) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization .
conditions or alternatively, under lower stringency, conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described .polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention. ~ .
[82] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table .1 B and the S' 10 polynucleotides of another sequence in column 6 corresponding to the same row are directly contiguous. In preferred embodiments, the 3' 10 polynucleotides of one of the sequences delineated in column ~6 of Table 1B is directly contiguous with the S' 10 polynucleotides of the next sequential exon delineated in Table IB, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polyriucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[83] . Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Accordingly, for each contig sequence (SEQ ID NO:X) listed in the fourth column of Table 1 A, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 and the final nucleotide minus I S of SEQ
ID NO:X, b is an integer of I S to the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14: More specifically, preferably excluded are one or more polynucleotides, comprising a nucleotide sequence described by the general formula of a-b, where a and b are integers as defined in columns 4 and 5, respectively, of Table 3. In specific embodiments, the polynucleotides of the invention do not consist of at least one, two, three, four, five, ten, or more of the specific polynucleotide sequences referenced by the Genbank Accession No. as disclosed in column 6 of Table 3 (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequences) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).
In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.
SEQ -ID EST Disclaimer lone ID NO: ontig Range NO: Z X ID: of a ccession #'s Range of b HE8UL90 12 942749 1 - 77S 15 - 789 D80247, D80022, D51060, D80195, D58283, D80043, D8039I, D59787, D80196, D59467, D80522, D57483, 014389, 014331, D80439, D802S3, . D80302, D59889, D80166, D59619, D80210, D80164, D80240, D59859, D59502, 014014, AA305409, D51423, D51799, D59275, D80038, D80227, D80366, D51022, D81026, D81030, 006015, D80212, D80219, D80269, D80268, D80188, D80248, D50979, D50995, D80024, D59927, 015076, AA514186, AA305578, D80133, D59610, D80193, D80045, ' D80157, AA514188, T11417, AW360811, D80378, D51103, 014429, AW177440, D80251, D51759, 075259, D80241, AW178893, 003092, D59653, H67866, T03269, AW377671, AW375405, D4S260, H67854, AA809122, AI525923, AW366296, AW 178906, AW360844, AW3608I7, C0S695, AW179328, 014227, T48593, D60010, AW37S406, AW378534, AW179332, AW377672, AW179023, AW178905, D59373, AW177731, AW378528, AW178762, AW179019, F13647, AW378532, D80014, D51221, T03116, AWI77501, 014344, AW177511, AW352170, D58246, AW360834, D80258, D81111, . - AI525917, AW179020, D59S03, D59317, AW 179024;
D80064, ' AW377676, AW352171, D80168, AW378533, 014973, AW178907, AW178908, 233452, AWI77505, AW178980, D59474, D51250, AW177733, AI535686, AW360841, AW352120, AI525920, AI525227, AI557774, AA514184, AW 178775, 014046, AW367950, AW178909, AW177456, 014407, 014957, AW179004, DS9551, D58101, AW 179329, AW 176467, AW178986, AW178914, AW178774, AW178754, AW179018, AW3521~8, AW352117, AI52S242, D60214, AI525235, AI557751, AW179009, AW179012, 016955, AW178911, AW378543, AW378525, ~
AW378540, AW177722, AW352163, AWI77734, AI52S912, T02974, AA285331, H67858, AI525925, AI525215, AW378539, AW177728, D59695, 221582, D45273, D80949, D59627, D51213, AW378542, 005763, F13796, AI525222, 004682, AW178781, ' AW360855, 014077, AI525237, . AI910186, 014298, D51053, AI905856, T02868, AW369651, 230160, 013958, AI525928, D80314, AA305720, D51231, D31458, D50981, N66429, AI525228, AI525216, AW177508, AW177497, T03048, AI525238, AI535961, AI535959, AF154671, .
A62300, AF058696, AR018138, AB002449, A82595, AR060385, A84916, A62298, AB028859, AJ132110, AR008278, I50126, I50132, I50128, I50133, AR054175, AR016514, I14842, X67155, AR060138, A45456, Y17188, A94995, D26022, A26615, AR052274, Y12724, A25909, AR066488, Y17187, Y09669, A43192, A43190, AR038669, A67220, D89785, A78862, D346I4, AR008443, AR066487, A30438, A63261, AR008277, AR008281, AR062872, A70867, D88547, AR016691, AR016690, U46128, D50010, AR008408, A64136, A68321, X82626, I79511, X68127, D13509, AR025207, AR060133, AF123263, X72378, and AR032065.
HETKR83 13 9632741 - 114115 - 1155 AW195777, AW269932, AI829559, AI571060, AI083491, AA905071, AW118125, AI049799, AI376671, N90902, W27632, AI273588, AI890622, AI393483, AA040604, . ~ W38638, ~W37154, W22119, AA.904910, N92239, AT194027;
W27681, W27896, AW367713, 014616, 002576, 014877, RS5809, AA897696, AA364393, AA298658, AA017680, W23093, W27851, . H83295, W22553, D81988, 855894, AW086128, W27371, W27944, D60284, W23268, AA040705, AF122922, AF122923, and AC044883.
_ HFIZB56 14 9556181 - 666 I S - 680 AA298425.
HFKHD91 15 9512591 - 644 15. - 658 AA422028, W79191, and AB023193.
HFKKE19 16 9474181 - 276 15 - 290 D87469, AL031597, AL157901, and AL356389.
HFOXL77 17 9106981 - 602 15 - 616 W22070, AB033063, and AR065869.
HHENW06 18 9713101 - 12571S - 1271 AW387854, 860430, H15208, AA454508, AA194058, H06926, T66357, AA159059, F06846, W40388, AA453713, AA160862, F08019, AA704013, AL117551, . L40459, and AR012385.
HKGDI91 19 9272221 - 433 15 - 447 AI952995, AI884982, AW071872, AW297176, AW131657, AI871511, AI568544, AI887085, AI214075, AW295595, AI356220, AW297665, AW006191, AI423713, AI383368, AW294321, AI363919, AF084576, ACOI 1500, and ACOI
1500.
HLCMP75 20 9447221 - 949 15 - 963 AA430329, H19258, H26693, AA853085, AW361580, AA747653, AA595199, AA730621, AI339553, AI041943, H12557, AI284951, 855547, and AI652283.
HLHCR16 21 910123I - 379015 - 3804 AA402528, AI379350, AA7I6107, AI123557, AI127175, AA234106, AA234698, AI039768, N77999, AI580137, AA424560, AA419490, AI334141, 871349, AI224976, AI417798, AI080508, N58410, AI8I8475, AA424657, N91089, AA399612, AI144265, AA3991.37, . AI498363, AA410986, AA235306, AA399148, AW292497, AI249102, W86869, AA115407, H81257, AI077499, H00194, H45499, 801206, AW104245, AA234880, AA367417, AA852175, F05822, AA853077, AA852176, 882875, H81245, AA298421, 871350, 822096, H81585, AI985171, . T49265, H81591, AA115408, 864037, T54283, AI492930, AI492932, 882876, 881695, 833935, 834138, 825630, H81595, 832025, 881696, T49264, 895688, 801319, 822040, 221579, 833043, 238741, AA514393, T97662, T34993, AA705441, D79105, 832078, H45500, AA707178, 830943, H81576, H81250, AI032721, AL079279, and I76197.
HLKAB61 22 9480021 - 284 15 - 298 W70120, W99328, AI139986, AA135576, AA399431, N45043, AA226160, AA143555, AW024960, . AA190958, and M93661.
HPTZB93 23 9718421 - 836 15 - 850 AT279486, AW206040, AW138281, AI817720, AW205987, AW206016, AI694554, AA862263, AI860959, AI29is729, AI299747, AI216051, AW340960, AI703067, AI885693, AW341220, AI681397, AI802146, AI066735, AA923300, AI127856, AI279331, AI298540, AI912139, AI630777, AW140104, AI002211, AI634502, AI268259, AI796940, AI129532, AI299414, AI914342, AI692842, AA938376, AW 137441, AW207254, AI216530, AI268955, AI459037, AW136174, AI693815, AI299132, AI912208, AI301061, AI299900, AI298698, AI702631, AI298009, AI871768, AI810454, AI689870, AW016202, AI458645, AI222004, AI689871, AW102711, AI702711, AI689859, AI804311, AI732920, AI732919, AA995350, N68345, AI351290, AA825171, AA962534, AI476744, AA878309, AA974790, AA934499, AA528135, AI702851, N94016, AI187311, AW003096, AI670694, AI223259, AW139377, AA916697, AI791238, AA829735, AA885627, AI791237, AA910380, AW003320, AA911255, AA987456, AW024083, AA971615, AA916296, AA953807, and AA128824.
HRDBE43 24 894862 1 - 186815 - 1882 AI160324, AA420500, AW263972, AI206613, AI417744, AI953056, AA778217, AI863306, AI804393, AI432712, AI587218, AI218649, AI339726, AA780366, AI825578, H70209, AA470738, AI669249, H70546, AI611674, AI792038, 808140, T82124, AI791228, AI820643, AA847657, AI342131, AI342141, AA989292, AI631664, AI697858, AI697880, AI278251, and 884867.
HSSKD85 25 908141 1 - 113615 - 1150 AW024960, AI479960, AI052585, AI139986, AA135576, AW137104, AW205456, N30762, AI631818, AI187156, AA830014, AI452434, AA634216, AW088488, AA398256, AI859053, AI088449, AI159823, AA564062, AI494095, AA158516, AI311717, AA811798, AA226160, . AI873741, N25872, AA146624, AA761003, AA190958, AI066392, AA535733, AI278434, W92242, AA225625, N40580, H94758, AI382438, AI796467, W92243, . 859937, H94384, H94363, AI969940, W70120, AW338143, W99328, AA143555, AA399431, AA190865, N45043, AI718153, -' AI245054, AI906964, AI760352, AA135514, AA653523, AI708811, AA034276, W69970, AA156400, M93661, D32210, and U57368.
HTEOF80 26 847224 1 - 506 15 - 520 AF186084, and AL117610.
HTNBMOI 27 910705 1 - 638 15 - 652 AA298841, and 228624.
HTSHM38 28 972248 1 - 604 15 - 618 AI907172, C14389, C15076, D59467, D80164, D59787, D81026, D59610, D51799, D58283, C14331, D80022, D81030, D80043, D80241, D80038, D59502, D59859, D80227, D80166, D80195, D51423, D59619, D80210, D80391, D59275, D80240, D80253, D80212, D59927, D80196, D80188, D50995, D80219, D80269, C14429, D57483, D80366, D50979, DS9889, D80193, D80251, C14014, D52291, AW177440, AA305409, D80378, D80024, D51022, D80045, AA305578, AW378532, D8~522, T03269, AW178893, D51060, C75259, T11417, AA514188, AW 179328, D80248, AW369651, ' D51250, AW352158, D80134, r D58253, C14077, AW178762, AW1,78775, AW1~77501, AW177511, C14407, D80268, AA514186, D80133, AW176467, AW360811, F13647, AW377671, AW352117, C05695, AW375405, D80132, AW378540, AI910186, AI557751, AW366296, AW360844, AW360817, AW375406, AW352170, AW378534, AW179332, AW377672, AW179023, AW178905,~D51213, AI905856, D80247, D80302, AW352171, D80014, D80439, AW377676, AW178906, AW177505, AW177731, AW178907, AW179019, AW179024, D59373, C06015, 221582, AW179020, AW360841, AW178909, AW177456, AW179329, AW178980, AW177733, AW352174, AW378528, AW178908, AW178754, AW179018, AWT79012, AW360834, D81111, D51103, AW179004, D59627, ' T02974, AW178914, AA285331, AW378525, D80258, D51097, AW367967, C14227, D80157, AW177722, AW177728, ' , ~ ' AW179009, D51759, AW178774, AW178911, AW378543, AW352163, D58101, D58246, ' AI557774, AW178983;
D59503, ' AW352120, AW178781, T48593, AI535850, AW177723, D59653, AW177508, D45260, D80168, C14975, H67854, C03092, AW378533, H67866, AW367950, AI525920, AW177497, AA809122, C14344, D80064, AW178986, AI525923, D80228, D59551, AW177734, T03116, AI525917, D45273, D59317, D80949, D51079, C14973, D51231, D60010, D51221, ' D59474, AA514184, AI535686, C14046, AI535961, T03048, ' C14957, D60214, AI525227, 233452, AI525235, AW378539, C16955, AI525242, AI525912, , AW378542, AI525925, AI525215, AI525237, AA305720, C05763, D59695, AI525222, H67858, C14298, AW360855, A62300, AR018138, AJ132110, A84916, A62298, AF058696, A25909, X67155, Y17188, D26022, AR008278, A67220, D89785, A78862~ D34614, AB028859, D88547, X82626, Y12724,'A82595, AR025207, A94995, AR060385, ' ~ AB002449, AR008443, AB012117, I50133, AR066482, I50126, I50132, I50128, X68127, A85396, A4417I, ' AR066488, AR016514, A85477, AR060138, A45456, I19525, A26615, AR052274, A86792, U87250, X93549, AR054175, " Y09669, A43192, A43190, AR038669, AR066490, AR066487, A30438, I18367, D88507, I14842, D50010, Y17187, A63261, AR008277, AR008281, A70867, " AR008408, AR062872, AR01.6691, AR016690, U46128, D13509, AF135125, A64136, A68321, AR060133, 179511, AB023656, U79457, AF123263, AB033111, AR032065, X93535, AR008382, and 242460.
HUSXE73 29 953246 1 - 129615 - 1310 AW138763, AI968244, AI671228, AI146849, AI650986, AA974891, AI935406, AI375I39, AI632343, AI580312, AI190358, AI823383, AA758662, AW166381, AI816934, AI3621'70, AI307616, AI33951 I, AI092493, AI193719, AA676785, r AA701414, AI375073, AI090245, AI077483, AW003931, N70081, AI307365, AI991601, AI967935, AI990350, AI637874, AI825545, m~
AI621021, W67234, AI186726, AW206481, N30322, AW140070, AW338117, AA031644, AI095704, H00954, AI859068, AW136394, AW263085, 839467, AI984849, AW338430, AI219050, AI334231, 862632, AI241351, AI355851, AI334036, AA449686, AI336416, H72039, AI735518, AA699736, 839468, F26300, 830863, AII30689, W67345, AI620138, AA358091, N74688, AA704504, AI524317, AI183860, AI801924, 874316, AI933476, AI933484, AA448958, H16951, N56653, 862685, AI634964, AA031725, H72038, T19026, AI433512, H00953, 831133, AW192226, AW235028, AI888621, AW190428, AI805638, AW029072, AI539153, AI628292, AI921082, AI379711, AW029606, AW188491, AI583533, AW002174, AI091468, AI598113, AI636719, AI358455, AI620093, , AI566507, AI498579, AW168723, AI811192, AI207454, AW088899, AI366549, AL046463, AI866608, AI874410, AI611743, AW083804, AW118332,.AA830821, AI696626, AI589993, AI365256, AW085786, AI805769, AW26S004, AI677797, AI364788, AI648567, AW089801, AI636619, AI866786, AW051107, AI866082, N74355, AI282651, AW129271, AI863397, AI310155, AI952920, AI536557, AW172723, AA579232, AW403717, AI539771, AW131954, AA420722, AI919345, . AI805688, AI251830, AI565125, AI862324, AA807352, AW168373, 840432, AIS39632, AI470701, AI738867, AI312428, AI434242, AI371'228, AI801605, AW080080, AI610429, AW168425, AI432736, ' AI307736, AI473598, AI499986, AI273839; AA928539, AI872064, AI568870, AI868831, AI869750, W33163, AI874151, AI950664, AI436429, AW087901, AI470293, AI570966, AI867042, AW082040, AI929108, AI537837, AI573026, AI699862, AI859464, AI242646, , W46547, AI445430, AI249877, AI862144, AI689420, AI879693, AI249946, AW130863, AI922577, AA848053, AW059713, AW068845, AI345677, AI274769, AI554218, AW192375, AA572758, AI872051, AI375730, AW 161892, AI800152, AW 191844, AI917055, AW 151750, AI702301, AW088134, AI500146, AI699255, AI570384, AI633477, AI680498, AA176980, AI453413, AI370390, AL047344, AI499512, AW162194, AI889147, AI678411, AI636445, AI494201, AI561299, AA693347, N71180, AI686823, AI539071, AI537307, AI674838, AA761557, AT344935, AI886124, .
AI476077, N75771, AI885974, AL036718, AI872074, AI560010, AI537617, AI311892, AI623682, AF18611I, AR059958, AL137556, AL122121, AL136842, AL133093, AL122111, AL080127, A08910, A08909, A08908, I41145, E15324, AJ242859, AF113676, A08916, AL133645, I00734, A18777, AR019470, E00617, E00717, E00778, AL035458, I89947,148978, A08913, I89931, AL133077, r ~ . AF090896, I49625, A08912, AR038854, AB019565, AF093119, AL133104, X62580, I26207, AL137527, AF000145, AL080060, X52128, AL080158, AF051325, E08631, D44497, AC002467, X53587, AF119337, A90832, AF097996, X70685, AL133075, AL080137, AL133031, AL122123, AL137300, AF012536, L13297, X92070, A23630, AL133081, AL117585, AF004162, U72620, AL122050, AL137281, AL137648, U96683, AL133S68, AF085809, AL133080, AL122098, AF125949, AL137273, X93495, AF081197;
AF081195, I89934, I89944;
M86826, AL133067, I09360, I33392, AL049466, I68732, AL137665, 568736, AL080086, AF003737, AF110329, 272491, AF106827, L30117, U55017, AF017152, AF090886, AL133014, A12297, AL133072, X65873, AJ0064I7, E15569, AL049465, AL137429, E03348, AF031903, AL137557, ALI37258, U68233, I92592, E07108, AF158248, AL122118, AL110222, U91329, S76508, AL137479, X72889, AF113691, AL137463, S61953, AL122049, AF118064, AF118070, AJ000937, AL133098, AJ238278, D89079, AL133557, I30339, I30334, Y09972, v U49434, U80742, AL137705, AL133113, AF026816, AL117432, X81464, AF111112, AF162270, U00763, AL137283, AL049938, AL049283, U67958, AL049382, I42402, AFI 11851, AF210052, I17767, AF159148, AL137538, AL137529, AL117457, 577771, AL096744, AJ003118, AL050146, AF106862, AF067?90, Y10655, Y10080, E02253, Y10936, AR000496, U39656, AR038969, A49I39, 237987, U00686, AF040751, AL050108, S79832, AF022363, M92439, AL110218, A93016, AF113013, L04849, .
A08907, AF078844, AF091084, AL137526, X87582, E05822, AF132676, AF061836, AL117583, X67688, X84990, E06788, E06790, E06789, 109499, A45787, I66342, AL137294, AL050138, AL122110, AL137574, AL050277, E04233, AL 110196, AL049314, A~079763, A07647, AL137712, U68387, AL110225, AL117394, AF069506, AL050393, U42766, AL133565, AL133606, X63574, AF061573, AF057300, AF057299, U88966, AF 142672, A21103, AL
110197, AF028823, AF100931, AF113689, AF126247, L19437, Y11587, AL137478, AL080159, AL137640, AL133640, AB007812, AF061795, Y14314, AF151685, AL133016, AL117440, AF061981, U78525, AL080148, AF030513, and I48979.
HWAAE95 30 789051 1 - 15 - 489 29.8743.
HWH 825 31 947020 1 - 15 - 565 AC020661, and AC020661.
HGBG022 32 11249101 - 15 - 1468 AI927400, AI678696, 1454 AA947645, AW295277, AA884167, AW296908, AA947188, AI984289, T28629, 211502, X80208, and X80209.
HCECQ23 33 938398 1 - 15 - 810 AI480182, AI500178, 796 AI873131, AA322958, AA322718, AI936088, . 885125, 890888, H05353, AA338672, H51247, 835934, AW139057, H05303, 849451, 842549, H29245, AI654790, AI638508, 851648, AW149807, 844423, AI458144, AI419465, F02105, AI499775, 240525, F09051, 885080, H51217, AI569283, D29763, D64009, and D64010.
HFXBI19 34 1136133I - 15 - 2218 AI796164, AF034611, 2204 and AF022247.
HMVCP64 36 11761521 - I S - 1422 AI215040, AW013830, 1408 AA425778, N30265, N41988, AA629285, AI867286, AI252688, AI251390, AI254897, AI252025, AI252700, AI254872, AA426241, and 297832.
HSXBV89 37 971821 1 - 15 - 652 890750, 887845, 825677, AJ245820, AJ245821, and AJ245822.
HTXAAI 38 11727351 - 1.5 - 2477 AA814365, AA648502, S 2463 H11953, 820002, AA7461 I7, H06599, AA374468, and W60570.
HWHQR10 39 915008 1 - 15 - 326 AC004235, AC004235, 312 AC004235, and AC004235.
HMZAD58 40 10021331 - 1'S - 2734 N26584, W94986, N38905, AI075815, AI367921, AI184158, AA830019, AI033601, N54995, AA830021, N27197, AA918808, AI539580, AI273730, AI262545, AA587088, AA779942, AA262747, AA676908, AA287348, AA730411, W86602, H16056, AA303482, AW 179318, W91912, AA670033, AA705754, AW300038, AA887595, AA703588, AI219099, N46479, AA312749, AA903850, AA495756, AA351345, AA887468, AA287308;
AW451932, AW197479, AI890907, AI538850, AI887308, AA761557, AL039132, AI873638, AI590686, AL036638, AI242931, AI627988, AI689702, AA848053, AW022699, AI922550, AI697372, AI440263, AI249946, AI801325;
AI683072, AI335426, AI348777, AI863382, AI818574, AL079960, AI281867, AI446538, AI567612, AI590423, AL041150, AA911767, AL038445, AI288050, AL037454, AL045626, . AI619426, AI354998, AI858827, AA502794, AI973152, AA908294, AI683395, AW161579, AI866090, AI082740, AW265004, AI619607, AI559632, AI698391, AI679506, AL048427, AW198090, AI868740, AW161156, AI680388, AI280747, AI344935, AI161279, AI537677, AI494201, AL047100, AI491852, AW051088, AA641818, AI873644, AI470293, AW023859, AI540458, AI916419, AI470648, AL119863, AI961286, AW167385, AI536685, AI570884, AI498067, AI538116, AI950664, AI624529, AI687166, AI9277S5, H89138, AI554343, AI241923, AI677824, AI473799, AI670009, AW243886, AL036631, AI913082, AI648408, AA225339, AI696612, AW059828, AI637748, AI932458, AL036772, AL036396, AAOOI397, AI872I84, AI872423, AL036980, AW029611, AW079336, AI969655, AI540674, AI251221, AW022682, AA568405, AI863321, AI174394, AI817552, AW020095, F37471, AI623941, AI500706, AI648508, AI469674, AI538342, AI345745, AI473536, AI887430, AW057937, AI500077, AI631216, AW149878, AW131428, AL046618, AI345347, AL079963, AW302924, AI538218, AI765469, AI499263, AI953562, AW192712, AI539687, AA572758, AI766348, AI868204, AI890507, AI537261, AI863191, AI269696, AI432040, AI918449, AI620284, AI263331, AI242248, AI801523, AI358701, AI445992, AI568138, AI445990, AI624293, , AW080746, AL036673, AI868931, AI783504, AI784230, AW 149925, AI568114, AI434741, AI419650, AI866770, AI343059, AI433157, AW172723, AW131999, AI702073, AI538764, AI554821, AW129271, AI612750, AW028416, AI283760, AI349933, AL039086, AI281757, AL038605, D63482, AFI24491, ' AF112366, ALI 10296, AL137574, 237987, I48978, AF146568, AL1335S7, AL133606, I89947, A08913, A08912, AL122093, AR013797, AF1I I 112, AF113694, AL137271, A089I6, A65341, A08910, AL122049, A08909, Y10080, A08908, AR029490, AL117578, AL133016, AF106657, E02221, AR038854, E08631, E05822, X83508, I89931, L19437, I48979, I41145,149625, AL050172, A93350, AF000301, AL023657, AL050I08, AF057300, AF057299, A18777, AL049283, AF153205, AF090900, ALI22110, Y16645, Y09972, AF113689, AR034830, AL050024, Y14314, 561953, AF 113019, AF090934, X79812, I33392, E02349, AF026I24, AL110225, S68736,'AL137521, AF097996, AF106862, AL133665, AF177401, AF090896, AL050155, S76508, AL133067, Y11254, AL137550, AL122100, AL137558, AL137548, AJ012755, I96214, AF113699, 282022, AF079763, AL117460, AL080158, AF008439, AL,120280, I68732, AL137476, . AL137463, A08911, X82434, AL110197, AF113691, E01614, 1m E13364, X5587, AL080074, I89934, I89944, AF118070, X70685, AL117585, AL050116,~U78525, AL137294, AL137292, I00734, AR020905, I26207, AF017437, AL049300, 272491, AL1374S9, AF017152, A23630, E00617, E00717, E00778, AL117394, AL050393, D83032, AF003737, A08907, E02253, AL117440, S78214, AF139986, AF137367, U35846, AL133560, I80064, AF090903, AR038969, U95114, A77033, A77035, AL049430, ' E06743, AL049452, AJ238278, U87620, AF125948, Y07905, AL117435, X65873, X96540, E12747, AL050277, AL080086, S36676, AL137557, AF067728, AF087943, AR000496, AL133645, AL133640, U39656, AL080154, AF111849, E07108, AL137533, U68387, AF185576, AF090901, AL133113, AL050092, AF032666, AJ005690, AF061943, AL080124, AL137429, AF028823, AF100931, AL050149, L31396, A08915, AL080I37, AF030513, AF079765, L3I397, X72889, A58524, A58523, 575997, AL133093, AL137478, AL080159, AF051325, X80340, A90832, U58996, ALI22098, ALI37529, AF061795, AF151685, AL096744, AL096720, AF06198I, A52563, AL080148, AL133010, U42766, AL 117432, AL 122121, AB019565, AF162270, AL133104, ' AL049466, AL122050, X62580, I03321, AL049314, AL133081, . M30514, D16301, AB007812, ' U68233, I92592, AF158248, . E15569, AL133565, AJ006417, U91329, U00763, E04233, AF065135, AL137560, AL117416, AL117583, AF183393, AL122045, . AL110222, AL080060, AF119337, .
I09360, X87582, Y11587, , AL110196, AJ000937, AL133080, AL049382~ AF118090, AF106827, . I42402, and AL137648.
HWDAE40 41 947007 1 - 216615 - 2180 AF150174, AI417513, AI698235, 856970, AA471187, AC008917, AC0089I7, and AC016605.
HADFCS1 42 10021421 - 392415 - 3938 AA203426, AI300I88, AW139439, AI379134, AA815253, AI568548, AW 134634, AA484158, N50870, AA687654, AW072618, N48188, AI304847, AA464588, N62844, N53335, AA043385, W80785, AI248256, AI206713, AI719303, AI630905, N77751, AI339657, AA932565, AA115602, 244436, . AW193024, AA525284, AI281971, AI298245, 854464, AI041687, 849127, AI091286, AI918287, AI887073, 240366, 806527, AA995384, M85459, AI125279, N26208, H97245, AA115137, W80892, AA043384, AA331941, AA523243, AW149086, AA902505, H02196, C21424, and AL008628.
HAWAM69 43 9431041 - 190115 - 1915 AA430300, AA541688, AA776700, AW385785, AA679037, AA573270, _ AA126614, AL045796, AA682186, AI268236, AI963606, AW192904, AI926591, AI924827, AI922590, AI032288, AI375804, AA705I72, AW081541, AA6945i4, AII30883, N25288, AA931725, AI800450, AI270687, AI366906, AW058362, AI683319, AA436891, W69578, AI597744, AI446542, 859176, AW453004, AI911821, AA687634, AI095665; AI130013, W69579, AA722782, AI191864, AI587015, AA398533, AA676733, AI476374, AA115447, AA554327, AA759328, AW242281, AA042956, AI139766, AA135916, AA886732, AA664356, AA358590, H71919, AI565897, AW304844, AA916086, AA618576, AA363371, 244808, AA430199, AI370031, AA320329, AA393105, AW452852, AA135927, AI004I40, AA135926, AA042816;
H44791, T35731, AI865731, AA813424, 842647, 827785, T32691, AI934183, AA115446, AI857286, AW008428, AI631988, AA678468, AW075384, H44790, AI569918, AI918635, AA603858, AA601518, AI745618, H42641, AI445766, 827874, AI939990, AA677131, AW364938, AI569374, AW029062, C01947, and AA732827.
HBGMG39 44 9714141 - 471 15 - 485 AA024454, AA024670, , AI458409, AI740930, AI742565, AI743686, AI066465, AI168481, AI379125, AI569972, AW069135, AI497641, AW192429, AW084071, AW339039, AW102701, AI061450, AI693756, AI991329, AW003414, AI342244, AI870883, AW007899, AI609020, AI453165, AI248142, AI129686, AI992036, AI761292, AI623708, AI864435, AW151858, AI362058, AW044270, AI378430, AW008808, AI479128, AL036585, AI923881, AI963067, AI740972, . AL045227, AI352617, AI334039, AI452903, AI700412, AI475537, AI469546, AI187911, AI066744, AI190677, AI812069, AI081231, AI050026, AI890929, AA551905, . AI080156, AI554840, AW073739, AI926062, AA577674,_AW190499, AW250073, AI524714, AI884727, AW168902, AI096904, AI369151, AI858574, AI422058, AA304774, AI890721, AI052823, AI683215, AI087154, AA682804, AW080685, AW004991, AA912478, AW079985, AA054275, AA461453, AI668789, AA970861, AA010790, AW085591, AI819302, AI217784,AI017023, AI584163, AI627434, AA709077, AA984939, AA446048, AI564659, AI000961, AI095374, AI581899, AA532435, AA864689, AW087352, AW057507, AW132118, AA744639, N53410, AA969103, AA918799, AA862362, AI672825, AA779825, AA931463, AI193638, AW 191953, N20167, AI569968, AW150032, AA612750, AI619904, AA429899, W02113, AA969104, W48745, AA827905, AW190663, AA780072, N50640, AA533847, W32574, AI640737, AI587533, AW182184, AI933617, AW073566, AI469425, AL041483, AA765028, AI952283, AI961508, AI095341, AA937508, N48059, AA228075, AI869852, ~
. N78603, AA485535, AA506502, AA877348, AA252354, AAOlOIOI, _ AA011219, T23065,'AW444850, AI917215, AA506948, AW297832, AW204879, N53257, AI917614, AI167689, AI937775, AI858564, AW043696, AW168023, AI263865, AI885655, AA450098, AWI39189, AA883859, E12258, AF153686, E12259, E12260, and AL390719.
HCEHD66 45 959160 1 - 15 - 1325 AI968437, AI824971, . 1311 AW104052, AI762197, AI598138, AI088543, AI492390, AI827280, AA058923, AW007187, AW135225, AI391466, AI808139, AA534403, AW028554, AI369729, AA460467, AA135928, AW006062, AW138526, AA507443, AI479413, AI400940, AW 137272, AW381735, AA135929, AA085774, W811S3, AA918755, C03738, 885039, AI472852, AI937792, AI867512, AA599118, N62215, . AI864402, H41491, N62216, D44882, H14329, W30972, H40979, AA4.63408, AI744140, H40980, N62166, AA319197, AI766568, N62223, AF186409, AF020184, L27421, L27420, and AC006241.
HCESP56 46 8276711 - 500 15 - 514 AW247740, AW247029,-AW204207, W39269, AA325536, 814422, W52568, Y16752, AL022170, and 265186.
HCHAT01 47 8672091 - 154215 - 1556 817167, and AB014576.
HCHMW40 48 9515181 - 870 15 - 884 AI732539, AI791495, AI791325, ' AA709067, AW082062, AI791964, . AI732667, AA505923, AI909857, AI909862, AA601601, AW057561, and AI909853.
HCUEV29 49 8160651 - 491 15 - SOS AW410192, AI570209, AA583494, AW087991, AW337550, T30350, T24722, AW246233, and ' AL031283.
HDQID90 50 8319761 - 953 15 - 967 AA767219; AI809238.,.AI219470, AA767092, AA114887, T71487, AA464762, AA504439, 225261, N87679; D57415, AA278335, AW300598, W46278, AA669095, D56990, D54675, AI797687, AI948608, AA909071, AW236181, AI718165, AB033082, AF132480, AF132479, and AC002350.
HE8PY29 51 8878621 - 741 15 - 755 AI27I550, AI753504, AA809220, AW081079, W78099, AW386283, AI264068, AI219556, AW082138, AA455733, AI382746, AA548778, AA431230, AF0921.37, AF100751, ' AF040252, AC009948, AC009948, ' and AC009948.
HE8QZ34 52 9522831 - 107015 - 1084 835313, AA210809, W28575, ' AA112I26, 886156, AA286.753, AW405566, AA385668, AA384297, H25863, H50786, 825032, 246079, . AA334931, AA490204, and AW367213. ' HE9TD31 54 8158451 - 941 15 - 955 AI475682, AI439613, AA815076, ' AB033082, AF132480, and AF 132479.
HELHB88 55 8119351 - S54 15 - S68 AI750406, AI580905, AA024853, AF114488, AF114487, AF064243, .
AP000116, AF064244, AP000049, AP000311, AF132672, AF127798, AF132478, and AF132481.
HEOPP67 56 8276301 - 436 15 - 450 AA641653, 299396, AF181972, and AF 181973.
HGBDG55 57 8158581 - 522 15 - 536 AA368408, and AL360268.
HHFOC79 58 9354061 - 1024_ AI569931, AA450162, 15 - 1038 AA405198, ' H26214, H14443, N28528, 873380, AW015358, 848456, AI750978, AA358230, 219130, AA359395, AA744173, H26831, and AC008745.
HHGAE47 59 9221941 - 705 15 - 719 AW025529, AW026010, AA657904, AA662803, AA886335, AA158820, AI475932, AW050607, AI885090, AI056120, AI244837, AA485S66, AI375435, AA922036, AA878578, AA643750, AI056614, AW449834, AW 197722, AI393408, AA485405, AI560410, AA161103, AI749095, AI72093I, AW058170, AI446208, N57590, AI268967, AI832600, AI913781, AA910277, N52768, AI277003, AI914599, AI192693, N57604, AI673692, AW050712, AA631339, N52783, and AI919380:
HKAOV71 60 8276791 - 743 15 - 757 AF123303, and AF004161.
HKGDE58 61 945039I - 925 15 - 939 T75535, AA287162, AA448686, ' 813858, H12041, 246111, T94956, F07044, H05358, AA053290, ' AA454139, F05720, F05717, AL122084, and AL049611.
HMCGL45 62 9221951 - 114115 - 1155 AW025529, AI475932, AW026010, ' AA886335, AA662803, AI056120, . ~ AWOS0607, AI885090, AI375435, AW449834, AA161103, AI244837, . AA878578, AA922036, AW197722, AI056614, AI393408, AW058170, AA643750, AI560410, AI749095, AI720931, AI446208, N52768, AI913781, AI277003, AI268967, AA910277, AI914599, AI192693, . ' N52783, AW050712, AA48S405, AI673692, AA631339, AA657904, AA485566, AA158820,~AI619710, AI560351, AI919380, N57590, .
AI832600, N57604, T25136, AW198090, AI499963, AW023338, AI638644, AI890214, AI538850, N75779, AI633125, AI686817, AI499570, AI500061, AI860027, AI473536, AI925164, AW163834, AI690813, AW162194, AA641818, AI684244, AI469505, AI376425, AI802542, AI673363, AI670009, AI498067, AW082532, AI433I57, AI886055, AW080700, AI702073, AI884318, AW128834, AI633198, AI289310, AI620056, AI590043, AW103928, AI890907, AW152182, AI589428, AI961S89, AI679550, AI961414, AI698391, AI479292, ' AI701097, AI570861, AI147292, AW169604, AL045413, AW081383, AI345688, AI245008, AI539800, AI950729, AI927233, AL079799, AI491775, AI872423, AI538980, AI288050, AI635634, AI440239, AA805434, AW161579, AW151893, AW148363, AI866465, AI538564, AI571439, AW083374, AL041150, AI973152, AI445611, AL047100, AI538116, AI687362, AI281757, AI241923, AA580663, AI440399, AI095003, AW022808, AI540674, AI954475, AI499890, AI471282, AI648494, AI927755, AI621341, AI445829, AI432030, AI915291, AI932503, AI341690, AI613038, AI866040, AW073865, AI699823, AW190194, AI55961.9, AL037582, AL037602, AI627988, AI685005, AL039086, AI348901, 841605, AI932794, AI345415, AI636588;
AW163554, AI572096, AI612852, AW050998, AI580436, AI627893, AI568138, AA587590, AI869377, AL046466, AL118781, AI819545, AI270183, AI912434, AI567513, AI524179, AA830709, AW051088, I48978, I48979, AL137550, AF061573, A15345, I89947, I68732, A65340, AL117460, A77033, A77035, X63-162, AF153205, AR029490, AL023657, D16301, AL117587, U78525, AL050366, E12747, AF067728, AF115392, AF090900, AR038854, A76335, ' E02349, AF102578, I33392, A58524, A58523, A08910, A08909, A6S341, AJ005690, AL080159, A08908, 282022, A58545~
AL137294, AL080074, AL137533, A52563, AL137479, U373S9, X70685, A93350, AF047716, , A03736,.AL,122100, A08913, U49908, AL122110, X82434, AF183393, AF094480, 237987, AL050393, A08912, Y10080, S54890, A08911, A08907, AL133049, AF113690, AF100931, AL137271, U35846, AL096744, 213966, Y14314, AR034821, S76508, AF026816, AL136884, AF215669, AL049347, AR068753, AL050149, AF177401, AF039138, AF039137, AF146568, AL117435, X72889, X53587, AL050155, AL133112, AL133637, AF031147, US5017, X67688, AF090903, AL137480, D83032,. AL050277, AF097996, AF200464, AL117457, AL133665, AL080163, 297214, AL133623, AR020905, AF017437, AL122049, AF113699, ALI10158, AF158248, AL137656, AL133010, A21103, AF 111849, AL
110221, AF061981, AL080148, X81464, L04849, A18777, S36676, AL137530, I25049, E01614, E13364, AF115410, AC002471, AC005374, S75997, I89931, AF145233, AL049339, A07588, AF087943, U95114, AL080110, AL080139, AL137529, L04504, S77771, AL117392,I49625, AF107847, AL117416, AL137459, Y09972, AF058921, I09499, - . AL137476, AL137537, AF114170, AL137526, AF090943, AL133558, E05822, I33391, Y11254, AJ000937, AL133080, AL122104, AF141289, U58996, AL133075, AL050116, S53987, AL137488, U72621, AF030513; AL133113, AL133072, .
- AF032666, AJ012755, X83508, AF061943, A17115, A18079, A08916, AF106945, I89934;
AL133067, U83980, Y10823, . U90884, AL117585, X66862, X66871, AL050024, 582852, AF176651, X84990, I03321, U96683, AL137256, AF180525, A12297, AL137521, AF106862, AF057300, AF057299, AL110225, AL110218, AR011880, A18788, AF113694, AB016226, A08456, A86558, A76337, AF118092, E 12806, AF026124, AF061795, AF15I685, U73682, I32738, AF090901, AL122093, AL050092, AL050138, and AL137292.
HMSOL52 63 921126 1 - 15 - 1304 AI911515, AI360955, 1290 AW028045, < - AI796049, AI609712, AW195544, AI184337, AI470056, AI361065, N34939, AI017177, AI038779, AI440241, AI651451, AA789292, AA854683, AI765258, AI702748, and AA384884.
HMTBB 64 950884 1 - 15 - 514 AA582539, AI963340, 17 500 AI097093, - AI460219, AA286856, AI761614, AI149781, AI032670, AI819154, AI636161, AI089302, AI811219, .
H12042, T95010, AA836993, AW271462, H05308, 837000, AI001803, AA904906~
AA743196, - AI015200, AA453607, F05000, AA578803, AI241466, AI033193, AA037601, AA330970, F03322, F01968, T75492, N47542, AW183219, AI288171, AA651907, AA054759, F01965, AL122084,~
and AL049611.
HOEET48 65 963290 1 - 15 - 1480 AI797684, AW239200, 1466 AA456267, _ AI478733, AI751749,AI990902, AA427646, AI379565, AI970534, W95460, AA788855, AA405402, AW068453, AW294114, AI751750, AA594137, AA947297, AW177719, AI057073, AA427487, AI341112, AA232452, AA041304, AW068711, H73236, AA041328, W95567, AW 167569, AA853047, AI652166, ' W02069, H74.164, 834003, AI341381, AW176526, AA580289, D30965, D31 I76, AA367502, and AR035969. ' HOUHL51 66 815891 1 - 15 - 647 AA431822, AA037543, 633 AI656610, AA431419, AA974280, AA815270, AA037457, AI341790, AW295199, AI651702, AW292290, AA583011, AI208605, AI419858, AA620408, AA417333, AA4I7321, W28051, AA251183, AI917695, W28536, and _AI024754.
HSIA078 67 889498 1 - 15 - 829 AA377072, AA298640, 815 AA453038, AC002302, AC012317, AC012185, and AC002302.
HSOBC04 68 927280 1 - 15 - 1220 AA115298, AI741325, 1206 ~ AI688227, AI819333, AA452504, AI9256.64, AI742595, AI174,530, AA115338, ' AI567500, AA563582, AA461615, AI142563, AA807844, N94422, AI095261, AA569395, W58424, ' AA687480, AA479551, AA582573, AI081428, AA779677, AI280806, N24393, AA988617, AI863187, AA834079,. AW302361, AI362861, AW273442, AA150123, AA553678, AI752480, AI312661, WS2661, AI298150, AA463418, W72509, AA024450, W72139, AI037968, W79868, AI028169, AA477651, H39596, W02690, AI198327, AI952450, AA926794, AI087245, W74236, AW004736, AI334346, AI870989, H98040, AI689546, AI332748, W76066, AAI50031, 840403, AI349417, AA595996, . W80872, H99144, AW166280, W52767, AA496878, H2,5985, AI357863, 855375, AA363023, AW 104147, AA378409, AI979074, AI376184, AI687489,T32290, N26307, H97338, N95244, W77880, AI917258, W2S604, AI536791, AA024802, AA577352, AA328156, AA359865, AA367475, AA461442, AA358275, W80763, T09474, AA987427, AI611160, AA888165, AA595303, AI9I8172, W30769, AI201782, AA187662, W21074, AA4119S5, AA935961, AA090719, AA4119S6, AA451977, AI371307, AW074526, N79974, AI635472, N39751, AI612934, AA478489, AA102215, AI802295, AI750502, AA496836, AL133116, L07063, and AC012192.
HTEIL07 69 953803 1 - 15 -_459 H55431, and AL031843.
HTEKS20 70 846714 1 - IS - 1077 AI936596, AA868353, 1063 AI797296, AA725553, AI221970, AA429551, AA428462, AA629305, AA629047, AA431190, AI073397, AW235895, AI123443, AI808267, AA609412, AI914363, AA953895, AI214385, AA431516, AA911681, AA781953, AI825106, AA298758, AI215028, AA909534, AA723768, D10393, 563991, AL137023, and AL137023.
HTEON29 71 815852 1 - 15 - 992 AW004028, AI968030, 978 AW237673, AA432290, AW138422, AA112090, AA428635, AI143780, AI143791, and AA861634.
HTLEN77 72 772363 1 - 15 - 424 T89583.
HTTEK47 73 573649=1 - 15 - 410 AA400005, AW205244, 396 AW 134723, and AI689951.
HTTJW49 74 948107 1 - 15 - 662 AA199865, AI769428, 648 AI061340, AW268880, AA707168, AI970984, AI884812, AW444872, AI479954, AI356088; AI701720, AI765045, AA722812, AW236544, AA410516, AI267987, AA005114, AI298592, AI865503, AI633370, AA878382, AW389168, AF118838, Y17571, AF164632, AF164526, AF164527;
AC004458, AF164528, and AF 164525.
HUJCT05 75 929264 1 - 15 - 639 AI124874, and AC003962.
HWBEG18 76 909798 1 - 15 - 954 AA906863, AW408789, 940 AW452373, .
AA352977, AA732349, AI269653, - AI492098, arid AB020653.
HWLFG75 77 916563 1 - 15 - 1269 AI356559, AW163067, 1255 AA443325, AW005140, C18386, 815375, 817389, 860462, AA442531, H16941, AA740299, AA025666, AA443338, R42I16, 860229, 842625, AW444512, AW450707, AW157098, AA724594, AA978110, AI810652, AA927875, AI924004, H16834, AI886594, AI376913, AA609873, AW173645, AA578062, AA578362, AA467933, AI147260, 852646, AI672253, and AI347103.
HWNCY05 78 928789 1 - I5 - 1380 AA210942, W40569, AW025860, .
D63226, AA362635, AA334307, AA211707, AI613267, and AC006928.
Code Descri tion Tissue Or an Cell DiseaseVector Line AR022 a_Heart a_Heart AR023 a_Liver a_Liver AR024 a_mammar gland a_mamma oland AR025 a_Prostate ~ a_Prostate AR026 a_smail intestinea_small intestine AR027 a_Stomach a_Stomach AR028 Blood B cells Blood B cells AR029 Blood B cells Blood B cells activated activated AR030 Blood B cells Blood B cells resting resting AR031 Blood T cells Blood T cells activated activated AR032 Blood T cells Blood T cells resting i:estin~
AR033 brain brain AR034 breast breast AR035 breast cancer breast cancer AR036 Cell Line CAOV3Cell Line AR037 cell line PA-1 cell line AR038 cell line transfomnedcell line transformed AR039 colon colon AR040 colon (9808co65R)colon (9808co65R) AR041 colon (9809co15)colon (9809co15) AR042 colon cancer colon cancer AR043 colon cancer colon cancer (9808co64R) (9$08co64R) AR044 colon cancer colon cancer 9809co14 9809co14 AR045 corn clone 5 corn clone AR046 corn clone 6 corn clone _ AR047 corn clone2 corn clone2 AR048 com clone3 corn clone3 AR049 Corn Clone4 Corn Clone4 AR050 Donor II B CellsDonor II
24hrs B Cells 24hrs AR05I Donor II B CellsDonor II
72hrs B Cells 72hrs AR052 Donor II B-CellsDonor II
24 hrs. B-Cells ' hrs.
AR053 Donor II B-CellsDonor II
72hrs B-Cells 72hrs AR054 Donor II RestingDonor II,Resting.
B Cells B .
Cells ' AR055 Heart Heart AR056 Human Lung (clonetech)Human Lung (clonetech) AR057 Human Mammary Human Mammary ' (clontech) (clontech) AR058 Human Thymus Human Thymus (ctonetech) (clonetech) AR059 Jurkat (unstimulated)Jurkat (unstimulated) _ AR060 . Kidne Kidne AR061 Liver Liver AR062 Liver(Clontech)Liver(Clontech) AR063 Lymphocytes Lymphocytes chronic chronic I
1 m hoc tic m hoc tic leukaemia leukaemia AR064 Lymphocytes Lymphocytes diffuse large B cell lymphomadiffuse large B cell 1 m homa AR065 Lymphocytes Lymphocytes follicular 1 m homa follicular I m homa AR066 normal breast normal breast AR067 Normal Ovarian Normal Ovarian (4004901) (4004901) AR068 Normal Ovary Normal Ovary AR069 Normal Ovary Normal Ovary AR070 Normal Ovary Normal Ovary AR071 Ovarian Cancer Ovarian Cancer AR072 Ovarian Cancer Ovarian Cancer (97026001 ) (97026001 ) AR073 Ovarian Cancer Ovarian Cancer (97076029) (97076029) .
AR074 Ovarian Cancer Ovarian Cancer (98046011 ) (9804601 I ) AR075 Ovarian Cancer Ovarian Cancer (98066019) (98066019) AR076 Ovarian Cancer Ovarian Cancer (980760 I7) (98076017) AR077 Ovarian Cancer Ovarian Cancer (98096001 ) (98096001 ) AR078 ovarian cancer ovarian cancer ' 15799 AR079 Ovarian Cancer Ovarian Cancer AR080 Ovarian Cancer Ovarian Cancer AR081 Ovarian Cancer Ovarian Cancer AR082 ovarian cancer ovarian cancer AR083 Ovarian Cancer Ovarian Cancer AR084 Ovarian Cancer Ovarian Cancer AR085 Ovarian Cancer Ovarian Cancer AR086 ovarian cancer ovarian cancer AR087 Ovarian Cancer Ovarian Cancer AR088 Ovarian cancer Ovarian cancer 3rd C00 3rd AR089 Prostate Prostate AR090 Prostate (clonetech)Prostate (clonetech) AR091 rostate cancer rostate cancer AR092 prostate cancerprostate #15176 cancer #15176 AR093 prostate cancerprostate #15509 cancer #15509 AR094 prostate cancerprostate #15673 cancer #15673 AR095 Smalf IntestineSmall Intestine (Clontech) (Clontech) AR096 Spleen Spleen AR097 Thymus T cells Thymus T
activated cells activated AR098 Thymus T cells Thymus T
resting cells resting AR099 Tonsil Tonsil AR100 Tonsil geminal Tonsil geminal center centroblast center centroblast AR101 Tonsil germinalTonsil germinal centerB
cell center B
cell R102 Tonsil 1 m h Tonsil I
A node m h pode _ Tonsil memory Tonsil memory, AR103 B cell B
cell AR104 Whole Brain Whole Brain AR105 Xeno~raft ES-2 Xeno raft ~ ES-2 AR106 Xeno raft S'W626W626 Xeno~raft S
H0008 Whole 6 Week _ Uni-ZAP
Old .Embryo ~ XR
H0009 Human Fetal - Uni-ZAP
Brain XR
H0012 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney Kidney XR
H0013 Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old Emb o Emb o XR
r H0014 Human Gall BladderHuman Gall Gall Uni-ZAP
Bladder Bladder XR
HOOIS Human Gall Bladder,Human Gall Gall Uni-ZAP
Bladder Bladder fraction II XR
H0017 Human Greater Human Greaterperitoneum Uni-ZAP
Omentum Omentum XR
H0024 Human Fetal Human Fetal Lung Uni-ZAP
Lung III Lung XR
H0028 Human Old'Ovar ar Ovar Bluescri Human Old , t Ov H0030 Human Placenta _ Uni-ZAP
XR
H0031 Human Placenta Human PlacentaPlacenta Uni-ZAP
XR
H0036 Human Adult Human Adult Small Uni-ZAP
Small Small Int.
Intestine Intestine XR
H0038 Human Testes Human TestesTestis Uni-ZAP.
XR
H0039 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor Tumor XR
H0040 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor Tumor XR
H0041 Human Fetal Human Fetal . Bone , Uni-ZAP
Bone Bone XR
H0042 Human Adult Human Adult Lung Uni-ZAP
Pulmonary Pulmonar XR
H0046 Human EndometrialHuman EndometrialUterus diseaseUni-ZAP
Tumor Tumor ' XR
H0048 Human Pineal Human Pineal _ Uni-ZAP
Gland Gland XR
H0050 Human Fetal Human Fetal Heart Uni-ZAP
Heart Heart XR
H0051 Human HippocampusHuman Brain Uni-ZAP
.
Hi ocam us XR
H0052 Human CerebellumHuman CerebellumBrain Uni-ZAP
XR
H0056 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein, Endo. remake Vein Endothelialvein XR
Cells H0057 Human Fetal ~ ~ ~ ~ Uni-ZAP
Spleen XR
HOOS9 Human Uteiine Human UterineUterus, diseaseLambda Cancer Cancer ZAP 1I
H0063 Human Thymus Human ThymusThymus Uni-ZAP
XR
H0069 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells T-Celts Line XR
H007I Human Infant Human InfantAdrenal Uni-ZAP
Adrenal Gland Adrenal Glandland XR
H0081 Human Fetal Human Fetal Skin Uni-ZAP
Epithelium Skin (Skin) XR
H0083 HUMAN JURKAT Jurkat Cells Uni-ZAP
MEMBRANE BOUND XR
POLYSOMES
H0086 Human epithelioidEpithelioid Sk Muscle diseaseUni-ZAP
sarcoma Sarcoma, XR
muscle H0087 Human Th mus Human Th Bluescri mus t H0090 Human T-Cell T-Cell LymphomaT-Cell diseaseUni-ZAP
Lymphoma XR
HO100 Human Whole Human Whole Embryo Uni-ZAP
Six Week Six Old Emb o Week Old XR
Embr o HO101 Human 7 Weeks Human Whole Embryo Lambda Old 7 Embryo, subtractedWeek Old ZAP II
Embryo HOIOS Human Fetal Human Fetal Heart pBluescript Heart, Heart subtracted H0116 Human Thymus Human ThymusThymus pBluescript Tumor, subtracted Tumor H0122 Human Adult Human SkeletalSk Muscle Uni-ZAP
Skeletal -Muscle Muscle XR
HOI23 Human Fetal Human Fetal Brain Uni-ZAP
Dura Mater Dura Mater XR
H0124 Human Human Sk Muscle diseaseUni-ZAP
Rhabdom osarcomaRhabdom osarcoma XR
H0125 Cem cells cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line treated Treated Cem, XR
Jurkat, Ra'i, and Su t H0130 LNCAP untreatedLNCAP Cell ProstateCell Uni-ZAP
Line Line XR
H0131 LNCAP + o.3nM LNCAP Cell ProstateCell Uni-ZAP
81881 Line Line XR
H0134 Raji Cells, CyclohexamideBlood Cell Uni-ZAP
cyclohexamide Line treated Treated Cem, ' XR
Jurkat, Ra'i, and ' Su t H013S Human Synovial Human Synovial_ Uni-ZAP
Sarcoma Synovium Sarcoma XR
H0141 Activated T-Cells,Activated Blood Cell Uni-ZAP
12 hrs. T-Cells Line XR
H0144 Nine Week Old 9 Wk Old Embryo Uni-ZAP
Early Early -Sta a Human Sta~e Human XR
H0149 7 Week Old EarlyHuman Whole Embryo Uni-ZAP
Stage 7 Human, subtractedWeek Old XR
Embr o HO1S0 Human EpididymusEpididymis Testis Uni-ZAP
XR
H0156 Human Adrenal Human AdrenalAdrenal diseaseUni-ZAP
Gland Tumor Gland Tumor Gland XR
H0163 Human Synovium Human SynoviumSynovium Uni-ZAP
XR
H0169 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer, Stage C fractionCancer, stage - XR
C
H0170 12 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old Human Earl Sta~e XR
Human H0171 12 Week Old Twelve Week Emb o Uni-ZAP
Earl Stage Old Human, II Earl Sta~e XR .
Human H0179 Human NeutrophilHuman NeutrophilBlood Cell Uni-ZAP
Line XR
H0181 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast Cancer Breast Cancer XR
H0188 Human Normal Human NormalBreast Uni-ZAP
Breast Breast _ XR
H0192 Cem Cells, cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line treated, subtraTreated Cem, ~ XR
Jurkat, Ra'i, and _ Su t H0194 Human Cerebellum,Human CerebellumBrain . pBluescript subtracted H0205 Human Colon Human Colon Colon pBluescript Cancer, differential Cancer H0208 Early Stage Human Fetal Lung pBluescript Human Lung, Lung subtracted H0213 Human Pituitary,Human Pituitary Uni-ZAP
subtracted , XR
' H0214 Raji cells, CyclohexamideBlood Cell pBluescript cyclohexamide Line treated, subtractedTreated Cem, Jurkat, Ra'i, and Su t H0231 Human Colon, Human Colon Bluescri subtraction t H0244 Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old Emb o, subtractedEmbr o XR
H0251 Human ChondrosarcomaHuman Cartilage diseaseUni-ZAP
Chondrosarcoma XR
H0252 Human OsteosarcomaHuman Bone diseaseUni-ZAP
Osteosarcoma XR
H0253 Human adult Human Adult Testis Uni-ZAP
testis, large Testis inserts XR
H0255 breast lymph Breast LymphLymph Lambda node CDNA Node Node libr ZAP II
H0261 H: cerebellum, Human CerebellumBrain Uni-ZAP
Enzyme , subtracted XR
H0264 human tonsils Human TonsilTonsil ~ Uni-ZAP
XR
H0265 Activated T-CellT-Cells Blood Cell Uni-ZAP
Line ( l2hs)/Thiouridine _ XR
labelledEco H0266 Human MicrovascularHMEC Vein Cell Lambda Line Endothelial ZAP II
Cells, Tract.
A
H0267 Human MicrovascularHMEC Vein Cell Lambda Line Endothelial ZAP II
Cells, fract.
B
H0270 HPAS (human Human PancreasPancreas Uni-ZAP
pancreas, subtracted) XR
H0271 Human Neutrophil,Human NeutrophilBlood Cell Uni-ZAP
- Line Activated Activated XR
H0284 Human OB MG63 Human Bone Cell Uni-ZAP
control Line fraction I Osteoblastoma XR
MG63 cell line H0286 Human OB MG63 Human Bone Cell Uni-ZAP
treated Line ( 10 nM E2) Osteoblastoma XR
fraction I
MG63 cell line H0288 Human OB HOS Human Bone Cell Uni-ZAP
control Line fraction I Osteobiastoma XR
HOS
cell line H0290 Human OB HOS Human Bone Cell Uni-ZAP
treated Line ( 1 nM E2) fractionOsteoblastoma XR
I HOS
cell line H0294 Amniotic Cells Amniotic PlacentaCell Uni-ZAP
- TNF Cells - Line induced TNF induced XR
H0295 Amniotic Cells Amniotic PlacentaCell Uni-ZAP
- Primary Cells - Line I Culture Prima Culture XR
H0309 Human Chronic Synovium, Synovium diseaseUni-ZAP
Synovitis Chronic Synovitis/ XR
Osteoarthritis H0316 HUMAN STOMACH Human StomachStomach Uni-ZAP
XR
H0318 HUMAN B CELL Human B CellLymph diseaseUni-ZAP
Node LYMPHOMA L m homa H0327 human corpus Human CorpusBrain Uni-ZAP
colosum Callosum XR
H0328 human ovarian Ovarian CancerOvary diseaseUni-ZAP
cancer _ XR
H0329 DermatofibrosarcomaDermatofibrosarcomSkin diseaseUni-ZAP
Protuberance a Protuberans XR
H0331 Hepatocellular HepatocellularLiver diseaseLambda Tumor .
Tumor ' ZAP II
H0333 HemangiopericytomaHemangiopericytomBlood diseaseLambda vessel a ZAP II
H0334 Kidney cancer Kidney CancerKidney diseaseUni-ZAP
XR
H0341 Bone Marrow Bone Marrow Bone Cell Uni-ZAP
Cell Line Cell Marrow Line (RS4; I 1 j Line R_S4;11 XR
H0352 wilm"s tumor Wilm"s Tumor diseaseUni-ZAP_ XR
H0355 Human Liver Human Liver, , pCMVSport normal Adult 1 H0369 H. Atrophic Atrophic Uni-ZAP
Endometrium Endometrium XR
and myometrium H0370 H. Lymph node Lymph node . diseaseUni-ZAP
breast with Cancer Met. Breast XR
Cancer H0386 Leukocyte and Human LeukocytesBlood Cell pCMVSport Lung; 4 , Line screens I
H0391 H. Meniin~ima, Human MeninQimabrain S ortl H0392 H. Menin~ima, Human MeninQimabrain S ortl MI
H0393 Fetal Liver, Human Fetal Liver Bluescri subtraction Liver t II
H0402 CD34 depleted CD34 DepletedCord ZAP Express Buffy Coat Blood (Cord Blood), Buffy Coat re-excision (Cord Blood) H0409 H. Striatum Human Brain,Brain pBluescript Depression, subtracted Striatum Dl: ression H0411 H Female Bladder,Human FemaleBladder pSportl Adult Adult Bladder H0412 Human umbilicalHUVE Cells 'UmbilicalCell pSportl vein Line endothelial vein cells, IL-4 induced H0413 Human UmbilicalHUVE Cells UmbilicalCell pSportl Vein Line Endothelial vein Cells, uninduced H0416 Human Neutrophils,Human NeutrophilBlood Cell pBluescript - Line Activated, re-excisionActivated H0418 Human Pituitary,Human Pituitary pBluescript subtracted VII
H0421 Human Bone Marrow,Bone Marrow pBluescript re-excision H0423 T-Cell PHA 24 T-Cells Blood Cell pSportl hrs Line H0424 Human Pituitar Human Pituita Bluescri ,~subt IX t H0427 Human Adipose Human A_ pSportl dipose, left hi 1i oma H0428 Human Ovary _ Ovary pSportl Human Ovary Tumor H0434 Human Brain, Human Brain, Bluescri striatum, t re-excision Striatum H0435 Ovarian Tumor Ovarian Tumor,_ Ovary pCMVSport OV3S0721 2.0 H0436 Resting T-Cell T-Cells Blood Cell pSportl.
Library,II Line H0438 H. Whole Brain Human Whole ZAP Express #2, re- Brain excision #2 H0441 H. Kidney Cortex,Kidney cortexKidney _ pBluescript subtracted H0445 Spleen, Chronichuman Spleen,Spleen diseasepSportl CLL
1 m hoc tic leukemia _ H0448 Salivary gland,Human SalivarySalivary Lambda subtracted Gland gland ZAP II
H0455 H. Striatum Human Brain,Brain pBluescript Depression, subt Striatum De ression H0457 Human Eosino Human Eosino S ortl hils hils H0483 Breast Cancer Breast Cancer pSportl cell line, Cell MDA 36 line, MDA
H0484 Breast Cancer Breast Cancer pSportl Cell line, Cell angiogenic line, Angiogenic, , H0486 Hodgkin"s LymphomaHodgkin"s diseasepCMVSport II
L m homa 2.0 II
H0488 Human Tonsils, Human Tonsils ~ pCMVSport Lib 2 2.0 H0494 Keratinocyte Keratinocyte pCMVSport , 2.0 H0497 HEL cell line HEL cell HEL pSportl-line 92.1.7 H0505 Human Astroc Human Astroc S ortl to to H0506 Ulcerative ColitisColon Colon S ortl H0509 Liver, HepatomaHuman Liver,Liver diseasepCMVSport He atoma, 3.0 atient 8 H0510 Human Liver, Human Liver,Liver pCMVSport normal normal, Patient ' 3.0 # 8 H0518 pBMC stimulatedpBMC stimulated pCMVSport w/ poly I/C with of IIC 3.0 H0519 NTERA2, controlNTERA2, pCMVSport Teratocarcinoma 3.0 cell line H0520-NTERA2 + retinoicNTERA2, ' pSportl acid, 14 days Teratocarcinoma cell line H0521 Primary DendriticPrimary Dendritic pCMVSport Cells, lib 1 cells 3.0 H0522 Primary DendriticPrimary Dendritic , pCMVSport cells,frac 2 cells 3.0 H0529 Myoloid ProgenitorTF-1 Cell pCMVSport Cell Line;
Line Myoloid progenitor 3.0 ' cell line H0538 Merkel Cells Merkel cellsL m h S octl node H0539 Pancreas Islet Pancreas Pancreas diseasepSportl Cell Tumor Islet Cell Tumour H0542 T Cell helper Helper T pCMVSport I cell 3.0 H0543 T cell helper HelperT cell pCMVSport II
3.0 H0544 Human endometrialHuman endometrial pCMVSport stromal cells stromal cells 3.0 H0545 Human endometrialHuman endometrial ' pCMVSport stromal cells-treatedstromal cells-treated 3.0 with ro~esterone with roe H0546 Human endometrialHuman endometrial CMVS
ort stromal cells-treatedstromal cells-treated~ 3.0 avith estradiol ~ with estra -H0547 NTERA2 teratocarcinomaNTERA2, pSportl cell line+retinoicTeratocarcinoma acid ( 14 da s) cell line H0549 H. Epididiymus,Human Uni-ZAP
caput &
corpus Epididiymus, XR
captit and co us H0550 H. Epididiymus,Human Uni-ZAP
cauda E ididiymus, XR
cauda H0551 Human Thymus Human Thymus pCMVSport Stromal Cells Stromal Cells 3.0 H0553 Human Placenta Human Placenta pCMVSport 3.0 H0555 Rejected Kidney,Human Rejected. Kidney diseasepCMVSport lib 4 Kidne 3.0 H0556 Activated T- T-Cells Blood Cell Uni-ZAP
Line cell( 12h)/Thiouridine-re- XR
excision H0559 PMA 4H, re- HL-60 Cells,Blood Cell Uni-ZAP
HL-60 PMA Line , stimulated XR
excision 4H
H0560 KMH2 , KMH2 pCMVSport 3.0 H0561 L428 L428 pCMVSport 3.0 H0562 Human Fetal Human Fetal pCMVSport Brain, Brain normalized c5-11-26 2.0 H0572 Human Fetal Human Fetal pCMVSport Brain, Brain normalized AC5002 2.0 H0574 Hepatocellular ' HepatocellularLiver , diseaseLambda Tumor; re- .
excision Tumor ZAP II
H0575 Human Adult Human Adult Lung Uni-ZAP
Pulmonar ;re-excisionPulmonar XR
H0576 Resting T-Cell;T-Cells Blood Cell ~ Lambda re- Line ZAP II
excision H0579 Pericardium Pericardium Heart S ortl ' H0580 Dendritic cells,Pooled dendritic pCMVSport pooled cells 3.0 H0581 Human Bone Marrow,Human Bone Bone Marrow pCMVSport treated Marrow 3.0 H0583 B Cell lymphomaB Cell LymphomaB Cell diseasepCMVSport . ~
3.0 H0586 Healing groin healing groingroin diseasepCMVSport wound, 6.5 hours post incisionwound, 6.5 f 3.0 hours ost incision H0587 Healing groin Groin-2!19/97groin diseasepCMVSport wound; 7.5 hours ost incision 3.0 .
H0590 Human adult Human Adult Small .
small Small Int. Uni-ZAP
intestine,re-excisionIntestine XR
H0591 Human T-cell T-CeII LymphomaT-Cell diseaseUni-ZAP
lymphoma;re-excision XR
H0592 Healing groin HGs wound diseasepCMVSport wound- healing zero hr post-incisionproject; , 3.0 abdomen (control) H0593 Olfactory Olfactory pCMVSport epithelium epithelium;nasalcavityfrom roof - 3.0 of left nasal cacit H0594 Human Lung Cancer;re-Human Lung Lung diseaseLambda Cancer ZAP II
excision H0595 Stomach cancer Stomach Cancer diseaseUni-ZAP
= , (human);re-excision5383A (human) XR
H0596 Human Colon Human Colon Colon ' Lambda Cancer;re-excision ' _ Cancer ZAP II
HOS98 Human Stomach;re-Human StomachStomach Uni-ZAP
excision ' XR
HOS99 Human Adult Human Adult Heart . Uni-ZAP
Heart;re- Heart excision XR
H0600 Healing AbdomenAbdomen diseasepCMVSport wound;70&90 3.0 min post incision _ H0601 Healing AbdomenAbdomen diseasepCMVSport Wound;l5 days 3.0 post incision H0606 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast Cancer;re-excisionBreast Cancer XR
H0610 H. Leukocytes, H.Leukocytes pCMVSport normalized cot 1 SA
H0616 Human Testes, Human TestesTestis Uni-ZAP
Reexcision XR
H0617 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast-Cancer ReexcisionBreast Cancer XR
H0618 Human Adult Human Adult Testis , Uni-ZAP
Testes, Testis ' Lame Inserts, XR
Reexcision H0619 Fetal Heart Human Fetal Heart Uni-ZAP
Heart XR .
HOf20 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney; Kidney Reexcision XR
H0622 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor;
Reexcision Tumor ' XR
H0623 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein;
Reexcision Vein Endothelialvein XR
Cells H0624 12 Week Early Twelve Week Embryo Uni-ZAP
Stage Old Human II; ReexcisionEarly Stage XR
Human H062S Ku 812F BasohilsLineKu 812F Baso S ortl hits H0626 Saos2 Cells; 5aos2 Cell pSportl Untreated Line;
Untreated H0627 Saos2 Cells; Saos2 Cell pSportl Vitamin D3 Line;
Treated Vitamin D3 Treated H0628 Human Pre-DifferentiatedHuman Pre- Uni-ZAP
Adipocytes Differentiated ' XR
Adipocytes .
H0631 Saos2, DexamethosomeSaos2 Cell pSportl Line;
Treated Dexamethosome Treated H0632 Hepatocellular HepatocellularLiver Lambda Tumor;re-excision Tumor ZAP II
H0633 Lung Carcinoma TNEalpha diseasepSportl A549 activated TNFalpha activatedAS49--Lung Carcinoma H0634 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor, re-excision Tumor XR
H063S Human ActivatedActivated Blood. Cell Uni-ZAP
T-Cells, T-Cells Line re-excision XR
H0638 CD40 activated CD40 activated pSportl monocyte dendridic cellsmonocyte dendridic cells H0641 LPS activated LPS activated , pSportl derived ' dendritic cellsmonocyte d derive dendritic cells H0644 Human Placenta Human PlacentaPlacenta Uni-ZAP
(re- .
excision) XR
H064S Fetal Heart, human Fetal Heart . Uni-ZAP
re-excision Heart XR
i H0646Lung, Cancer Metastatic pSportl (4005313 A3): 'Invasive squamous Poorly cell lung Differentiated carcinoma, Lung poorly di Adenocarcinoma, H064$ Ovary, Cancer: Papillary diseasepSportl (4004562 Cstic B6) Papillary neoplasm , Serous of low Cystic Neoplasm,malignant Low potentia Ivlali~nant Pot H0649 Lung, Normal: Normal Lung pSportl (4005313 Bl) H0650 B-Cells B-Cells pCMVSport 3.0 H06S Ovary, Normal: Normal Ovary pSporti (9805C040R) H06S2 Lung, Normal: Normal Lung pSportl (4005313 B1) H0653 Stromal Cells Stromal Cells S ortl H0656 B-cells (unstimulated)B-cells pSportl (unstimulated) H0657 B-cells (stimulated)B-cells (stimulated) S ortI
H0658 Ovary, Cancer 9809C332- Ovary diseasepSportl Poorly &
(9809C332): differentiateFallopian Poorly differentiated Tubes adenocarcinoma H0659 Ovary, Cancer Grade II ' Ovary ' , diseasepSportl - ' Papillary (15395A1F): Carcinoma, Grade II Ovary Papillary Carcinoma' H0660 Ovary, Cancer: Poorly differentiated diseasepSportl (15799A1F)Poorlycarcinoma, ovary differentiated carcinoma H0661 Breast, Cancer:Breast cancer diseasepSportl (4004943 AS) H0662 Breast, Normal:Normal BreastBreast pSportl -(400552282) #4005522(B2) H0664 Breast, Cancer:Breast CancerBreast diseasep5portl (9806C012Rj H0665 Stromal cells Stromal cells 5 ortl 3.88 3.88 H0667 Stromal cells(HBM3.18)Stromal cell(HBM pSportl 3.18) H0668 stromal cell stromal cell pSportl clone 2.5 clone 2.5 H0670 Ovary, Cancer(4004650Ovarian Cancer- pSportl A3): Well-Differentiated4004650A3. ' .
Micropapillary Serous Carcinoma H0672 Ovary, Cancer: Ovarian Ovary ' pSportl (4004576 Ag) Cancer(4004576A8) H0673 Human Prostate Human ProstateProstate Uni-ZAP
Cancer, Stage B2; re-excisionCancer, stage XR
H0674 Human Prostate Human ProstateProstate Uni-ZAP
Cancer, . Stage C; re-excissionCancer, stage XR
C
H0675 Colon, Cancer: Colon Cancer pCMVSport (9808C064R) 9808C064R 3.0 H0677 TNFR degenerateB-Cells PCRII
olio H0682 Serous Papillaryserous papillary pCMVSport Adenocarcinoma adenocarcinoma 3.0 (9606G304SPA3B) H0683 Ovarian Serous Serous papillary pCMVSport Papillary Adenocarcinoma adenocarcinoma, 3.0 stage 3C
(9804601 H0684 Serous PapillaryOvarian Cancer-Ovaries pCMVSport Adenocarcinoma 98106606 3.0 H0686 Adenocarcinoma Adenocarcinoma pCMV
of of Sport Ovary, Human Ovary, Human 3.0 Cell Line Cell Line, # SW-626 H0687 Human normal Human normalOvary pCMVSport ova (#9610G21S)ova (#9610G21S) 3.0 H0688 Human Ovarian Human Ovarian pCMVSport Cancer(#98076017)cancer(#9807G017), 3.0 mRNA from Maura Ru H0689 Ovarian Cancer Ovarian Cancer, pCMVSport #98066019 3.0 H0690 Ovarian Cancer,Ovarian Cancer, pCMVSport #
97026001 #97026001 - 3.0 H0693 Normal ProstateNormal Prostate pCMVSport #ODQ3958EN Tissue # 3.0 H069S mononucleocytesmononucleocytes pCMVSport from patient from patient 3.0 at Shad Grove Hos it S0001 Brain frontal Brain frontalBrain Lambda cortex cortex ZAP II
50002 Monocyte activatedMonocyte-activatedblood CeILLine Uni-ZAP
XR
S0003 Human OsteoclastomaOsteoclastomabone diseaseUni-ZAP
XR
SOOOS Heart Heart-left Heart CDNA
ventricle 50007 Early Stage Human Fetal Uni-ZAP
Human Brain Brain XR' S0010 Human Amygdala Amygdala Uni-ZAP
' XR
50011 STROMAL - Osteoclastomabone diseaseUni-ZAP
.
OSTEOCLAS'TOMA XR
50022 Human OsteoclastomaOsteoclastoma Uni-ZAP
Stromal Cells Stromal Cells XR
-unamplified S0026 Stromal cell stromal cellBone Cell Uni-ZAP
TF274 marrow Line XR
S0027 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
serum Line treated arter XR
S0028 Smooth muscle,controlSmooth musclePulmanaryCell Uni-ZAP
Line arter XR
50029 brain stem Brain stem brain Uni-ZAP
XR
50030 Brain pons Brain Pons Brain Uni-ZAP
XR
S0031 Spinal cord Spinal cord spinal Uni-ZAP
cord XR
S0032 Smooth muscle-ILbSmooth musclePulmanaryCell Uni-ZAP
Line induced arter XR
50036 Human SubstantiaHuman Substantia Uni-ZAP
Nigra Ni ~ra XR
S0037 Smooth muscle, Smooth muscle.PulmanaryCell Uni-ZAP
IL16 Line induced arter XR
50038 Human Whole Human Whole ZAP Express Brain #2 - Brain Oli o dT > 1.SKb#2 50040 Adipocytes , Human Adipocytes , Uni-ZAP
from Osteoclastoma XR
S004S Endothelial Endothelial endothelialCell ~ Uni-ZAP
cells-control cell Line cell-lun XR
S0046 Endothelial-inducedEndothelial endothelialCell Uni-ZAP
cell Line cell-lung XR
50049 Human Brain, Human Brain, Uni-ZAP
Striatum Striatum ~ XR
S0051 Human Human diseaseUni-ZAP
Hypothalmus,SchizophrenHypothalamus,, . XR
is Schizo hrenia 50052 neutrophils human neutrophilsblood Cell Uni-ZAP
control Line XR
S0053 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS Line induced induced XR
50112 Hypothalamus Brain Uni-ZAP
XR
S0114 Anergic T-cell Anergic T-cell Cell Uni-ZAP
Line XR
50116 Bone marrow Bone marrow Bone marrow Uni-ZAP
XR
S0126 Osteoblasts Osteoblasts Knee Cell Uni-ZAP
Line XR' 50132 Epithelial-TNFaAirway Epithelial Uni-ZAP
and INF
induced XR
50134 Apoptotic T-cellapoptotic Cell Uni-ZAP
cells Line XR
50144 Macrophage (GM-CSFMacrophage Uni-ZAP
(GM-treated) CSF treated) ~ XR
S0146 prostate-editedprostate Prostate Uni-ZAP
BPH
XR
SO150 LNCAP prostate LNCAP Cell Prostate Cell Uni-ZAP
cell line Line Line XR
50152 PC3 Prostate PC3 prostate Uni-ZAP
celi line cell line XR
S0174 Prostate-BPH Human Prostate pBluescript subtracted II
BPH
S0182 Human B Cell Human B- Uni-ZAP
8866 Cell 8866 XR
50192 Synovial FibroblastsSynovial pSportl Fibroblasts (control ) S0194 S novial h oxiaS novial S ortl Fibroblasts 50196 Synovial IL-1/TNFSynovial pSportl Fibroblasts , stimulated S0206 Smooth Muscle- Smooth musclePulmanaryCell pBluescript HASTE Line normalized arter S0210 MessanQial cell,MessanQial S ortl frac 2 cell 50212 Bone Marrow Bone Marrow pSportl Stromal Cell, untreatedStromal Cell ,untreated S0216 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS ~ Line induced induced ~ XR
S0222 H. Frontal H. Brain, Brain diseaseUni-ZAP
Frontal cortex,epileptic;re-Cortex, Epileptic XR
excision S0242 Synovial FibroblastsSynovial p5portl Fibroblasts (I11/TNF), subt 50250 Human OsteoblastsHuman OsteoblastsFemur diseasepCMVSport II
2.0 S0260 Spinal Cord, Spinal cord spinal Uni-ZAP
re-excision cord XR
50276 Synovial hypoxia-RSFSynovial Synovial pSportl fobroblasts subtracted (rheumatoid)tissue S0278 H Macrophage Macrophage Uni-ZAP
(GM-CSF (GM-treated),re-excisionCSF treated) XR
S0280 Human Adipose Human Adipose Uni-ZAP
Tissue, re-excision Tissue XR
50282 Brain Frontal Brain frontalBrain Lambda Cortex, re- cortex ZAP II
excision 50294 Larynx tumor Larynx tumorLarynx,vocal diseasepSportl cord 50300 Frontal lobe,dementia;re-Frontal LobeBrain Uni-ZAP
excision . dementia/Alzheimer' XR
's S0312 Human Human diseasepSportl osteoarthritic;fractionosteoarthritic' ' II
cartilage S0316 Human Normal Human Normal p5portl Cartilage,FractionCartilage I
S0328 Palate carcinomaPalate carcinomaUvula diseaseSor_tl _ S0330 Palate normal Palate normalUvula S ortl S0332 Pha nx carcinomaPhar nx carcinomaH o har S ortl nx S0338 Human OsteoarthriticHuman diseasepSportl Cartilage Fractionosteoarthritic III
cartilage S0340 Human OsteoarthriticHuman diseasepSportl Cartilage Fractionosteoarthritic IV
cartilage ' S0342 Adipocytes;re-excisionHuman Adipocytes Uni-ZAP
from Osteoclastoma XR
50344 Macrophage-oxLDL;macrophage- blood Cell - Uni-ZAP
re- Line excision oxidized , XR
LDL
treated 50346 Human Amygdala;re-Amygdala Uni-ZAP
excision XR
S0354 Colon Normal Colon NormalColon S ortl II
50356 Colon CarcinomaColon CarcinomaColon diseaseS ortl 50358 Colon Normal Colon NormalColon ' _S o_rtl III
S0360 Colon Tumor Colon Tumor Colon disease_S ortl II
S0364 Human Quadrice Quadrice pSportl s s muscle S0366 Human Soleus Soleus Muscle S ortl S0370 Lar nx carcinomaLar nx carcinoma diseaseS ortl II
S0374 Normal colon Normal colon S ortl S0376 Colon Tumor Colon Tumor diseaseS ortl S0378 Pancreas normalPancreas pSportl PCA4 Normal No PCA4 No S0380 Pancreas Tumor Pancreas diseasepSportl PCA4 Tu Tumor PCA4 Tu S0386 Human Whole Whole brain Brain ZAP Express Brain, re-excision ' S0388 Human Human . diseaseUni-ZAP
Hypothalamus,schizophreHypothalamus, XR
re-excision Schizo hrenia .
nia, 50390 _ Smooth musclePulmanaryCell Uni-ZAP
Smooth muscle, Line control;
re-excision arter XR
~
50392 Salivary Gland Salivary pSportl , gland;
normal 50400 Brain; normal Brain; normal S ortl 50404 Rectum normal Rectum, normal S ortl 50406 Rectum tumour Rectum tumour S ortl S0408 Colon, normal Colon, normal 5 ortl S0410 Colon,tumo Colon, tumour S ortl ur 50418 _ CHME Cell pCMVSport CHME Cell Line;treatedLine;
hrs treated 3.0 S0420 CHME Cell . CHME Cell pSportl line, .
Line,untreated untreatetd ' S0422 Mo7e Cell Line Mo7e Cell pCMVSport GM-CSF Line treated (lng/ml)GM-CSF treated 3.0 ( 1 n~/ml) S0424 TF-1 Cell Line TF-1 Cell pSportl GM-CSF Lane Treated GM-CSF Treated 50426 Monocyte activated;Monocyte-activatedblood, Cell Uni-ZAP
re- Line excision XR
50428 Neutrophils human neutrophilsblood Cell Uni-ZAP
control; re- Line ' excision XR
50434 Stomach Normal Stomach Normal diseaseS ortl 50436 StomachTumour Stomach Tumour diseaseS ortl ~
S0438 Liver Normal Liver Normal p5portl MetSNo MetSNo S0440 Liver Tumour Liver Tumour S ortl Met 5 Tu S0442 Colon Normal Colon Normal S ortl 50444 Colon Tumor Colon Tumour diseaseS ortl S0456 Ton~ue Normal Ton~ue Normal S ortl S0458 Thyroid Normal Thyroid normal pSportl (SDCA2 No) S0460 Th roid Tumour Th roid Tumour S ortl S0468 Ea.h .926 cell Ea.h .926 S ortl line cell Tina S0472 Lun MesotheliumPYBT S ortl S0474 Human blood Platelets Blood Other platelets .
platelets S3012 Smooth Muscle Smooth musclePulmanaryCell ~ pBluescript Serum Line Treated, Norm arter ' S3014 Smooth muscle, Smooth musclePulmanaryCell pBluescript serum Line induced,re-exc arter 56016 H. Frontal Cortex,H. Brain, Brain diseaseUni-ZAP
Frontal E ile tic Cortex, E ~ XR
ile tic S6022 H. Adipose TissueHuman Adipose Uni-ZAP
Tissue XR
S6024 Alzheimers, Alzheimer"s/SpongyBrain diseaseUni-ZAP
spongy chan a chance XR
S6026 Frontal Lobe, Frontal LobeBrain Uni-ZAP
Dementia dementialAlzheimer' XR
's S6028 Human Manic Human Manic Brain diseaseUni-ZAP
Depression Tissue de ression XR
tissue T0003 Human Fetal Human Fetal pBluescript Lung Lung SK-.
T0004 Human White Human White pBluescript Fat Fat SK-T0006 Human Pineal Human Pinneal pBluescript Gland Gland SK-T0010 Human Infant Human Infant Other Brain Brain T0039 HSA 172 Cells Human HSA ~ pBluescript 172 cell line SK-T0040 HSC172 cells SA172 Cells pBluescript SK-T0041 JurkatT-cell JurkatT-cell pBluescript G1 phase SK-T0048 Human Aortic Human Aortic' pBluescript .
- Endothelium Endothilium ~ SK-T0049 Aorta endothelialAorta endothelial pBluescript cells +
TNF-a cells SK-T0060 Human White Human White pBluescript Adipose Fat SK-T0067 Human Thyroid Human Thyroid pBluescript SK-T0068 NormalOvary, Normal Ovary, pBluescript Premeno ausal Premeno ausal SK-T0069 Human Uterus, Human Uterus, pBluescript ~ normal normal SK-T0082 Human Adult Human Adult pBluescript Retina Retina SK-.
T0109 ~ Human (HCC) Bluescript ~ cell line liver (mouse) SK-metastasis, remake TO Human colon pBtuescript I carcinoma IO
(HCC) cell line, SK-remake L0004 ClonTech HL
1065a L0005 Clontech human aorta of A+ mRNA (#6572) L0021 Human adult (K.Okubo) L0022 Human adult .
lung 3"
directed MboI
cDNA
L0103 DKFZ ham 1 am ~dala L0109 Human brain brain cDNA
L0142 Human placenta placenta cDNA
(TFu'iwara) L0143 Human placenta placenta polyA+
(TFu'iwara) _ Human fetal brain L0157 brain (TFu'iwara) L0163 Human heart . heart cDNA
(YNakamura) .
L0351 Infant brain, , BA, M13-Bento Soares derived L0352 Normalized infant BA, M
brain, 13-Bento Soares derived L0362 Stratagene ovarian Bluescript cancer (#937219) SK-_ L0364 NCI_CGAP_GC5 germ cell Bluescript tumor SK-L0366 Stratagene schizoschizophrenic Bluescript brain brain S11 ' S-I1 frontal SK-lobe L0367 NCI CGAP Schl Schwannoma Bluescript _ _ tumor SK- .' L0368 NCI_CGAP_SS synovial Bluescript 1 sarcoma SK-L0369 NCI CGAP_AA1 adrenal adenomaadrenal Bluescript' -' gland SK-L0370 Johnston frontalpooled frontalbrain Bluescript cortex lobe SK-L0372 NCI_CGAP_Col2 colon tumor colon Btuescript SK-L0374 NCI CGAP_Co2 tumor colon Bluescript SK-L0375 NCI CGAP_Kid6 kidney tumorkidney Bluescript -SK-L0378 NCI_CGAP_Lul lung tumor lung Bluescript SK-L0381 NCI_CGAP_HN4 ' squamous pharynx Bluescript cell carcinoma SK-L0383 NCI_CGAP_Pr24 invasivetumor(cellprostate Bluescript line) ' SK-L0384 NCI_CGAP_Pr23 prostate prostate Bluescript tumor ~
SK-L0438 normalized infanttotal brain brain lafmid brain BA
cDNA
L0439 Soares infant whole~brain Lafmid brain 1NIB BA
L0455 Human retina retina eye lambda cDNA gtl0 randomly primed sublibrary L0456 Human retina retina eye lambda cDNA gtl0 Tsp509I-cleaved subtibrary L0471 Human fetal Lambda heart, Lambda ZAP Ex ~ ZAP Ex ress ress L0480 Stratagene cat#937212 Lambda (1992) ZAP, p$luescript SK(-) L0~183Human pancreatic Lambda islet ZAPII
L0485 STRATAGENE Humanskeletal leg muscle Lambda muscle skeletal muscle ZAPII
cDNA
library, cat.
#936215.
L0517 NCI_CGAP_Prl AMP10 L0518 NCI_CGAP Pr2 AMP10 ~
L0519 NCI CGAP_Pr3 - AMP10 L0520 NCI_CGAP_Alvl alveolar pAMPlO
rhabdomyosarcoma L0521 NCI_CGAP_Ervl Ewin~"s sarcoma AMP10 L0522 NCI CGAP_Kidl kidne AMP10 L0526 NCI_CGAP_Prl2 metastatic pAMPlO
prostate bonelesion L0527 NCI_CGAP_Ov2 ovary pAMPlO
L0533 NCI_CGAP_HSC1 stem cells bone AMP10 marrow L0540 NCI_CGAP_PrlO invasive prostate pAMPlO
prostate tumor L0542 NCI_CGAP_PrII normal prostaticprostate pAMPlO
a ithelial cells LOS46 NCI_CGAP_Prl8 stroma rostate AMPIO
L0564 Jia bone marrowbone man-orv Bluescri stroma stroma t LOS65 Normal Human Bone . Hip pBluescript Trabecular Bone ' Cells ~
L0590 Stratagene fibroblast pBluescript ' (#937212) SK-L0591 Stratagene HeLa pBluescript cell s3 L0592 Stratagene hNT pBluescript neuron (#937233) SK-L0593 Stratagene pBluescript neuroepithelium SK-(#937231) L0595 Stratagene NT2 neuroepithelialbrain pBluescript neuronal cells ~
recursor 937230 SK-L0596 Stratagene colon colon pBluescript (#937204) ~ SK-L0597 Stratagene corneal cornea pBluescript stroma (#937222) ' SK-L0598 Morton Fetal cochlea ear pBluescript Cochlea SK-L0599 Stratagene lung lung p131uescript (#937210) SK-L0600 Weizmann Olfactoryolfactory nose ~ p$luescript epithelium E ithelium ' SK-L0601 Stratagene pancreas pancreas pBluescript (#937208) SK-L0602 Pancreatic Isletpancreatic pancreas~ pBluescript islet SK-L0603 Stratagene placenta placenta~ pBluescript (#937225) SK-L0604 Stratagene musclemuscle skeletal pBluescript muscle SK-L0605 Stratagene fetalfetal spleenspleen pBluescript spleen (#937205) SK-L0608 Stratagene lunglung carcinomalung NCI-H69 pBluescript carcinoma 937218 ~SK-I L0617Chromosome 22 pBluescriptII
exon KS+
L0622 HM1 pcDNAII
(Invitro~en) L0623 HM3 pectoral pcDNAII
muscle (after mastectom (Invitro~en) ) L0629 NCI_CGAP_Mel3 metastatic bowel pCMV-(skin melanoma rima SPORT4 to bowel ) L0631 NCI_CGAP_Br7 breast pCMV-L0635 NCI_CGAP_PNS1 dorsal root peripheral pCMV-ganglion nervous SPORT4 s stem L0636 NCI CGAP_Pitl four pooled brain pCMV-pituitary adenomas SPORT6 L0637 NCI_CGAP_Brn53 three pooledbrain pCMV-menin~iomas SPORT6 L0638 NCI_CGAP_Brn35 tumor, 5 brain pCMV-pooled (see descri lion) SPORT6 L0639 NCI_CGAP_Brn52 tumor, 5 brain pCMV-pooled (see descri lion) SPORT6 L0640 NCI_CGAP_Brl8 four pooled breast pCMV-high-grade tumors, SPORT6 includin two rima L0641 NCI_CGAP_Col7 juvenile colon pCMV-granulosa tumor ~ SPORT6 L0642 NCI_CGAP_CoI8 moderately colon pCMV-differentiated SPORT6 adenocarcinoma L0643 NCI_CGAP_Col9 moderately colon pCMV-differentiated' SPORT6 adenocarcinoma L0644 NCI_CGAP_Co20 moderately colon pCMV-differentiated SPORT6 adenocarcinoma L0646 NCI_CGAP_Col4 moderately- colon pCMV-differentiated SPORT6 adenocarcinoma L0647 NCI_CGAP_Sar4 five pooled connective pCMV-sarcomas, tissue SPORT6 including myxoid hposarcoma L06~18~NCI_CGAP Eso2 squamous esophagus pCMV-cell carcinoma SPORT6 L0649 NCI_CGAP_GLJ1 2 pooled genitourinary pCMV-' high-grade transitionaltract SPORT6 cell tumors L0650 NCI_CGAP_Kidl3 2 pooled kidney pCMV-, Wilms"
tumors, one SPORT6 primary and one metast L0651 NCI_CGAP Kid8 renal cell kidney pCMV-tumor L0653 CGAP_Lu28 two pooled lung , pCMV-NCI
_ squamous SPORT6 cell carcinomas L0654 NCI CGAP_Lu31 lung, pCMV-cell line L0655 CGAP_Lyml2 lymphoma, lymph pCMV-NCI node _ follicular SPORT6 mixed small and large cell L0656 CGAP_Ov38 normal epitheliumovary pCMV-NCI
_ SPORT6 L0657 CGAP_Ov23 tumor, S ovary ' pCMV-NCI pooled (see _ description) SPORT6 L0658 NCI_CGAP_Ov35 .tumor, 5 ovary pCMV-- pooled (see descri tion) SPORT6 L0659 NCI_CGAP_Panl . adenocarcinomapancreas pCMV-L0662 NCI_CGAP_Gas4 poorly differentiatedstomach pCMV-adenocarcinoma SPORT6 with signet r L0663 CGAP Ut2 moderately- uterus pCMV-NCI
_ differentiated SPORT6 endometrial adenocarcino L0664 CGAP_Ut3 poorly-differentiateduterus pCMV-NCI
_ endometrial SPORT6 adenocarcinoma, L0665 CGAP_Ut4 serous papillaryuterus pCMV-NCI
_ carcinoma, SPORT6 high Qrade, 2 ooled t L0666 CGAP_Utl well-differentiateduterus pCMV-NCI
_ endometrial SPORT6 adenocarcinoma, L0667 NCI_CGAP_CML1 myeloid cells,whole pCMV-18 blood pooled CML SPORT6 cases, BCRIABL rearra L0717 Gessler Wilms SPORTI
tumor L073I Soares_pregnant_uterus_' uterus pT7T3-Pac NbHPU
L0738 Human colorectal pT7T3D
cancer L0740 Soares melanocytemelanocyte pT7T3D
2NbHM (Pharmacia) with a modified of linker L0741 Soares adult - brain pTTT3D
brain N2b4HB55Y (Pharmacia) with a modified of linker L0742 Soares adult brain pTTT3D
brain N2b5HB55Y (Pharmacia) with a modified polyiinker L0743 Soares breast breast pTTT3D
2NbHBst (Pharmacia) - with a modified of linker L0744 Soares breast breast pTTT3D
3NbHBst (Pharmacia) with a . ~ modified of linker L0745 Soares retina retina eye pTTT3D
N2b4HR
(Pharmacia) with a modified of linker L0746 Soares retina retina eye pTTT3D
N2b5HR
(Pharmacia) with a modified of linker L0747 Soares fetal_heart heart T7T3D
NbHH
19W (Pharmacia) with a modified of linker L0748 Snares fetal Liver pTTT3D
liver spleen and 1NFLS Spleen (Pharmacia) with a modified of linker L0749 Soares_fetal Liver pTTT3D
liver_spleen and 1NFLS_S 1 Spleen (Pharmacia) with a modified of linker L0750 Snares fetal lung pTTT3D
lung_NbHLI
9W (Pharmacia) with a modified ' ~ of linker L07S1 Snares ovary ovarian tumorovary pTTT3D
tumor NbHOT ~ (Pharmacia) with a modified of linker L0752 Soares_parathyroidparathyroid parathyroid pTTT3D
tumor tumor NbHPA gland (Pharmacia) - with a modified polylinker .
L0753 5oares_pineal_gland_N3H pineal pTTT3D
gland pG (Pharmacia) with a modified of linker L0754 Snares placenta placenta pTTT3D
Nb2HP
(Pharmacia) with a modified of linker L0755 Soares_placenta_8to9wee~ placenta pTTT3D
ks_2NbHP8to9W (Pharmacia) with a modified polylinker L0756 Soares_multiplemultiple pTTT3D
sclerosis sclerosis 2NbHMSP lesions (Pharmacia) - with a . modified polylinker V_TYPE
L0757 Soares_senescent_fibroblasenescent pTTT3D
fibroblast sts NbHSF - (Pham~acia) - ' with a modified polylinker V_TYPE
L0758 Soares_testis pTTT3D-Pac NHT
(Pharmacia) with a modified of linker L0759 Soares_total_fetus_Nb2H pT7T3D-Pac F8 9w (Pharmacia) - with a ' modified - of linker L0761 NCI_CGAP_CLL1 B-cell, chronic pT7T3D-Pac lymphotic (Pharmacia) leukemia with a modified of linker L0762 NCI_CGAP_Brl.l breast pT7T3D-Pac (Pharmacia) with a modified of linker L0763 NCI_CGAP_Br2 breast pT7T3D-Pac (Pharmacia) with a modified of linker L0764 NCI_CGAP_Co3 colon pT7T3D-Pac (Pharmacia) vYith a modified of linker L0766 NCI_CGAP_GCB1 germinal ~ pT7T3D-Pac centerB
cell (Pharmacia) with a modified of linker LD767 NCI_CGAP_GC3 pooled germ pTTT3D-Pac cell tumors (Pharmacia) with a modified ' polylinker L0768 NCI_CGAP_GC4 pooled germ pT7T3D-Pac cell tumors . (Pharmacia) with a modified of linker L0769 NC1 CGAP_Brn25 anaplastic brain pT7T3D-Pac _ oligodendroglioma Pharmacia) ( with a modified of linker L0770 NCI_CGAP_Brn23 glioblastomabrain pTTT3D-Pac ~ .
(pooled) (Pharmacia) ' with a modified polylinker L0771 NCI_CGAP_Co8 adenocarcinomacolon pTTT3D-Pac (Pharmacia) with a modified of linker L0772 NCT_CGAP_CoIO colon tumor colon pT7T3D-Pac RER+ .
(Pharmacia) with a modified of linker L0773 NCI_CGAP_Co9 colon tumor colon pT7T3D-Pac RER+
(Pharmacia) with a modified polylinker L0774 NCI CGAP_Kid3 kidney pT7T3D-Pac (Pharmacia) with a modified of linker L0775 NCI_CGAP_KidS 2 pooled kidney pTTT3D-Pac tumors (clear cell (Pharmacia) type) with a modified of linker L0776 NCI_CGAP_Lu5 carcinoid lung pTTT3D-Pac (Pharmacia) with a modified of linker L0777 Soares_NhHMPu_SPooled humanmixed pTTT3D-Pac 1 (see melanocyte, below) (Pharmacia) fetal heart, and with pregnant a modified of linker L0778 Barstead pancreas pancreas pTTT3D-Pac HPLRB 1 (Pharmacia) . with a modified ' of linker L0779 Soares NFL T_GBC_S pooled pTTT3D-Pac -(Pharmacia) with a modified polylinker L0780 Soares NSF_F8_9W_OT pooled pTTT3D-Pac PA P_S1 (Pharmacia) with a modified of linker L0782 NCI CGAP Pr21 normal prostateprostate pTTT3D-Pac - (Pharmacia) with a modified of linker .
L0783 NCI CGAP_Pr22 normal prostateprostate pTTT3D-Pac ..
- (Pharmacia) with a - modified polylinker L0785 Barstead spleen spleen pTTT3D-Pac (Pharmacia) with a modified _ of linker L0786 Soares NbHFB whole pTTT3D-Pac brain - ~ (Pharmacia) with a modified of linker L0788 NCI_CGAP_Sub2 . pTTT3D-Pac (Pharmacia) with a modified of linker L0789 NCI_CGAP_Sub3 pTTT3D-Pac (Pharmacia) with a modified of linker L0790 NCI_CGAP_Sub4 pT7T3D-Pac ~
(Pharmacia) with a modified of linker L0791 NCI_CGAP~SubS pTTT3D-Pac (Phannacia) with a modified oI linker L0792 NCI_CGAP_Sub6 pT7T3D-Pac (Pharmacia) with a modified of linker L0793 NCI_CGAP_Sub7 ~ pTTT3D-Pac (Pharmacia) with a modified polylinker L0794 CGAP_GC6 pooled germ pT7T3D-Pac NCI cell _ tumors . (Pharmacia) with a modified of linker L0800 NCI CGAP Col6 colon tumor,colon pTTT3D-Pac RER+
- (Pharmacia) with a modified of linker L0803 NCI CGAP_Kid kidney pTTT3D-Pac - (Pharmacia) with a modified polylinker L0804 NCI_CGAP_Kidl2 2 pooled kidney pT7T3D-Pac tumors (clear cell (Pharmacia) type) with a modified of linker L0805 NCI CGAP_Lu24 carcinoid lung pT7T3D-Pac - ( . Pharmacia) with a modified of linker L0806 CGAP_Lul9 squamous lung pT7T3D-Pac NCI cell ~
_ carcinoma, (Pharmacia) poorly differentiated with (4 a modified ' ' of linker L0807 CGAP_Ov I8 fibrotheoma ovary pTTT3D-Pac NCI
_ (Pharmacia) with a modified of linker L0809 NCI CGAP Pr28 prostate pT7T3D-Pac - ~
(Pharmacia) with a modified of linker OMIM ~ Description Reference 120160 Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420, 120160 Osteo orosis, idio athic, 166710 120160 Ehlers-Danlos s ndrome, a VIIA2, 130060 120160 Marfan syndrome, aty ical 126650 Chloride diarrhea, con enital, Finnish e, 214700 126650 Colon cancer 129900 EEC s ndrome-1 147450 Am tro hic lateral sclerosis, due to SOD I deficienc , 105400 154276 Mali nant h erthermia susce tibili 3 173360 Thrombo hilia due to excessive lasmino en activator inhibitor 173360 Hemorrha is diathesis due to PAI1 deficienc 176261 Jervell and Lan e-Nielsen syndrome, 220400 183600 S lit hand/foot malformation, a 1 187680 6-merca to urine sensitivi 253270 Multi 1e carbox lase deficienc , biotin-res onsive 300000 O itz G s ndrome, a I
300066 . Deafness, X-linked 6, sensorineural 300077 Mental retardation, X-linked 29 300310 A aroma lobulinemia, a 2, X-linked 301220 Partin ton s ndrome II
302350 Nance-Horan s ndrome 304050 Aicardi s ndrome 3041 I0 Craniofrontonasal d s lasia 306100 Gonadal d s enesis, XY female a 309530 Mental retardation, X-linked I, non-dysmo hic 309585 Mental retardation, X-linked, syndromic-6, with gynecomastia and obesit ~
312040 N s ndrome, 310465 601 Platelet disorder, familial, with associated m 399 eloid mali nanc _ Refsum disease, infantile, 266510 602136 Zellwe er s ndrome-1, 214100 _ Adrenoleukod stro h , neonatal, 202370 602447 Coronary artery disease, susce tibilit to PolynZrcleotide and Polypeptide Variants [84] The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide sequences encoding the polypeptide of SEQ. ID NO:Y, the nucleotide sequence of SEQ ID
NO:X
encoding the polypeptide sequence as defined in column 7 of Table 1A, nucleotide sequences encoding the polypeptide as defined in column 7 of Table 1A, the nucleotide sequence as defined in columns 8 and 9 of Table 2, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9, of Table 2, the nucleotide sequence as defined in column 6, of Table 1B, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in column 6 of Table 1B, the cDNA sequence contained in Clone ID NO:Z, and7or nucleotide sequences encoding the polypeptide encoded by the cDNA sequence contained in Clone ID NO:Z. .
[85] The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y, the polypeptide sequence as defined in column 7 of Table 1A, a polypeptide sequence encoded by the polynucleotide .sequence. in SEQ ID NO:X, a polypeptide sequence encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as ,defined in column 6 of Table 1B, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO.:X, and/or a polypeptide sequence encoded by the cDNA
sequence contained in Clone ID NO:Z.
[86j "Variant" refers to a polynucleotide. or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are ovexall closely similar, arid, in many regions, identical to the polynucleotide or polypeptide of the present.invention.
[87] . Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting o~ (a) a nucleotide sequence described in SEQ ID NO:X
or contained in the cDNA sequence of Clone ID NO:Z; (b) a nucleotide sequence in SEQ
ID NO:X or the cDNA in Clone ID NO:Z which encodes the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid_sequence encoded by the cDNA in Clone ID
NO:Z; (c) a nucleotide sequence in SEQ ID NO:X or the cDNA in Clone ID NO:Z
which encodes a mature polypeptide; (d) a nucleotide sequence in SEQ ID NO:X or the cDNA
sequence of Clone ID NO:Z, which encodes a biologically active fragment of a polypeptide;
(e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of Clone ~ ID
NO:Z, which encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence encoding a polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDN.A in Clone ID NO:Z; (g) a nucleotide sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID
NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (h) a nucleotide sequence encoding a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (i) a nucleotide sequence encoding an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.
j88] The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (fj, (g), (h), (i), or (j) above, the nucleotide coding sequence in SEQ
ID NO:X or the complementary strand thereto, the nucleotide coding sequence of the cDNA
contained in Clone ID NO:Z or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding a,polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide.
sequence encoded by the complexrient of the polynucleotide sequence in SEQ ID NO:X, a nucleotide sequence encoding the. polypeptide encoded by the cDNA contained in Clone ID
NO:Z, the nucleotide coding sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, the nucleotide coding sequence in SEQ ID
NO:B as defined in column 6 of Table IB or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto, the nucleotide sequence in SEQ ID NO:X encoding the polypeptide sequence as defined in column 7 of Table 1A or the complementary strand thereto, nucleotide sequences encoding the polypeptide as defined in column 7 of Table 1A or the complementary strand thereto, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides and nucleic acids.
j89] In a preferred embodiment, the invention encompasses nucleic acid molecules .
which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent hybridization conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (fj, (g), (h), or (i), above, as are polypeptides encoded by these polynucleotides. In- another preferred embodiment, ~polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions, or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[90] In another embodiment, the invention provides a purified protein comprising, or alternatively consisting of, a polypeptide having an amino acid' sequence selected from the group consisting of: (a) the complete amino acid.sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (b) the amino acid sequence of a mature form of a polypeptide having the amino acid sequence of SEQ ID
NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (c) the amino acid sequence of a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; and (d) the amino acid sequence of an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded ~by the cDNA in Clone ID NO:Z. .
[91] The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the amino acid sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid sequence .
encoded by the cDNA contained in Clone ID NO:Z, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B, the amino acid sequence as defined in column 7 of Table 1A, an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are also provided (e.g., those fragments described herein). Further proteins encoded by polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these amino acid sequences under stringent hybridization, conditions or alternatively, under lower astringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins.
(92] ~By a nucleic acid having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to ~5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referred to irx Table 1A or 2 as the ORF (open reading frame), or any fragment specified as described herein.
(93] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention.can be determined conventionally using known~computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). .
In a sequence alignment the query and subject sequences are both DNA
sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, 153 .
k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=S, Gap Size Penalty O.OS, Window Size=S00 or the length of ' the subject nucleotide sequence, whichever is shorter.
[94) If the subject sequence is shorter than the query sequence because of S' or 3' deletions, not because of internal deletions, a manual correction must be made to the results.
This is because the FASTDB program does not account for S' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the S' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are S' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the present invention. Onlybases outside the S' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the~percent identity score.
[95] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the S' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment'of the first 10 bases at S' end. The 10 unpaixed bases represent 10% of the sequence (number of bases at the S' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the S'. or 3' of the subject sequence which are not matched/aligned with the query. In this case.the percent identity calculated by FASTDB is not manually corrected. Once again, only bases S' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
[96] By a polypeptide having an amino acid sequence at least, for example, 9S~%
"identical" to a query amino acid sequence of the present invention, it is intended. that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 9S% identical to a query amino acid sequence, up to S% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur .
at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
(97) As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 9S%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a polypeptide referred to in Table 1A (e.g., the amino acid sequence identified in column 6) or Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the polynucleotide sequence defined in columns 8 and 9 of Table 2) or a fragment thereof, the amino acid sequence of the polypeptide encoded by the polynucleotide sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or a fragment thereof, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment thereof, or the amino acid sequence of the polypeptide encoded by cDNA contained in Clone ID
NO:Z, or a fragment thereof, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-24S (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM
0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group' Length=0, Cutoff Score=I, Window Size=sequence length, Gap Penalty=S, Gap Size Penalty=0.05, Window Size=S00 or the length of the subject amino acid sequence, whichever is shorter.
[98) If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results.
This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global~percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity, calculated by the above FASTDB
program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and Ci-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N-and C- terminal residues of the subject sequence..
[99] For example, a 90 amino acid residue subject sequence is aligned with a residue query sequence to determine percent identity. The deletion occurs at the N-terminus of'the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matchedltotal number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence., This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment, which are not matched/aligned with the query sequnce axe manually corrected for. No other manual corrections are to made for the purposes of the present invention.
[100] The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
Moreover, polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, I-5, or 1-2 amino. acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced fox a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
(101] Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.
(Genes II, Lewin, B., ed., John Wiley & Sons, New York (I985)). These allelic variants can vary at either the polynucleotide and/or polypeptide' level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. -[102] . Using known methods of protein engineering and recombinant DNA
technology, variants may be generated to impxove or alter the characteristics of the polypepfides .of the present invention. For instance, one or more amino acids can be deleted fxom the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem.,268: 2984-2988 (1993)) xeported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).) .
[103] Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring. protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1 a. They used random mutagenesis to generate over 3,500 individual IL-1 a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity]." In fact, only 23 unique amino acid sequences, out of more than 3;500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
[104] ~ Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide . results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N-or C-terminal residues of a protein retains such immunogeriic activities can readily be determined by routine methods described herein and otherwise known in the art.
[105] Thus, the invention further includes polypeptide variants which show a functional activity (e.g., biological activity) of the polypeptides of the invention.
Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
[106] The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95°,%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide having the amino acid sequence of an N
and/or C
terminal deletion), irrespective of whether they encode a polypeptide having functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having functional activity include, inter alias (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the gene, as described~in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA
expression in specific tissues (e.g., normal or diseased tissues); and (4) in situ hybridization '(e.g., histochemistry) for detecting mRNA expression ~in specific tissues (e.g., normal or diseased tissues).
[107] Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, which do, in..fact, encode a polypeptide having functional activity. By a polypeptide having "functional activity" is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein of the invention. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an anti-polypeptide of the invention antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for' a polypeptide of the invention.
[108] The functional activity of the polypeptides, and fragments, variants and derivatives of the invention, can be assayed by various methods.
[109] For example, in one embodiment where one is assaying for the ability to bind or compete with a full-length polypeptide of the present invention for binding to an anti-polypetide antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western. blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
[110] In another embodiment, where a ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of a ~polypeptide o~ the present invention to bind to a substrates) of the polypeptide of the invention can be routinely assayed using techniques known in the art.
[111] In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the present invention and fragments, variants and derivatives thereof to elicit polypeptide related biological activity (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.
[112] Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acid sequence of the cDNA contained in Clone ID NO:Z, the nucleic acid sequence referred to in Table 1A (SEQ ID NO:X), the nucleic acid sequence disclosed in Table 2 (e.g,. the nucleic acid sequence delineated in columns 8 and 9) or fragments thereof, will encode polypeptides "having functional activity." In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
[113] For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., "Deciphering the Message in Protein Sequences:
' Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
[114] The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast; the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
[115] The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. . See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
[116] As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the. protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and 11e; replacement of the hydroxyl residues Ser and Thr; replacement, of the acidic residues Asp and Glu;
replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitutions with one or more of the amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) fusion of the polypeptide with additional amino acids, such as, for example, an 1gG Fc fusion region peptide, serum albumin (preferably human serum albumin) or.a fragment thereof, or leader or secretory sequence, or a sequence facilitating purification, or (v) fusion of the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)).
Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
[117J For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance- due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Bobbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
[118] A further embodiment of the invention relates to polypeptides which comprise the amino acid sequence of a polypeptide having an amino acid sequence which contains at least one amino acid substitution, but not more than~50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions from a polypeptide sequence disclosed herein. Of course ~ it is highly preferable fox a polypeptide to have an amino acid sequence which comprises the ~arnino acid sequence of a polypeptide of SEQ ID NO:Y, an~ amino acid sequence encoded by SEQ
ID NO:X, an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2, an amino acid sequence encoded by the complement of SEQ
ID NO:X, and/or an amino acid sequence encoded by cDNA contained in Clone ID
NO:Z
which contains, in order of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
[119] In specific embodiments, .the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of a reference amino acid sequence selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof (e.g., the mature form and/or other fragments described herein); (b) the amino acid sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the complement of SEQ ID NO:X or fragments thereof; (d) the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns, 8 and 9 of Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA contained in Clone ID
NO:Z or fragments thereof; wherein the fragments or variants have 1-5, 5-10, 5-25, 5-S0, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Polynucleotide aid Polypeptide Fragments .
(120] The present invention is also directed to polynucleotide fragments of the polynucleotides (nucleic acids) of the invention. In the present invention, a "polynucleotide fragment" refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in Clone ID NO:Z or the 'complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide encoded by the cDNA
contained in Clone ID NO:Z or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ
ID NO:X as defined in columns 8 and'9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence in SEQ ID NO:X or the complementary strand thereto; is a polynucleotide sequence encoding a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence encoding a portion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide sequence encoding a portion of a polypeptide encoded by the complement of the polynucleotide sequence in SEQ ID NO:X; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:B as defined in column 6 of Table 1B or the complementary strand thereto; or is a portion of the polynucleotide sequence of SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto.
[121] The polynucleotide fragments of the invention are preferably at least about 15 nt, and more-preferably at least about 20 nt, still more preferably at least about 30 nt, arid even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at Least about 150 nt in length. A fragment "at least 20 nt in length,'-' for example, is intended to include 20 or more contiguous bases from the, cDNA sequence contained in Clone ID
NO:Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto. In this context "about" includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at.both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 160, I70, 180, 190, 200, 250, 500, 600, 1000, or 2000 nucleotides in length ) are also encompassed by the invention.
[I22] ~ Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number I-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 45I-500, 50I-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1400, 1401-1450, 1451-1500, 1501-1550, 1551-'1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551.-3600, 3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400,'5401-5450, 5451-5500,.
5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 58b1-5850, 5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, .6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of SEQ ID NO:X, .or the complementary strand thereto. In this context "about"
includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein.
Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or. alternatively, under lower stringency ~
conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[123] ' Further representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 175I-1800, I80I-1850, 1851-1900, 1.901-1950, 1951-2000, 2001-2050, 2051-2I00;
2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of the cDNA sequence contained in Clone ID NO:Z, or the complementary strand thereto.
In this context "about" includes the particularly recited range or a range larger or smaller by several (5, 4,'3, 2, or 1) nucleotides, at either terminus or at both termini.
Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity).
More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[124] Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist .of, a nucleic acid sequence comprising one, two, three, four, five,' six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence delineated in Table 1B column 6. Additional, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described' polynucleotide fragments of the invention in combination with a polynucleotide sequence that is the complementary strand ,of a sequence delineated in column 6 of Table 1 B. In further embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID
NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated Table 1B, column 6, and have a nucleic acid sequence~which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally;
fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[12S] In additional specif c embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1 B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[126] In additional specific embodiments; polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ
ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are .also encompassed by the invention.
[127] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in the same row of column 6 of Table 1B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defned in Table 1A or 1B) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[128] In additional specific embodiments, polynucleotides of the invention comprise, :or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of the sequence,of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also.
encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention. ' [129] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein) are directly contiguous Nucleic acids which hybridise to the complement of these 20 contiguous polynucleot'ides under stringent hybridization conditions ox alternatively, 'under lower stringency conditions, are also encompassed by the invention. ~Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and po~lypeptides are also encompassed by the invention.
[130J In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also .
encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[I3IJ In specific embodiments, .polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 are directly contiguous. In preferred embodiments, the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1 B is directly contiguous with the 5'' 10 polynucleotides of the next sequential exon delineated in Table 1B, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[132) ~ In the present invention, a "polypeptide fragment" refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y, a portion of an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2, a portion of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID
NO:X, a portion of an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, and/or a portion of an amino acid sequence encoded by the cDNA contained in Clone ID NO:Z. Protein (polypeptide) fragments may be "free-standing," or comprised within a larger polypeptide of which~the fragment forms a part or region, most preferably as a single continuous region. Representative examples of .
polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700; 701-720, 721-740, 741-760, 76I -780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of cDNA. and SEQ ID NO: Y. In a preferred embodiment, polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140,.141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID NO:Y. Moreover, polypeptide fragments of the invention may be at least v about 10, 15, 20, 25, 30, 35, 40, 45, 50, S5, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about" includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at Goth extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
[133j Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the maj ority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether- a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such.
immunblogic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
[134] Accordingly, polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the. carboxy terminus, or both. For eXample,..any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form.
Similarly, any number of amino acids, ranging from I-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
[135] The present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptid~e_ of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1B, and/or a polypeptide, encoded by the cDNA contained in Clone ID NQ:Z). In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ID NO:Y, or the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
[136] The present invention further provides polypeptides having one or more residues from the caxboxy terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ
ID
NO:X as defined in columns 8 and 9 of Table 2, and/or a polypeptide encoded by the cDNA
contained in Clone ID NO:Z). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where n corresponds to the position of amino acid residue, in a polypeptide of the invention.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[137] In addition, 'any of the above 'described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. 'The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide disclosed as SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA~ contained in Clone ID NO:Z, and/or the complemexlt thereof, where n and m are integers as described above.
Polynucleotides encoding these polypeptides are also encompassed by the invention:
[138] Also as mentioned above, even if deletion of orie~ or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. Fox example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or~mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
[139] The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions.
Polynucleotides encoding these polypeptides are also ericoinpassed by the invention.
[140] Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for example, in Tables 1A and 2), the cDNA contained in Clone ID NO:Z, or the polynucleotide sequence as defined in column 6 of Table IB, znay be analyzed to determine certain preferred regions of the polypeptide. For example, the amino acid sequence of a polypeptide encoded by a polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ ID NO:Y
and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2) or the cDNA contained in Clone ID NO:Z may be analyzed using the default parameters bf the DNASTAR computer algorithm (DNASTAR, Inc., 1228. S. Park St., Madison, WI 53715 USA; http://www.dnastar.com~.
[I41] Polypeptide regions that may be routinely obtained using the DNASTAR
computer algorithm include, but are not limited to; Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle hydrophilic regions and hydrophobic regions;
Eisenberg alpha-and beta-amphipathic regions; Karplus-Schulz .flexible regions; Emini surface-forming regions; and Jameson-Wolf regions of high antigenic index. Among highly preferred polynucleotides of the invention in this regard are those that encode polypeptides comprising regions that combine several structural features, such as several (e.g., l, 2, 3 or 4) of the features_set out above.
[142] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from data by DNASTAR
analysis by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
[143] Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a functional activity (e.g.
biological activity) of the polypeptide sequence of which the amino acid sequence is a fragment. By a polypeptide displaying a "functional activity" is meant a polypeptide capable of one or more known functional activities associated' with a full-length protein, such as, for example, biological activity, antigenicity, immunogenicity, and/or multimerization, as described herein.
[144] Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or, a decreased undesirable activity.
[145] In preferred embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the antigenic fragments. of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides encoding these polypeptides are also encompassed by the invention.
j146] The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y; a polypeptide sequence encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the portion of SEQ ID NO:B
as defined imcolumn 6 of Table 1B or the complement thereto; the polypeptide sequence encoded by the cDNA contained in Clone ID NO:Z; or the polypeptide sequence encoded by a polynucleotide that hybridizes to the sequence of SEQ ID NO:X, the complement of the sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, or the cDNA sequence contained in Clone ID NO:Z
under stringent hybridization conditions or alternatively, under lower stringency hybridization as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or alternatively, under lowei stringency hybridization conditions defined supra.
(147] The term "epitopes," as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses- a polypeptide comprising an~ epitope, as well as the polynucleotide encoding this polypeptide. An "immunogenic epitope," as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983)). The term "antigenic epitope," as used herein, is defined as a portion of .a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[148] Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135.
(1985) further described in U.S. Patent No. 4,631,211.) [149] In the. present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 1~1,, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids.
Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 3S; 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of -two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
[150] Non-limiting examples of epitopes of polypeptides that card be used to generate .
antibodies of the invention include a polypeptide comprising, or alternatively consisting of, at least one, two, three, four, five, six or more of the portions) of SEQ ID
NO:Y specified in column 7 of Table 1A. These polypeptide fragments have been determined to bear antigenic epitopes of the proteins of the invention by the analysis of the Jameson-Wolf antigenic index which is included in the DNAStar suite of computer programs.
By "comprise" it is intended that a polypeptide contains at least one, two, three, four, five, six or more of the portions) of SEQ ID NO:Y shown in column 7 of Table 1A, but it may contain additional flanking residues on either the amino or carboxyl termini of the recited portion. Such additional flanking sequences are preferably sequences naturally found adjacent to the portion; i.e., contiguous sequence shown in SEQ ID NO:Y. The flanking sequence may, however, be sequences from a heterolgous polypeptide, such as from another protein described herein or from a heterologous polypeptide not described herein.
In. particular embodiments, epitope portions of a polypeptide of the invention comprise one, two, three, or more of the portions of SEQ ID NO:Y shown in column 7 of Table 1A.
[151] Similarly, immunogenic epitopes can be used, for example, to~-induce antibodies -according to methods well known in the art. See, for instance, Sutcliffe et al., supra;
Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985): Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well-as any combination .of two, three, four, five or .more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have -been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
[152] Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo ' immunization, in vitro immunization, and phage display methods. -See, e.g., Su'tcliffe et al., supra; Wilson et al., supra, and-Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer rnay be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide. ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ~.g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide ,antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
[153] As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including fragments or variants thereof), may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides.
By way of another non-limiting example, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S.
Patent No.
5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP
Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Patent 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N.- or'C-terminal end of the heterologous protein (e.g., immurioglobulin Fc ~polypeptide or human , serum albumin ~polypeptide).
Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.
[154) Such fusion proteins as those described above may facilitate purification and may increase half life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of 'the heavy or light chains of mammalian immunoglobulins. See, e.g., EP
394,827;
Traunecker et al., Nature, 331:84-86 (I988). Enhanced.delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT
Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been~found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-(1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows 'for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Fusion Proteins [155] Any polypeptide of the present invention can be used to generate fusion proteins.
For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
Moreover, because secreted proteins target cellular locations based on trafficking signals, polypeptides of the present invention which are shown to be secreted can be used as - targeting molecules once fused to other proteins.
[156] Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
[157] - In certain preferred embodiments, proteins of the invention are fusion proteins comprising an amino acid sequence that is an N , and/or C- terminal deletion of a polypeptide of the invention. In preferred embodiments, the invention is directed to a fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence.of the invention.
Polynucleotides encoding these proteins are also encompassed by the invention.
[158] Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to. final preparation of the polypeptide.
The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
[159] As one of skill in the art will appreciate that, as discussed above, polypeptides of the present invention, and epitope-bearing fragments thereof, can be combined with heterologous polypeptide sequences. For example, the ,polypeptides of the present invention may be fused with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHl, CH2, CH3, and any combination thereof, including both. entire domains and portions thereof), or albumin (including, but not limited to, native or recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. For example, EP-A-O 464 S~3 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy. and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP-A
0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the, Fc portion may hinder therapy -and diagnosis if the fusion protein is used as an antigen for immunizations.
In drug discovery, for example,' human proteins, such as hIL-S, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-S. See, D. Bennett et al., J. Molecular Recognition 8:52-S8 (1995); K. Johanson et al., J. Biol.
Chem. 270:9459-9471 (1995).
[160] Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a polypeptide which'facilitates purification of the fused polypeptide. In ' .preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, .Inc., 9259 Eton Avenue, Chatsworth, CA, 91.311), among others, many of which are commercially available: As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. .Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)).
[161] Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;
5,830,721; 5,834,252;
and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997);
Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);
and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each of these patents and publications are hereby incoiporated ~ by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In. another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, . , etc. of one or more heterologous molecules.
[162] Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
Recombinant and Synthetic Production of Polyp~tides of the Invention [163] The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective.
In the latter case, viral propagation generally will occur only in complementing host cells.
[164] The polynucleotides of the invention may be joined ~to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vecfor, is a virus, it may be packaged in vitro using an appropriate packaging cell line and then txansduced into host cells.
[165] The polynucleotide insert should be operatively linked ~ to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, imthe transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
[166] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, 6418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal ,cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178));
insect cells such as Drosophila S2 and Spodoptera S~ cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
[167] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNHl6a, pNHlBA, p~1H46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG
available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
Preferred expression vectors for~use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S 1 ~ pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogeri, Carlbad, CA). Other suitable vectors will be readily apparent to the skilled artisan.
[168] Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative.
Glutamine synthase expression systems can also functiom in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine~ synthase expression system and components thereof are detailed in PCT publications: W087/04462;
W086/05807;
W089/01036; W089/10404; and W091/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using a GS expression system in marine myeloma cells is described in Bebbington et al., Bioltechr~ology 10:169(1992) and in Biblia and Robinson Biotech~ol.
Prog. 11:1 (1995) which are herein incorporated by reference.
[169] The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic~cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
[170] Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector. .
[171] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, ~e.g., US Patent Number 5,641,670, issued June 24, 1997; International Publication Number WO 96/29411;
International Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad.
Sci. USA
86:8932-8935 (1989); and Zijlstra et al., Nature 32:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
[172] Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or canon exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
[173] Polypeptides of the present invention can also be recovered from:
products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal. process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
[174] In one embodiment, the yeast Pichia pastoris is used to express polypeptides of the invention in a eukaryotic system. Pichia pasto~is is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using OZ. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for 02. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOXI) is highly active. In the presence of methanol, alcohol oxidase produced from the ADXI gene comprises up to approximately 30% of the total soluble protein in Pichia pastor~is. See Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al., Yeast 5:167-77 (1989); Tschopp, J.F., et al:, Nucl. Acids Res. 15:3859-76 (1987).
Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the A OXl regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
[175] In one example, the plasmid vector pPIC9K is used to express DNA
encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J.
Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOXl promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
[176) Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDI, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in the art would readily appreciate, as Long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
[177] . In another embodiment,. high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol. ' [178] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucl~eotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polyriucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent No.
5,641,670, issued June 24, 1997; International Publication No. WO 96/29411', published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:.435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
[179] In addition, polypeptides of the invention can. be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman' & Co., N.Y., and Hunkapiller et al., Nature, 310:105-('1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, .if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common .amino acid°s, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic .acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, .
norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary).
[180] The invention encompasses polypeptides of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V 8 protease, NaBH4; acetylation, formylation, oxidation, reduction;
metabolic synthesis in the presence of tunicamycin; etc.
[181] Additional post-translational modifications encompassed by the .
invention include, for example, e.g., N-linked or 0-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
[182] Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples ~of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine (1211, i23I,.izsl, i3il), carbon (14C), sulfur (3sS), tritium (3H), indium (111In, llzln, 113mIn, llsmln), technetium (99Tc,99mTc), thallium (2olTi), gallium (68Ga, 67Ga), palladium (lospd), molybdenum (991VIo), xenon (133Xe), fluorine (18F), ls3Sm,, 177Lu, ls9Gd, 149Pm~ la°La~ 17s~' 166Hp~ 9oY~ 47SC~ ls6Re~ ls8Re~ 142Pr~ lose and 97Ru.
[183] In specific embodiments, a polypeptide of the present invention or, fragment or variant thereof is attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 177Lu, 9oY, 166Ho, and ls3Sm, to polypeptides.
In a preferred 'embodiment, the radiometal ion associated with the macrocyclic chelators is 111In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is 9°Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via a 186 _ linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art - see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
[184] As mentioned, the proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide.
Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et aL, Meth.
Enzymol.
182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[185] Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at-random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
[186] The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used,- depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).-For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,S00, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
[187j As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in IJ.S. Patent No.
5,643,575;
Morpurgo et al., Appl. Biochem. Biotech~zol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioco~jug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
[188] The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to-those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having ar free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol. molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
[189] As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g.., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
[190] One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated 'moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modif cation may be accomplished by reductive alkylation which exploits differential reactivity of-different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
[191] _ As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev.
Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S.
Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98132466, the disclosures of each of which are incorporated herein by reference. _ [192] One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the, modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (CISOzCH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[193) Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Patent No. 5,612,460, the entire disclosure of which is incozporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to, the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described' in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of.the invention.
[194] The number of polyethylene glycol moieties attached to each protein bf the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, S, 6, 7, 8, 9, 10, 12, 1 S, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-S, 4-6, S-7, 6-8, 7-9, 8-10, 9-LI, 10-I2, 11-13, I2-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule.
Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Theca. Drug Carrier Sys. 9:249-304 (1992).
[195] The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are .not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC".) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
[196] The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly; the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trirt~ers, or at least tetramers.
[197] Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer refers to a multimer containing only polypeptides corresponding to a protein of the invention (e.g., the amino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID
NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or an amino acid sequence encoded by cDNA contained in Clone ID
NO:Z (including fragments, variants, splice variants, and fusion proteins, corresponding to these as described herein)). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence.
In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specif c embodiments, the multimer of the invention is a homodimer (e.g., containing two polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing three polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
[198] As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
[199] Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked by, for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, lieteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ
ID NO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or encoded by the cDNA contained in Clone ID NO:Z). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein. In one example, covalent associations are~between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a Fc fusion protein of the invention (as described herein). In another specific.example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO:
WO
98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
[200] Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
[201] Trimeric polypeptides of the invention may offer.the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S.
patent application Ser. No. 08/446,922, hereby incorporated by reference.
Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides 'of the invention.
[202] In another example, proteins of the invention are associated by interactions between Flag~ polypeptide sequence contained in fusion proteins of the invention containing Flag~ polypeptide sequence. In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in .
Flag~ fusion proteins of the invention and anti-Flag~ antibody.
[203] The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques, known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-Iinks between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US
Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is~ herein incorporated by reference' in its entirety).
[204] Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, .
which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., ,US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). ' Antibodies [205] Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of the invention (e.g., a polypeptide or fragment or variant of the.
amino acid sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in Clone ID
No:Z, and/or an epitope, of the present invention) as determined by immunoassays well known in the art for assaying specific antibody-antigen binding. Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of.the above. The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The irnmunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the immunoglobulin molecules of the invention are IgGl . In other preferred embodiments, the immunoglobulin molecules of the invention are IgG4.
[206) Most preferably the antibodies are human antigen-binding antibody fragments of the present invention.and include, but are not limited to, Fab, Fab' and.F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable regions) alone or in combination with the' entirety or a portion of the following: hinge region, CH1; CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable regions) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibodies include antibodies having the amino acid sequence'of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.~
J
[207] The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92105793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5;601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[208] Antibodies of the present invention may be described or,specif ed in terms of the epitope(s) or portions) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portions) may be specified as described herein, e.g., by N-terminal and C-terminal positions, or by size in contiguous amino acid residues, or listed in the Tables and Figures. Preferred epitopes of the invention include the predicted epitopes shown in column 7 of Table 1A, as well as polynucleotides that encode these epitopes. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the .
same.
(209] Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind'any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, Less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, Iess than 55%, and Less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combinations) of 2, 3, 4, 5,. or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affnities include those with a dissociation constant or Kd less than 5 X 10'2 M, 10'2 M, 5 X 10'3 M, 10'3 M, 5 X 10' 4 M, 10'4 M, 5 X 10'5 M, 10'5 M, 5 X 10'6 M, 10'6M, 5 X 10'7 M, 107 M, 5 X
10'8 M, 1 b'8 M, 5X10'9M,10'9M,5X10'1°M,10'1°M,5X10'11M,10'11M,SX10''2M,10''2 M,SX
10'I3M, 10'13M,SX 10'14 M, 10'14M,SX10'lSM,orlO'15M.
[210J The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
[211] Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation.. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. ' For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for examphe, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the.activity in absence of the antibody.
[212J . The invention also features receptor-specific antibodies which .both prevent ligand binding and receptor activation as well as antibodies that recognize the.receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are. neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the Iigand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT
publication WO
96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et aL, Cancer Res. 58(15):3209-3214 (I998); Yoon et aL; J.
Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998);
Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997);
Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
[213] Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have utility in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
incorporated by reference herein in its entirety.
[~14] As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be rscombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules-useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT
publications WO 92/08495; WO 91114438; WO 89112624; U.S. Patent No. 5,314,995;
and EP 396,387; the disclosures of which are incorporated herein by reference in their entireties.
[215] The antibodies of the invention include derivatives that are modifed, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, arnidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by~ known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic. synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical amino acids. ~ ' [216] The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc.
to induce the production of sera containing polyclonal antibodies specific for the antigen.
Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG, (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
[217] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-(Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
[218] Methods for producing and screening for specif c antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and' cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
(219] Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
(220] Another well known method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr Virus (EBV). Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference. The source of B cells for transformation is commonly human peripheral blood, but B cells for. transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues.
Tissues axe generally made into single cell suspensions prior to EBV
transformation.
Additionally, steps maybe taken to either physically remove or inactivate T
cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
[221J In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast micxoscopy, transformed cells may appear large, clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV
lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV
transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SILO-007, RPMI 8226, and KR-4). Thus, the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
[222] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) ox pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
[223] For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected ox identified with antigen, e.g., using labeled antigen or antigen bound-or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.
Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994);
Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994);
PCT application No. PCTlGB91/01134; PCT publications WO 90/02809; WO 91/10737;
WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.
Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
[224] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
[225] Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos.
5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., ~ by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al:, PNAS 91:969-973 (1994)), and chain shuffling (0.S.
Patent No. 5,565,332).
[226j Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111;
and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO
96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
[227] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma_ technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int.
Rev.
Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92101047; WO 96/34096;
WO
96/33735; European Patent No. 0 598 877; U.S. Patent Nos. 5,413,923;
5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771;
5,939,598;
6,075,181; and 6,114,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, CA) arid Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
[228] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Biotechnology 12:899-903 (1988)).
[229] Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic". polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona, FASEB J.
7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby block its biological activity. Alternatively, antibodies which bind to and enhance polypeptide multimerization and/or binding, and/or receptor/ligand multimerization, binding and/or signaling can be used to generate anti-idiotypes that function as agonists of a polypeptide of the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens as agonists of the polypeptides of the invention or its ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its.
ligand(s)/receptor(s), and thereby promote or enhance its biological activity.
j230] Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen et al., Hum. Gene Ther. 5:595-601 (1994);
Marasco, W.A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev.
Microbiol.
51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999);
Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999);
Zhu et al., J. Immunol. Methods 231:207-222 (1999); and references cited therein. .
Poly~ueleotides Encoding Antibodies [231] The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ
ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or to a polypeptide encoded by the cDNA contained in Clone ID
NO:Z.
[232] The polynucleotides. may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
[233] Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR
amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody.
Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
[234] Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
[235] In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA
techniques, one or more of the CDRs may be inserted within framework regions, e..g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
[236] In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable .region derived from a murine rilAb and a human immunoglobulin constant region, e.g., humanized antibodies.
[237] Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778; Bird Science 242:423- 42 (1988); Huston et al., Proc. Natl.
Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) cari be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. colt may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
Methods ofP~oducingAntibodies [238] The antibodies of the invention can be produced by any method known in the art 'for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Methods of producing antibodies include, but are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below.
[239] Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody.molecule may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an . antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an~antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a-promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S.
Patent No. 5,122,464) and the variable domain of the antibody may be cloned- into such a vector for expression of the entire heavy or light chain.
[240] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
[241] , A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule: For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 ( 1990)).
[242] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX
vectors may also be used to express . foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin,or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
[243] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
(244] . In mammalian host cells, a number of viral-based expression systems 'may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)).
Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences.
Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons, can be of a variety of origins, both natural and synthetic. The efficiency of expression may -be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods, in Enzymol. 153:51-544 (1987)).
[245] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e:g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
[246] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which~contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of. the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasniid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and ,grow to form foci which in turn can be cloned and' expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
[247] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxaxithine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad.
Sci. USA
48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes:
dhfr, which confers resistance _to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.
32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.
Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).
Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY
(1990); and in Chapters 12 and' 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981); which are incorporated by reference herein in their entireties.
[248] The expression levels of an .antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplif canon for the expression of cloned genes in mammalian cells in DNA
cloning, Vol.3. ~ (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Grouse et al., Mol. Cell. Biol. 3:257 (1983)).
[249] Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drubs methinninP c»inhnximinP pr mPthntraxatP
reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suplliers, including, for example Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using a GS expression system in marine myeloma cells is described in Bebbington et al., Bioltechnology 10:169(1992) and in Biblia and Robinson Biotechnol.
Prog. 1 1:l (1995) which are incorporated in their entirities by reference herein.
[250] The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986);
Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
[251] Once an antibody molecule of the invention has been produced by an,animal, chemically,synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
[252] The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does ' not necessarily need to be direct, but may occur through linker sequences. , The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232;,EP
439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981;
Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
[253] The present invention fizrther includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other thar~
the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain' or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053;
5,447,8SI;
5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 9-1/06570;
Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:1OS3S-1OS39 (1991); Zheng et al., J.
Immunol. 1S4:SS90-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA
89:11337-11341 (1992) (said references incorporated by reference in their entireties).
[254] As discussed, sZrpra, the polypeptides corresponding to a polypeptide,_ polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to ~
facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See EP 394,827; and Traunecker et 214:
al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. See, for example, Fountoulakis et al., J.
Biochem. 270:3958-3964 (1995). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. See, for example, EP A 232,262. Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).
[255] Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al,, Cell 37:767 (1984)) and the "flag" tag.
[256] The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part' of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferi~n, and aequorin; and examples of suitable radioactive material include 125I, 131I, 11 lIn or 99Tc.
[257] Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for,example, 213Bi. A
cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol~, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin~ etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, Iidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, rrielphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthrac~clines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
[258] The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent .or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, 13-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator,.an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as~
for example, lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
[259] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
[260] Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985)? Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.
(Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", I-mmunol. Rev. 62:119-58 (1982).
[261] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety. ' [262] An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factors) and/or cytokine(s) can be used as a therapeutic.
Immunophenotyping [263] The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. Translation products of the gene of the present invention may be useful as cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Patent 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
[264] These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and "non-self' cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
Assays Fot~ Antibody Binding.
[265] The antibodies of the invention may be assayed for imrnunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). .Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
[266] Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hburs) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds., (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.16.1.
[267] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non=fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g:, an anti-human antibody) conjugated to .an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane . in wash buffer, and detecting the presence of the antigen., One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York, section 10.8.1.
[268] ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, (1994), Current Protocols. in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 11.2.1.
[269] The binding affinity of an antibody to an antigen and the off rate of an antibody=
antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
[270] Antibodies of the invention may be characterized using immunocytochemisty methods on cells (e.g., mammalian .cells, such as CHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art. Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.
Therapeutic Uses , [271] The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
[272] In a specific and preferred embodiment, the present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more diseases, disorders, or conditions, including but not limited to: neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions., and/or as described elsewhere herein.
Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g., antibodies directed to the full length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a polypeptide of the invention (such as, for example, a predicted linear epitope shown in column 7 of Table 1A; or a conformational epitope, including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
[273] A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g.
as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary sleill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
[274] The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as; e.g., IL-2, IL-3 and IL-7), for example, which serve o increase the number or activity of effector cells which interact with the antibodies.
[275] The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
[276] It is preferred to use high affinity and/or potent i~ vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof.
Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10'2 M, 10'2 M, S X 10'3 M, 10'3 M, S X 10-4 M, 10'4 M, S X 10'' M, 10'5 M, S X 10'6 M, 10'6 M, S X
10'7 M, 10'7 M, S X 10-8 M, 10'$ M, S X 10-9 M, 10'9 M, S X 10'1° M, 10-1° M, S X 10-11 M, 10-11 M, S X 10-12 M, 10-12 M, S X 10'13 M, 10' 13 M, S X 10'14 M, 10-14 M, S
X 10'15 M, and lp-ls M.
222.
Gene Therapy [277] In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an'expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
[278] Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
[279) For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-SOS (1993); Wu and Wu, Biotherapy 3:87-9S (1991); Tolstoshev, Ann.
Rev. Pharmacol. Toxicol. 32:573-S96 (1993); Mulligan, Science 260:926-932 (1993); and Moigan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH
11 (S):1 S S-21 S ( 1993 ). Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY ( 1990).
[280] In a preferred embodiment, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In-another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 ( 1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
(281] Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
[282] In a specific embodiment, the nucleic acid sequences are directly administered ih vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; W092/20316; W093/1418$, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Roller and Smithies, Proc. Natl.
Acad. Sci.
USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
[283] In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
[284] Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia.
Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. I~ozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991);
Rosenfeld et al., Cell 68:143- 155 (1992); Mastrangeli et al., J. Clin.
Invest. 91:225-234 (1993); PCT Publication W094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995).
In a preferred embodiment, adenovirus vectors are used.
[285] Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).
[286] Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells axe then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
[287] ~ In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.
217:599-618 (1993); Gohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac.
Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide.for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
[288] The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
[289) Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
[290] In a preferred err~bodiment, the cell used for gene therapy is autologous to the patient.
[291] In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered i~
vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used.
Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992);
Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc.
61:771 (1986)).
[292] In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by the presence or absence of an appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity [293] The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
Ther-~apeuticlProphylactic Administration and Composition [294] The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably a polypeptide or antibody of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that Iimit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
[295] Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above;
additional appropriate formulations and routes of administration can be selected ,from among those described herein below.
[296] Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
[297] In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or b'y means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
[298] In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Larger, Science 249:1527-1533 (1990);
Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 3S3- 36S (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) [299] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Larger, supra;
Sefton, CRC Crit. Ref. Biomed. Erg. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Larger and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);
Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). ' [300] Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
[301] In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector 'and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.
USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
[302] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium_stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
[303] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[304] The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[305] The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention 'of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
[306] For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to mg/kg of the patient's body weight. Generally, human antibodies have a longer half life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
[307] The invention also provides a pharmaceutical pack or kit comprising one or more containers f lled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. - .
Diagnosis and Imaging [308] Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
[309] The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the 1 presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
[310] Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J.
Cell . Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (I25I, 12II), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[3I1] One facet of the invention is the detection and diagnosis of a disease or. disorder s associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds' to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
[312] It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S.W.
Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments."
(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.
A.
Rhodes, eds., Masson Publishing Inc. (1982)).
[313] Depending on several variables, including the type of label used and'the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or to 10 days.
[314] In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
[315] Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used.
Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
[316] In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S.
Patent No. 5,441,050). Imanother embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
Kits [317] The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specif cally immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound;
an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
[318] ~ In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recbmbinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
[319] In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
[320] In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody.
The detecting means of the kit may include a second, labeled monoclonal antibody.
Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
[321] In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
[322] The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
[323] Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
Uses of the Polynucleotides [324] Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
[325] The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), axe presently available. Each sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome, thus each polynucleotide of the present invention can routinely be used as a chromosome marker using techniques known in the art. Table 1A, column 9 provides the chromosome location of some of the polynucleotides of the invention. .
[326] Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably at least 15 by (e.g., 15-25 bp) from the sequences shown in SEQ ID
NO:X.
Primers can optionally be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR
screening of somatic cell hybrids containing individual human chromosomes.
Only those hybrids containing the human gene corresponding to ~SEQ ID NO:X will yield an amplified fragment.
[327] Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, preselection by hybridization to construct chromosome specific-cDNA
libraries, and computer mapping techniques (See, e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is hereby incorporated by reference in its entirety).
[328] Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread.
This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 by are preferred. For a review of this technique, see Verma et al., "Human Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York (1988).
[329] For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes).
[330] Thus, the present invention also provides a method for chromosomal localization which involves (a) preparing PCR primers from the polynucleotide sequences in Table 1A
and/or Table 2 and SEQ ID NO:X and (b) screening somatic cell hybrids containing individual chromosomes.
[331] The polynucleotides of the present ~ invention would likewise be useful for radiation hybrid mapping, HAPPY mapping, and long range restriction mapping.
For a review of these techniques and others known in the art, see, e.g. Dear, "Genome Mapping:
A Practical Approach," IRL Press at Oxford University Press, London (1997);
Aydin, J.
Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry 3:483-492 (1998);
Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol.
62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999) each of which is hereby incorporated by reference in its entirety.
[332] Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis.
Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library)). Column 10 of Table 1A provides an OMIM reference identification number of diseases associated with the cytologic band disclosed in column 9 of Table 1A, as determined using techniques described herein and by reference to Table 5.
Assuming I
megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
[333] Thus, once coinheritance is established, differences in a polynucleotide of the invention and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene .
from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.
[334] Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using the polynucleotides of the invention. Ariy of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker. Diagnostic and prognostic methods, kits and reagents encompassed by the present invention are briefly described below and more thoroughly elsewhere herein (see e.g., the sections labeled "Antibodies", "Diagnostic Assays", and "Methods for Detecting Diseases").
[335] Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder. Additional non-limiting examples of diagnostic methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., Example 12).
[336] In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general embodiment, the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the invention, where each probe has one strand containing a 31'mer-end internal to the region. In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification.
[337] Where a diagnosis of a related disorder, including, for example, diagnosis of a tumor, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
[338] ~ By "measuring the expression Ievel of polynucleotides of the invention" is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA
level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the related disorder or being determined by averaging levels from a population of individuals not having a related disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
[339] By "biological sample" is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains polypeptide of the present invention or the corresponding mRNA. As indicated, biological samples include body fluids (such as semen, lymph, vaginal pool, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
[340] The methods) provided above may preferably be applied in a diagnostic method andlor kits in which polynucleotides and/or polypeptides of the invention are attached to a solid support. In one exemplary method, the support may be a "gene chip" or a "biological chip" as described in US Patents 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the invention attached may be used to identify polymorphisms between the isolated polynucleotide sequences of the invention, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, such as for example, in neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, digestive disorders, metabolic disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. Such a method is described in US Patents 5,858,659 and 5,856,104. The US Patents referenced supra are hereby incorporated by reference in their entirety herein.
[341] The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides of the invention are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA
analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by Nielsen et al., Science 254, 1497 (1991); and Egholm et al., Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than.DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA
hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (Tm) by 8°-20° C, vs. 4°-16° C for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
[342] The compounds of the present invention have uses which, include, but are not limited to, detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to:
acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.;
and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
[343j Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., "The Etiology of Acute Leukemia:
Molecular Genetics and Viral Oncology," in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types. (Gelmann et al., supra) Indeed, the, human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra) [344] For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated. (International Publication Number WO
91/15580). However, it has been shown that exposure of HL-60 cells to a DNA
construct that is complementary to the 5' end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells.
(International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci.
85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the present invention's usefulness is not be limited to treatment, prevention, and/or prognosis of proliferative disorders of cells and tissues of hematopoietic origin, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.
[345] In addition to the foregoing, a polynucleotide of the present invention can be used to control gene expression through triple helix formation or through antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem.
56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC
Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney_ et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding .of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix - see Lee et al., Nucl.
Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense - Okano, J.
Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC
Press, Boca Raton, FL (1988)). Triple helix formation optimally results in a shut-off of RNA
transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. The oligonucleotide described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of polypeptide of the present invention antigens. Both techniques are effective in model systems, and the information disclosed hereim can be used to design antisense or triple helix polynucleotides in an effort to treat disease, and in particular;
for the treatment of proliferative diseases and/or conditions. Non-limiting antisense and triple helix methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the section labeled "Antisense and Ribozyme (Antagonists)").
[346] Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner.
Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled "Gene Therapy Methods", and Examples 16, 17 and 18).
[347] The polynucleotides axe also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel.
This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.
[348] The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA
sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
[349] Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood,, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can be amplified using PCR'. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992)). Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA
corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.
(350] There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention, specific to tissues, including but not limited to those shown in Table 1A. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen 'tissue cultures for contamination. Additional non-limiting examples of such uses are further described herein.
[351] The polynuc~eotides of the present invention are also useful as hybridization probes for differential identification of the tissue(s)~or cell types) present in a biological sample. Similarly, polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissues) (e.g., immunohistochemistry assays) or cell types) (e.g., immunocytochemistry assays). In addition, for a number of disorders of the above tissues or cells, significantly higher or lower levels of gene expression of the polynucleotides/polypeptides of the present invention may be detected in certain tissues.(e.g., tissues expressing polypeptides and/or polynucleotides of the present invention, for eXample, those disclosed in column 8 of Table 1A, and/or cancerous and/or wounded tissues) or~bodily fluids (e.g., semen, lymph, vaginal pool, serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
[352] Thus, the invention provides a diagnostic method of a disorder, which involves:
(a) assaying gene expression level in cells or body fluid of an individual;
(b) comparing the gene expression level with a standard gene expression level, whereby an increase or decrease in the assayed gene expression level compared to the standard expression level is indicative of a disorder.
[353] In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip" or other support, to raise anti-DNA antibodies using DNA
immunization techniques; and as an antigen to elicit an immune response.
Uses of the Polypeptides [354] Each of the polypeptides identified herein can be used in numerous ways.
The following description should be considered exemplary and utilizes known techniques.
[355] Polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissues) (e.g., immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et al., J.
Histochem. Cytochem. 29:577-580 (1981)) or cell' types) (e.g., immunocytochemistry assays). ' [356] Antibodies can be, used to assay levels of polypeptides encoded by polynucleotides. of the invention in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J.
Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (131h lzsh lzsh izil)~ c~.bon (14C), sulfur (3sS), tritium (3H), indium (1 tsmln~ 113mIn, llzln, 111In), and technetium (99Tc, 99mTc), thallium (z°1Ti), gallium (68Ga, 67Ga), palladium (losPd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), is3Sm~ ~77Lu~ is9Gd~ ia9Pm~ i4oLa~ i7syb~ 166Ho~ goy~ a7Sc~ ia6Re~ lasRe~
i4zPr~ ios~~ 97Ru;
luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[357] In addition to assaying levels of polypeptide of the present invention in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
[358] A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for ~ example, ~31I, llzIn, 99m~Z~~~ (lslh i2sl~ lz3h izil)~ carbon (14C), sulfur (3sS), tritium (3H), indium (11'"'In, 113"'In, llzln, 111In), and technetium (99Tc, ~99mTc), thallium (zo.lTi), gallium (6~Ga, 67Ga), palladium (lo3pd), molybdenum (~9Mo), xenon (l3sXe), fluorine (18F, ls3Sm, 177Lu, is9Gd, la9Pm, laoLa, ms~,b~ 166H~~ 9oY.~ a.7Sc~ is6Re~ issRe, i4zPr~ lose 97Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which express the polypeptide encoded by a polynucleotide of the invention. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc.
(1982)).
[359] In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g., polypeptides encoded by polynucleotides of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell.
In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
[360] In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention in association with toxins or cytotoxic prodrugs.
[361] By "toxin" is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. "Toxin" also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, Zi3Bi, or other radioisotopes such as, for example, lo3Pd, 133Xe, i3ih 68Ge,'7Co, 6'Zn, 85Sr, 32P, 3'S, Soy, ls3Sm, ls3Gd, 169Yb, ~ICr, 54Mn, 75Se, 113Sn, 9oyttrium, 117Tin, lg6Rhenium, 166Holmium, arid 188Rhenium;
luminescent labels, such as luminol;~ and fluorescent labels, such as fluorescein and rhodamine, and biotin. In a specific embodiment, the invention,provides a method for the ' specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 9°Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope lllln. In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 1311.
[362] Techniques known in the art may be applied to label polypeptides of the invention (including antibodies). Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. PatentNos_ 5,756,065;
5,714,631;5,696,239;
5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560;
and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety).
[363] Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying,Vthe expression level of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
[364] Moreover, polypeptides of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
[365] Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease (as described supra, and elsewhere herein). For example, administration of an antibody directed to a polypeptide of the present invention can bind, and/or neutralize the polypeptide, and/or reduce overproduction of the polypeptide.
Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
[366] At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS=PAGE gels or.on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the biological activities described herein.
Diagnostic Assays [367] The compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described herein under the section heading "Biological Activities".
[368] For a number of disorders; substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding the polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression levels) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
[369] The present invention is also useful. as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
[370] In certain embodiments, a, polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognose diseases and/or disorders associated with the tissues) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[371] By "assaying the expression level of the gene encoding the polypeptide"
is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or, the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA
level in a second biological sample). Preferably, the polypeptide expression level or mRNA
level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
[372] By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof] or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mamriials are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
[373] Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S 1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the -ligase chain reaction (RT-LCR).
[374J The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of polypeptides of the invention compared to normal control tissue samples may be used to detect the presence of tumors. Assay techniques that can be used to determine levels of a polypeptide, such as a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying polypeptide levels in a biological sample can occur. using any art-known method.
[375J Assaying polypeptide levels in a biological sample can occur using antibody-based techniques. For example, polypeptide expression in tissues can be studied ' with classical immunohistological methods (Jalkanen et al., J. Cell. Biol.
101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (lzsl, izil), carbon (14C), sulfur (3sS), tritium (3H), indium (llzln), and technetium (99mTC), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[376] The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of inteest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.
[377] For example, antibodies, or fragments of antibodies, such as those described herein, may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
[378] In a preferred embodiment, antibodies, or fragments of antibodies directed to any one or all of the predicted epitope domains of the polypeptides of the invention (shown in column 7 of Table 1A) may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
[379] In an additional preferred embodiment, antibodies, or fragments of antibodies directed to a conformational epitope of a polypeptide of the invention may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection. .
[380] The antibodies (or fragments thereof), and/or polypeptides of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The antibody (or fragment thereof) or polypeptide is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the gene product, or conserved variants or peptide fragments, or polypeptide binding, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection. _ [381] Immunoassays and non-immunoassays for gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detestably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
[382] The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detestably labeled antibody or detectable polypeptide of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.
(383] By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylaxnides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc.
Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
[384] The binding activity of a given lot of antibody or antigen polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
[385] In addition to assaying polypeptide levels or polynucleotide levels in a biological sample obtained from an individual, polypeptide or polynucleotide can also be detected in vivo by imaging. For example, in one embodiment of the invention, polypeptides and/or antibodies of the invention are used to image diseased cells, such as neoplasms. In another embodiment, polynucleotides of the invention (e.g., polynucleotides complementary to all or a portion of an mRNA) and/or antibodies (e.g., antibodies directed to any one or a combination of the epitopes of a polypeptide of the invention, antibodies directed to a conformational epitope of a polypeptide of the invention, or antibodies directed to the full length polypeptide expressed on the cell surface of a mammalian cell) are used to image diseased or neoplastic cells.
[386] Antibody labels or markers for in vivo imaging of polypeptides of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR
and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. Where a in vivo imaging is used to detect enhanced levels of polypeptides for diagnosis in humans, it may be preferable to use human antibodies or "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using techniques described herein or otherwise known in the art. For example methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567;
Taniguchi et al., EP
171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO
8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 31=1:268 (1985).
[387] Additionally, any polypeptides of the invention whose presence can be detected, can be administered. For example, polypeptides of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.
[388] ~ A polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 13i1, nzln~ 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the antigenic protein. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc.
(1982)).
[389] With respect to antibodies, one of the ways in which an antibody of the present invention can be delectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et al., J. Clin.
Pathol. 31:507-520 (1978); Butler, J.E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotornetric, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody include, but. are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme.
Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
[390] Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect polypeptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Tiaining Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, 'but not limited to, a gamma counter, a scintillation counter, or autoradiography.
[391] It is also possible to label the antibody with a fluorescent compound.
When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
[392] The antibody can also be detectably labeled using fluorescence emitting metals such as ls2Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[393] The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
(394] Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Methods for Detecting Diseases [395] In general, a disease may be detected in a patient based on the presence of one or more proteins of the invention and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, urine, and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a disease or disorder, including cancer and/or as described elsewhere herein. In addition, such proteins may be useful for the detection of other diseases and cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding polypeptides of the invention, which is also indicative of the presence or absence of a disease or disorder, including cancer. In general, polypeptides of the invention should be present at a level that is at least three fold higher in diseased tissue than in normal tissue.
[396] There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, supra. In general, the presence or absence of a disease in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value. , [397] In a preferred embodiment, the assay involves the use of a binding agents) immobilized on a solid support to bind to and remove the polypeptide of the invention from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include polypeptides of the invention and portions thereof, or antibodies, to which the binding agent binds, as described above.
[398] The solid support may be any material known to those of skill in the art to which polypeptides of the invention may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
Alternatively, the support may be a bead or disc, such as glass fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No.
5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature.
In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support br may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for the suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 ug, and preferably about 100 ng to about 1 ug, is sufficient to immobilize an adequate amount of binding agent.
[399] Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
Gene Therapy Methods [400] Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate t~o the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of the polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, W090/11092, which is herein incorporated by reference.
[401] Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide of the present invention. Such methods are well-known in the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 ( 1993); Ferrantini, M.
et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J.
Immunology 153:
4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-(199.6); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3 : 31-3 8 ( 1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
(402] As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like).
The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
[403] In one embodiment, the polynucleotide of the present invention is delivered as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotide of the present invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
[404] The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVI~3, pBPV, pMSG and pSVL
available from Pharmacia; and pEFl/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
[405] Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter;
inducible promoters, such as the MMT promoter, the metallothionein promoter;
heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters;
viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter;
retroviral LTRs; the b-actin promoter; and human growth. hormone promoters.
The promoter also may be the native promoter for the polynucleotide of the present invention.
[406] Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
(407] The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,. cartilage, pancreas, kidney, gall bladder,. stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue.
Interstitial space bf the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising These cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
[408] For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection.
The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
[409] The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used,, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during. angioplasty by the catheter used in the procedure.
[410] The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called "gene guns". These delivery methods are known in the art.
[411] The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
[412) In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.
However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid.
Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA
(Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:607.7-6081, which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol.
Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in functional form.
[413] Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA
(1987) 84:7413-7416, which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
[414] Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092-(which is herein incorporated by reference) for a description of the synthesis of DOTAP
( 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA
liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc.
Natl. Acad. Sci.
USA 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.
[415] Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such' materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatid~l glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also, be mixed with the DOTMA and DOTAP starting materials in appropriate ratios.
Methods for making liposomes using these materials are well known in the art.
[416] For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following 261 .
day with deionized water. The sa~rrple is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size.
Other methods are known and available to those of skill in the art.
[417] The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527, which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water ar an isotonic buffer solution such as 10 mM. Tris/NaCI, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art.
Commonly used methods include Ca2+-EDTA chelafion (Papahadjopoulos et al., Biochim.
Biophys.
Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D.- and Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem.
Biophys.
Res. Commun. 76:836 (1977); Fraley et aL, Proc. NatI. Acad. Sci. USA 76:3348 (1979));
detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad.. Sci.
USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem.
255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978);
Schaefer-Ridder et al., Science 215:166 (1982)), which are herein incorporated by reference.
[418] Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10.
Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
(419J U.S. Patent No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice.
U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA
into cells and.
mammals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.
[420] In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding a polypeptide of the present invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, .avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and. mammary tumor virus.
[421] The retroviral plasrnid vector is employed to transduce packaging cell lines to form producer cell lines. Exarr~ples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 ( 1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO~ precipitation. In one alternative, the retroviral plasmid vector may-be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
(422] The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a polypeptide~ of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a polypeptide of the present invention.
[423] In certain other embodiments, cells are engineered, ex vivo or i~ vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses a polypeptide of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis.109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA
76:6606).
[424] Suitable adenoviral vectors useful in the present invention are described, for example, in I~ozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993);
Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692 (1993); and U.S. Patent No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the El region of adenovirus and constitutively express Ela and Elb, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are also useful in the present invention. .
[425] Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide ~of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E 1 a, E 1 b, E3, E4, E2a, or L 1 through L5.
[426] In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr.
Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb.
Methods for producing and using such AAVs are known in the art. See, for example, U.S.
Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377. .
[427] For example, an appropriate AAV vector for use in the present invention will include aII the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc.
Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct.
These viral particles are then used to transduce eukaryotic cells,. either ex vivo or in vivo. The transduced cells will contain: the polynucleotide construct integrated into its genome, and will express a polypeptide of the invention.
[428] , Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994;
Koller et aL, Proc. Natl. Acad. .Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), which are herein encorporated by-reference. This method involves the activation of a gene which is .present in the target cells, but .which is not normally expressed in the cells; or is expressed at a Lower level than desired.
[429] Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter.
Suitable promoters are described herein. The targeting sequence is suff ciently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5' end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to~the endogenous sequence upon homologous recombination.
[430] The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5' and 3' ends. Preferably, the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3' end of the amplified promoter. The 'amplified promoter and targeting sequences are digested and ligated together.
(431] The promoter-targeting sequence construct is delivered to the cells, either as .
naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles,' whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
[432] The promoter-targeting sequence construct is taken up by cells.
Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
[433] The polynucleotide encoding a.polypeptide of the present invention may contain a secretory signal sequence that facilitates secretion of the protein.
Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5' end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
[434] Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, . catheter infusion, biolistic injectors, particle accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e;g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. Fox example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).
[435] A preferred method of local administration is by direct injection.
Preferably, a recombinant molecule of the present invention completed with a delivery vehicle is administered by direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
[436] Another method of local administration is to contact a~polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
[437] Therapeutic compositions useful in systemic administration, include' recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.
In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising polypeptides of the invention for targeting the vehicle to a particular site.
[438] Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery: Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad.
Sci. USA
189:11277-11281, 1992, W hick is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal.
Examples of such carriers, include plastic capsules or tablets, such as those known in.the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into 'the skin.
[439] Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
(440] Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
Biolo ical Activities [441] Polynucleotides or polypeptides; or agonists or antagonists of the present invention, 'can be used in assays to test for one or more biological activities. If these polynucleotides or polypeptides, or agonists or antagonists of the present invention, do exhibit activity in a particular assay, it is likely that these molecules may be involved in the _ diseases associated with the biological activity. Thus, the polynucleotides and polypeptides, and agonists or antagonists could be used to diagnose, prognose, prevent and/or treat the associated disease.
[442] Members of the calcium-binding family of proteins are believed to be involved in biological activities associated with gene expression, cell proliferation, cell death, and secretion processes. Accordingly, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated with aberrant calcium-binding protien activity.
[443] In preferred embodiments, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders relating to blood disorders (e:g., blood coagulation disorders, fibrinolysis disorders, and/or as described under "Immune activity" and "Cardiovascular Disorders"
below) neoplastic disorders (e.g., inappropriate cell mitosis, aberrant cytokinesis, and/or as described under "Hyperproliferative Disorders" below), and neural transmission (e.g., disorders of neurotransmitter exocytosis, dysfunctions associated with neural excitation, and/or as described under "Neural Activity and Neurological Disorders" below), and digestive/endocrine disorders (e.g., hypercholesterolemia, and/or as described under "Gastrointestinal Disorders" and "Endocrine Disorders" below).
[444] In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognose diseases and/or disorders associated with the tissues) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[445] Thus, polynucleotides, translation products and antibodies of the invention are useful in the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated. with activities that include, but are not limited to, blood coagulation, exo- and ~endocytosis, cytokinesis, gene expression, neurotransmitter release, and cholesterol uptake.
[446] More generally, polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated with the following systems.
Immune Activity [447] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation,, differentiation, or mobilization (chemotaxis) of immune cells.
Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T
lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious. Moxeover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
[448] In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissues) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[449] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing,' diagnosing, and/or prognosing immunodeficiencies,. including both congenital and acquired immunodeficiencies.
Examples of B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinem.ia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA
deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with increased IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired), and transient hypogammaglobulinemia of infancy.
[450] In specific embodiments, ataxia-telangiectasia or conditions associated with ataxia-telangiectasia are treated, prevented, diagnosed, and/or prognosing using the polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof.
[451] Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion,. chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
[452] In specific embodiments, DiGeorge anomaly or conditions associated with DiGeorge anomaly are treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides of the invention, or antagonists or agonists thereof.
[453] Other immunodeficiencies that may be treated, prevented, diagnosed, andlor prognosed using polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, include, but are not limited to, chronic granulomatous disease, Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including Cl, G2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs:
[454] In a preferred embodiment, the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
[455] In a preferred embodiment polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among B cell and/or T
cell iminunodeficient individuals.
(456] The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing andlor prognosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of polynucleotides and polypeptides of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing,autoimmune disorders.
[457] Autoimmune diseases or disorders that may be treated, prevented, diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoirrimune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thromliocytopenia, idiopathic, thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.
[458] Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.
(459] Additional disorders that are likely to have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by ~glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).
[460] Additional disorders that may have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g.; by IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous, degenerative, and atrophic disorders.
(461) In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using for example, antagonists or agonists, polypeptides or polynucleotides, or antibodies of the present invention. In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
[462] In another specific preferred embodiment, systemic lupus erythematosus is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies; and/or agonists or antagonists of the present invention.
[463] In another specific preferred embodiment IgA nephropathy is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
[464] In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or. antagonists of the present invention (465] In preferred embodiments, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a iminuno,suppressive agent(s).
[466] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, prognosing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells.
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia.
Alternatively,~Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the piesent invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.
[467] Allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosed using polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof. Moreover, these molecules can be used to treat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility. -[468] Additionally, polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, may be used to treat, prevent, diagnose andlor prognose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema. In specific embodiments, polynucleotides, polypeptides;
antibodies, and/or agonists or antagonists of the present invention may be used to modulate IgE
concentrations in vitro or in vivo.
[469] ' Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or ' antagonists of the present invention have uses in the diagnosis, prognosis, prevention, and/or treatment of inflammatory conditions. For example, since polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic. inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy);
gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis;
ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative'disorders (e.g., Parkinson's disease and Alzheimer's disease); AIDS-related dementia; and prion disease);
cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional diseases, conditions, and disorders that are characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout,' trauma, pancreatitis, , sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus, diabetes mellitus, and allogenic transplant rejection).
[470j Because inflammation is a fundamental defense mechanism, inflammatory disorders can effect virtually any tissue of the body. Accordingly, polynucleotides, polypeptides, and antibodies of the invention, as well as agonists or antagonists thereof, have uses in the treatment of tissue-specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, _ osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis. .
[471] In specific embodiments, polypeptides, antibodies, .or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat organ transplant rejections and graft-versus-host disease. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.
[472] In other embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat immune complex diseases, including, but not limited to, serum sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.
[473] Polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the invention can be used to treat, detect, and/or prevent infectious agents. For example, by .
increasing the immune response, particularly increasing the proliferation activation and/or differentiation of B and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.
(474] In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen. In a specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance tumor-specific immune responses.
[475] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or~antagonists of the present invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In .specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment; the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, paxainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
[476] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
[477] In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B
streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, and Borrelia burgdorferi.
[478] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an ~adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania.
[479] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis;
for example, by preventing the recruitment and activation of mononuclear phagocytes.
f (480] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.
[481J In one embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, carriel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
[482] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B
cell responsiveness to pathogens.
(483] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an activator of T
cells.
[484] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.
[485] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to induce higher affinity antibodies.
j486] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to increase serum immunoglobulin concentrations.
[487J In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to accelerate recovery of immunocompromised individuals. ' [488] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among aged populations and/or neonates.
[489] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or,-agonists or antagonists of the present invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, .
prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
[490] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or .
agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function.
Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
[491J In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency.
Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious .
mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.
[492] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo. Moreover, in related embodiments, said enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.
[493] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to direct an individual's immune 'system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.
[494] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.
[495] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B
cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.
[496] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect.
In another.
specific embodiments polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in the pretreatment of bone marrow samples prior to transplant.
[497] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetencelimmunodeficiency such as observed among SCID patients.
[498] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present . invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.
[499] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of regulating secreted eytokines that are elicited by polypeptides of the invention.
[500] In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in one or more of the applications decribed herein, as they may apply to veterinary medicine.
[501] In another specific embodiments polypeptides, antibodies, polynucleotides and/or agonists or antagonists, of the present invention are used as a means of blocking various aspects of immune responses to foreign agents or self. Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimrnune disorders such as lupus, and arthritis, as well as immunoresponsiveness to. skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.
[502] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for preventing the B
cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erytheriiatosus and multiple sclerosis.
[503] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a inhibitor of B
and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
[504] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for chronic hypergammaglobulinemia evident in such diseases a's monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.
[505] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells,.in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes. .
[506] The polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.
[507] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit complement mediated cell lysis.
[508] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.
[509] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.
[510] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed to treat adult respiratory distress syndrome CARDS).
[511] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, agonists and antagonists of the invention may be used to stimulate the regeneration of mucosal surfaces.
[512] In a specific. embodiment, polynucleotides or polypeptides, and/or agonists thereof are used to diagnose, prognose, treat, and/or prevent a disorder characterized by primary or acquired immunodeficiency, deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, polynucleotides or polypeptides, and/or agonists thereof may be used to treat or prevent infections of the joints, bones, skin, andlor parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, andlor osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseases and disorders that may be prevented, diagnosed, prognosed, and/or treated with polynucleotides or polypeptides, and/or agonists of the present invention include,~but are not limited to, HIV
infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.
[513] In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention axe used to treat, and/or diagnose an individual having common variable immunodeficiency disease ("CVID"; also known as "acquired agammaglobulinemia" and "acquired hypogammaglobulinemia") or a subset of this disease.
[514] In a specific embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to diagnose, prognose, prevent, and/or treat cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms. Examples of cancers or neoplasms that may be prevented, diagnosed, or treated by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and disorders described in the section entitled "Hyperproliferative Disorders" elsewhere herein.
[515] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
[516] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
[517] In specific embodiments, the compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.
[518]. Antagonists of the invention include, for example, binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms of the polypeptides of the present invention (e.g., Fc fusion protein; see, e.g., Example 9). Agonists of the invention include, for example, binding or stimulatory antibodies, and soluble forms of the polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9). ,polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described herein.
[519] In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of.producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin.
molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. W098/24893, WO/9634096, WO/9633735, and . W019110741). Administration of polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention to such animals is useful for the generation of monoclonal antibodies against the polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention.
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(1996)).
[8J Several families of calcium-binding proteins bind calcium at EGF-like domains rather than EF-hands. The epidermal growth factor-like (EGF) domain is a widely distributed, independently folding protein module that is thought to play a general role in.
extracellular events such as adhesion, coagulation, and receptor-ligand interactions (Campbell and Bork, Current Opinions in Structural Biology 3: 38S-392 (1993)).
A distinct class of these domains has been identified containing a consensus sequence associated with calcium binding: D/N-x-D/N-E/Q-xm-D/N*-xn-Y/F (where m and n are variable and indicates possible -hydroxylation) (Reel et al., EMBO Journal 7:2053-2067 (1988);
Handford et al., Nature 351:.164-167 (1991); Mayhew et al., Protein Engineering S:_489-494 (1992)). The importance of the calcium-binding EGF domain is emphasized by its occurrence in functionally diverse proteins, including those involved iri extracellular matrix architecture (fibrillin-1,2, fibulin-1,2, nidogen), control of blood coagulation (factors IX and X, proteins C and S, thrombomodulin), cholesterol uptake (low density lipoprotein receptor [LDLR]), and specification of cell fate (Drosophila Notch, Delta, and Serrate). Genetic mutations causing amino acid changes in calcium-binding EGF domains from some of these proteins have been identified in patients with the Marfan syndrome,, familial hypercholesterolemia, hemophilia B (diseases caused by mutations in fibrillin-1, LDLR, and factor IX genes, respectively), and protein S deficiency (Dietz et al., Human Molecular Genetics 4: 1799-1809 (1995); Hobbs et al., Human Mutations 1: 45S-466 (I992);
Giannelli et al., Nucleic Acids Research 24: 103-118 (1996); Gandrille et al., Blood 8S:
(1995)).
[9] Further, annexins are a structurally conserved family of proteins characterized by reversible calcium-dependent membrane binding. High-resolution crystal structures of the soluble forms of several different annexins have been solved, and all structures share a common fold consisting of four domains, each of which contains five helices connected by short loops. The loop regions on the convex side of the protein are believed to account for the calcium-dependent binding of the protein to membranes by jointly coordinating calcium with phosphatidylserine. Membrane association is of critical importance for the proposed functions of annexins, which includes vesicular trafficking, membrane fusion, and ion-channel formation (Seaton, Annexins: Molecular Structure to Cellular Function, Landes, New York ( 1996)).
(10] Calcium is involved in a wide array of different biological events. New structural and biophysical studies of calcium-binding proteins have provided important information on the roles of calcium at the molecular level, however much remains to be done to establish links between structure, calcium levels, and biological function.
Accordingly, there is a clear need for identifying and exploiting novel calcium-binding proteins, such as those described above, that may contribute to diseases resulting from the aberrant calcium flux. Additionally, novel members of these protein families are useful as screening tools to identify antagonists and/or agonists that may enhance or block activities mediated by calcium-binding proteins. Further, blockers of these calcium-binding proteins may prove useful in the diagnosis, prevention, andlor treatment of disorders including, but not limited to, neurological diseases, immune dysfunction, digestive disorders, neoplastic diseases, blood disorders, and/or infectious disease.
Summary of the Invention [1l] The, present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention:
The present invention further relates to methods and/or compositions for inhibiting .or enhancing the production and function of the polypeptides of the pxesent invention.
Detailed Desct-iption Tables [12] Table 1A summarizes some of the polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) and contig nucleotide sequence identifier.
(SEQ ID NO:X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, "Clone ID NO:Z", for a cDNA clone related to each contig sequence disclosed in Table 1 A. The third column provides a unique contig identifier, "Contig ID:" for each of the contig sequences disclosed in Table 1A. The fourth column provides the sequence identifier, "SEQ ID
NO:X", for each of the contig sequences disclosed in Table 1A. The fifth column, "ORF
(From-To)", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineate the preferred open reading frame (ORF) that encodes the amino acid sequence shown in the sequence listing and referenced in Table IA
as SEQ ID NO:Y (column 6). Column 7 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID
NO:Y).
Identification of potential immunogenic regions was performed according to the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988)); specifically, the -Genetics Computer Group (GCG) implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE (Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wisc.). This method returns a measure of the probability that a given residue is found on the surface of the protein. Regions where the antigenic index score ~is greater than 0.9 over at least 6 amino acids are indicated in Table 1 A as "Predicted Epitopes". In particular embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table 1A. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly. Column 8, "Tissue Distribution" shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucIeotides of the invention. .The first number in column 8 (preceding the colon), represents the tissue/cell source identifier code corresponding to the key-provided in .
Table 4. Expression of these polynucleotides was not observed in.the other tissues and/or cell libraries .tested. For those identifier codes in which the first two letters are not "AR", the second number in column 8 (following the colon), represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID
NO:X) was identified in the tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are "AR" designate information generated using DNA array technology.
Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA
probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids, from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the Level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after "[array code]:"
represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissues) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression. Column 9 provides the chromosomal location of polynucleotides corresponding to SEQ ID NO:X. Chromosomal location was determined by finding exact matches. to EST and cDNA sequences contained in the NCBI
(National Center for Biotechnology Information) UniGene database. Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIMTM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD) 2000. World Wide Web URL:
http://www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM
identification number is disclosed in column 10 labeled "OMIM Disease References)". A key to the OMIM reference identification numbers is provided in Table 5.
[~3] Table 1B summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID
NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, "Clone ID NO:Z", for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, "SEQ ID NO:X", for each contig sequence. The third column provides a unique contig identifier, "Contig ID:" for each contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, "SEQ ID NO:B" for a fragment of the BAC clone identif ed in column four of the corresponding row of the table. The sixth column, "Exon From-To", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID
NO:B which delineate certain polynucleotides of the invention that are also exemplary .
members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
[14) ~ Table 2 summarizes homology and features of some of the polypeptides of the invention. The first column provides a unique clone identifier, "Clone ID
NO:Z", corresponding to a cDNA clone disclosed in Table IA. The second column provides the unique contig identifier, "Contig ID:" corresponding to contigs in Table 1A
and allowing for correlation with the information in Table 1A. The third column provides the sequence.
identifier, "SEQ ID NO:X", for the contig polynucleotide sequence. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. Comparisons were made between polypeptides encoded by the polynucleotides of the invention and either a non-redundant protein database (herein referred to as "NR"), or a database of protein families (herein referred to as "PFAM") as further described below.
The fifth column provides a description of the PFAM/NR hit having a significant match to a polypeptide of the invention. Column six provides the accession number of the PFAM/NR
hit disclosed in the fifth column. Column seven, "Score/Percent Identity", provides a quality score or the,percent identity, of the hit disclosed in columns five and six.
Columns 8 and 9, "NT From" and "NT To" respectively, delineate the polynucleotides in "SEQ ID
NO:X"
that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth and sixth columns. In specific embodiments polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence encoded by 'a polynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
(15] Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention. The first column provides a unique clone identifier, "Clone ID", for a cDNA clone related to contig sequences disclosed in Table l A. The second column provides the sequence identifier, "SEQ ID NO:X", for contig sequences disclosed in Table 1A. The third column provides the unique contig identifier, "Contig ID:", for contigs disclosed in Table 1A. The fourth column provides a unique integer 'a' where 'a' is any integer between 1 and the final nucleotide minus 15 of SEQ ID
NO:X, and the fifth column provides a unique integer 'b' where 'b' is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14.
For each of the polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be substituted into the general formula of a-b, and used to describe polynucleotides which may be preferably excluded , from the invention. In certain embodiments, preferably , excluded from the invention are at least one, two, three, four, five, ten, or more of the polynucleotide sequences) having the accession numbers) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC
clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequences) contained in the clones corresponding to at least one, two, three;
four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).
[16] Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1A, column 8. Column 1 provides the tissue/cell source identifier code disclosed in Table 1A, Column 8. Columns 2-5 provide a description of the tissue or cell source.
Codes corresponding to diseased tissues are indicated in column 6 with the word "disease". The use of the word "disease" in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the "disease"
designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissuelcell source is a library, column 7 identifies the vector used to generate the library.
[17] Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1A, column 10. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM.
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, MD) 2000. WorldWide Web URL: http://www.ncbi.nlm.nih.gov/omim/).
Column 2 provides diseases associated with the cytologic band disclosed in Table 1A, column 9, as determined using the Morbid Map database.
[18] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present application.
[19] ~ Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers and vector information relating to these cDNA libraries.
[20] Table 8 provides a physical characterization of clones encompassed by the invention. The first column provides the unique clone identifier, "Clone ID
NO:Z", for certain cDNA clones of the invention, as described in Table IA. The second column provides the size of the cDNA insert contained in the corresponding cDNA
clone. , Definitions [21] The following definitions are provided to facilitate understanding of certain terms used throughout this specification.
[22] ~ In the present invention, "isolated" refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the polyriucleotide. The term "isolated" does not refer to genomic or cDNA
libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
(23] As used herein, a "polynucleotide" refers to a molecule having a nucleic acid sequence encoding SEQ ID NO:Y or a fragment or variant thereof; a nucleic acid sequence contained in SEQ ID NO:X (as described in column 3 of-Table 1A) or the complement thereof; a cDNA sequence contained in Clone ID NO:Z (as described in column 2 of Table 1A and contained within a library deposited with the ATCC); a nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B or a fragment or variant thereof; or a nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1B or the complement thereof. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA
sequence, including the 5' and 3' untranslated sequences, the coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
Moreover, as used herein, a "polypeptide" refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-PhenyIalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).
(24] In the present invention, "SEQ ID NO:X" was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X is deposited at Hurnan Genome Sciences, Inc.
(HGS) in a catalogued and archived library. As shown, for example, in column 2 of Table 1A, each clone is identified by a cDNA Clone ID (identifier generally referred to herein as Clone ID NO:Z). Each Clone ID is unique to an individual clone and the Clone ID is all the information needed to retrieve a given clone from the HGS library.
Furthermore, certain clones disclosed in this application have been deposited with the ATCC on October 5, 2000, having the,ATCC designation numbers PTA 2574 and PTA 2575; and on January 5, 2001, having the depositor reference numbers TS-1', TS-2, AC-1, and AC-2. In addition to the individual cDNA clone deposits, most of the cDNA libraries from which the clones were derived were deposited at the Ariierican Type Culture Collection (hereinafter "ATCC").
Table 7 provides a list of the deposited cDNA libraries. One can use the Clone ID NO:Z to determine the library source by reference to Tables 6 and 7. Table 7 lists the deposited cDNA libraries by name and links each library to an ATCC Deposit. Library names contain four characters, for example, "HTWE." The name of a cDNA clone (Clone ID) isolated from that library begins with the same four characters, for example "HTWEP07".
As mentioned below, Table l A correlates the Clone ID names with SEQ ID NO:X..
Thus, .
starting with an SEQ ID NO:X, one can use Tables 1, 6 and 7 to determine the corresponding Clone ID, which library it came from and which ATCC deposit the library is contained in. Furthermore, it is possible to retrieve a given cDNA clone from the source library by techniques known in the art and described elsewhere herein. The ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC
deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
(25] In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.Skb, kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise alI or a portion of any intron. In another embodiment, the polynucleotides comprising~coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene ~of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
(26] A "polynucleotide" of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, or the complement, thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein),. the polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or cDNA sequences contained in Clone ID NO:Z (e.g.; the complement of any one, two, three, four, or more of the polynucleotide fragments, or the cDNA clone within the pool of cDNA
clones deposited with the ATCC, described herein), and/or the polynucleotide sequence delineated in column 6 of Table 1B or the complement thereof. "Stringent hybridization conditions" refers to an overnight incubation at 42 degree C in a solution comprising 50%
formamide, Sx SSC (750 mM NaCI, 75 mM trisodium citrate), SO mM sodium phosphate (pH 7.6), Sx Denhardt's solution, 10% dextran sulfate, and 20 ~g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C.
[27] Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions.
Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C in a solution comprising 6X SSPE (20X SSPE = 3M NaCI; 0.2M NaH2P04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e:g. 5X SSC).
(28j Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include .Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations: The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
[29] Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing}, or to a complementary stretch. of T {or U) residues, would not be included in the definition of "polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded. cDNA clone generated using oligo dT as a primer).
[30] The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid~molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA
or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified"
bases include, for example, tritylated bases and unusual bases such as inosine. A
variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
[31] The polypeptide of the present invention can be composed of amino acids joined to each., other by peptide bonds or' modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be.
modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic .texts and in more detailed monographs, as well as in a voluminous research literature. Modif canons can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that ,the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modif cations include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-Linking, cyclization, disulf de bond formation, demethylation, formation of covalent cross-Links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS -STRUCTURE AND' MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H.
Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT
MODIFICATION OF PROTEINS, B. C.. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646. (1990); Rattan et al., Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[32] "SEQ ID NO:X" refers to a polynucleotide sequence described, for example, in Tables lAor 2, while "SEQ ID NO:Y" refers to a polypeptide sequence described 'in column 6 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 4 of Table 1A.
The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. "Clone ID NO:Z" refers to a cDNA clone described in column 2 of Table 1 A.
[33] "A polypeptide having functional activity" refers to a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody which 'binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
(34], The polypeptides of the invention can be assayed for functional activity (e.g.
biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, one of skill in the art may routinely assay calcium binding polypeptides (including fragments and variants) of the invention for activity using assays as described in the examples section below.
[3S] "A polypeptide having biological activity" refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or .
without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and .most preferably, not more than about three-fold Iess activity relative to the polypeptide of the present invention).
(36] Table 1A summarizes some of the polynucleotides encompassed by the invention (including contig sequences (SEQ ID NO:X) and clones (Clone ID NO:Z) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby.
OAS
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j37] The first column in Table 1A provides the gene number in the application corresponding to the clone identifier. The second column in Table 1A provides a unique "Clone ID NO:Z" for a cDNA clone related to each contig sequence disclosed in Table 1A.
This clone ID references the cDNA clone which contains at least the 5' most sequence of the assembled contig and at least a portion of SEQ ID NO:X was determined by directly sequencing the referenced clone. The reference clone may have more sequence than described in the sequence listing or the clone may have less. In the vast majority of cases, however, the clone is believed to encode a full-length polypeptide. In the case where a clone is not full-length, a full-length cDNA can be obtained by methods described elsewhere herein.
[38] . The third column in Table 1A provides a unique "Contig ID"
identification for each contig sequence. The fourth column provides the "SEQ ID NO:" identifier for each of the contig polynucleotide sequences disclosed in Table 1 A. The fifth column, "ORF
(From-To)", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence "SEQ ID NO:X" that delineate the preferred open reading frame (ORF) shown in the sequence listing and referenced in Table 1A, column 6, as SEQ ID
NO:Y. Where the nucleotide position number "To" is lower than the nucleotide position number "From", the preferred ORF is the reverse complement of the referenced polynucleotide sequence.
[39] The sixth column in Table 1A provides the corresponding SEQ ID NO:Y for the polypeptide sequence encoded by the preferred ORF delineated in column 5. In one embodiment, the invention provides an amino acid sequence comprising, or alternatively consisting of, a polypeptide encoded by the portion of SEQ TD NO:X delineated by "ORF
(From-To)". Also provided are polynucleotides encoding such amino acid sequences and the complementary strand thereto. .
[40] Column 7 in Table 1A lists residues comprising epitopes contained in the polypeptides encoded by the preferred ORF (SEQ ID NO:Y), as predicted using the algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN
(Version 3.11 for the Power Macintosh, DNASTAR, Inc., 1228 South Park Street-Madison,-WI). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, at least one, two, three, four, five or more of the predicted epitopes as described in Table 1A.
58 ' It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly.
j41] Column 8 in Table 1A provides an expression profile and library code:
count for each of the contig sequences (SEQ ID NO:X) disclosed in Table 1A, which can routinely be combined with the information provided in Table 4 and used to determine the tissues, cells, and/or cell line libraries which predominantly express the polynucleotides of the invention.
The first number in column 8 (preceding the colon), represents the tissue/cell source identifier code corresponding to the code and description provided in Table 4.
For those identifier codes in which the first two letters axe not "AR", the second number in column 8 (following the colon) represents the number of times a sequence corresponding to the reference polynucleotide sequence was identified in the tissuelcell source.
Those tissue/cell source identifier codes in which the first two letters are "AR" designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA
probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of 33P ~dCTP, using oligo(dT) to prime reverse.
transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A
local background signal subtraction was performed. before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after "[array code]:" represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased. tissues) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.
j42] Column 9 in Table 1A provides a chromosomal map location for certain polynucleotides of the invention. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Tnformation) UniGene database. Each sequence in the UniGene database is assigned to a "cluster"; all of the ESTs, cDNAs, and STSs in a cluster are believed to be derived from a single gene. Chromosomal mapping data is often available for one or more sequences) in a UniGene cluster; this data (if consistent) is then applied to the cluster as a whole. Thus, .it is possible to infer the chromosomal location of a new polynucleotide sequence by determining its identity with a mapped UniGene cluster.
[43] A modified version of the computer program BLASTN (Altshul et al., J..MoI.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet. 3:266-272 (1993)) was used to search the UniGene database for EST or cDNA sequences that contain exact or near=exact matches to a polynucleotide sequence of the invention (the 'Query'). A
sequence from the UniGene database (the 'Subject') was said to be an exact match if it contained a segment of 50 nucleotides in length such that 4~ of those nucleotides were in the same order as found in the Query sequence. If all of the matches that met this criteria were in the same Uni.Gene cluster, and mapping data was available for this cluster, it is indicated in Table 1A under the heading "Cytologic Band". Where a cluster had been further localized to a distinct cytologic band, that band is disclosed; where no banding information was available, but the gene had been localized to a single chromosome, the chromosome is disclosed.
[44] Once a presumptive chromosomal location was determined for a polynucleotide of the invention, an associated disease locus was identified by comparison with a database of diseases which have been experimentally associated with genetic loci. The database used was the Morbid Map, derived from OMIMTM (supra). If the putative chromosomal location of a polynucleotide of the invention~(Query sequence) was associated with a disease in the Morbid Map 'database, an OMIM reference identification number was noted in column 10, Table 1A, labelled "OMIM Disease References)". Table 5 is a key to the OMIM
reference identification numbers (column 1), and provides a description of the associated disease in Column 2.
TABLE IB
Clone ID SEQ ID CONTIG BAC ID: SEQ ID EXON
A
NO:Z NO:X ID: NO:B From-To HWHQR25 31 947020 AC02066I. 173 1-342 ' 1234-1287 . 5504-5728 ' 606-683 HBGMG39 44 971414 AL390719 181 ~ 1-1344 ~
' 3205-3305 HE8PY29 51 887862 AC009948 183 _ _ _ ~
1249=1380 HSIA078 67, 889498 AC002302 189 1-303 _ 6990-7582 [45] Table 1B summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID
NO:X)), and genomic sequences (SEQ ,ID NO:B). The first column provides a unique clone identifier, "Clone ID NO:Z", for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, "SEQ ID NO:X", for each contig sequence. The third column provides a unique contig identifier, "Contig ID:" for each contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A" for the BAC,clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, "SEQ ID NO:B" for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, "Exon From-To", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID
NO:B which delineate certain polynucleotides. of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
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[46] Table 2 further characterizes certain encoded polypeptides of. the invention, by providing the results of comparisons to protein and protein family databases.
The f rst column provides a unique clone identifier, "Clone ID NO:", corresponding to a cDNA
clone disclosed in .Table 1 A. The second column provides the unique contig identifier, "Contig ID:" which allows correlation with the information in Table 1A. The third column provides the sequence identifier, "SEQ ID NO:", for the contig polynucleotide sequences.
The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. The fifth column provides a description of the PFAM/NR hit identified by each analysis. Column six provides the accession number of the PFAM/NR
hit disclosed in the fifth column. Column seven, score/percent identity, provides a quality score or the percent identity, of the hit disclosed in column five.
Comparisons were made between polypeptides encoded by polynucleotides of the invention and a non-redundant protein database (herein referred to as "NR"), or a database of protein families (herein referred to as "PFAM"), as described below.
[47] The NR database, which comprises the NBRF PIR database, the NCBI GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-redundant using the computer program nrdb2 (Warren Gish, Washington University in Saint Louis). Each of the polynucleotides shown in Table 1A, column 3 (e.g., SEQ TD NO:X or the 'Query' sequence) was used to search against the NR database. The computer program BLASTX
was used to compare a 6-frame translation of the Query sequence to the NR
database (for information about the BLASTX algorithm please see Altshul et al., J. Mol.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the sequence that is most similar to the Query sequence (the highest scoring 'Subject') is shown in column five of Table 2 and the database accession number for that sequence is provided in column six. The highest scoring 'Subject' is reported in Table 2 if (a) the estimated probability that the match occurred by chance alone is less than I.Oe-07, and (b) the match was not to a known repetitive element. BLASTX returns alignments of short polypeptide segments of the Query and Subject sequences which share a high degree of similarity; these segments are known as High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity between the Query and the Subject for each HSP as a percent identity in Column 7. The percent identity is determined by dividing the number of exact matches between the two aligned sequences in the HSP, dividing by. the number of Query amino acids in the HSP
and multiplying by 100. The polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that generates an HSP are delineated by columns 8 and 9 of Table 2.
[48] The PFAM database, PFAM version 2.1, (Sonnhammer et al., Nucl. Acids Res., 26:320-322, 1998)) consists of a series of multiple sequence alignments; one alignment for each protein family. Each multiple sequence alignment ~is converted into a probability model called a Hidden Markov Model, or HMM, that~ represents the position-specific variation among the sequences that make up the multiple sequence alignment (see, e.g., Durbin et al., Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998 for the theory of HMMs). The program HMMER
version 1.8 (Sean Eddy, Washington University in Saint Louis) was used to compare the predicted protein sequence for each Query sequence (SEQ ID NO:Y in Table 1A) to each of the HMMs derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to be a significant match to a polypeptide of the invention if the score returned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 score obtained with the most distantly related known member of that protein family. The description of the PFAM family which shares a significant match with a polypeptide of the invention is listed in column 5 of Table 2., and the database accession number of the PFAM hit is provided in column 6.
Column 7 provides the score returned by H1VIMER version 1.8 for the alignment.
Columns 8 and 9 delineate the polynucleotides of SEQ TD NO:X which encode the polypeptide sequence which show a significant match to a PFAM protein family.
[49] As mentioned, columns 8 and 9 in Table 2, "NT From" and "NT
To",.delineate the polynucleotides of "SEQ ID NO:X" that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth column. In one embodiment, the invention provides a protein comprising, or alternatively consisting of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2.
Also provided are polynucleotides encoding such proteins, and the complementary strand thereto.
[50] The nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, the nucleotide sequences of SEQ ID NO:X
are useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences, contained in SEQ ID NO:X or the cDNA contained in Clone ID NO:Z.
These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling immediate applications in chromosome mapping, linkage analysis, tissue identification and/or typing, and a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to these polypeptides, or fragments thereof, and/or to the polypeptides encoded by the cDNA clones identified in, for example, Table 1A.
[51] Nevertheless, DNA sequences generated by sequencing reactions can.contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9%
identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
[52] Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated.
nucleotide sequence identified as SEQ ID NO:X, and a predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA
containing cDNA
Clone ID NO:Z (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and having depositor reference numbers TS-1, TS-2, AC-1, and AC-2;
and/or as set forth, for example, in Table 1A, 6 and 7). The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. Further, techniques known in the art can be used to verify the nucleotide sequences of SEQ ID NO:X.
[53] The predicted amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence. .
R~4CE Protocol For Recovery of Full-Length Genes [54] Partial cDNA clones can be made full-length by utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M.A., et al., Proc. Nat'1.
Acad.
Sci. USA, 85:8998-9002 (1988). A cDNA clone, missing either the 5' or 3' end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start codon of translation, therefor. The following briefly describes a modification of this original 5' RACE
procedure. Poly A+ or total RNA is reverse transcribed with Superscript II
(Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA
is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL).
Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoI; SaII and CIaI) at the 5' end and a primer containing just these restriction sites. This double-stranded cDNA is PCR
amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA
is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or SaII, and ligated to a plasmid such as pBluescript SKII
(Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the' correct protein-coding inserts.
Correct 5' ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3' ends.
[55] Several quality-controlled kits are commercially available for purchase.
Similar reagents and methods to those above are supplied in kit form from Gibco/BRL
for both 5' and 3' RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure' are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
[56] An alternative to generating 5' or 3' cDNA from RNA is to use cDNA
library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
.RNA Ligase Protocol For Generating The S' or 3' End Sequences To Obtain Full Length Genes [57] Once a gene of interest is identified, several methods are available for the identification of the 5' or 3' portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5' and 3' RACE.
While the full length gene may be present in the library and can be identified by probing, a useful method for generating the 5' or 3' end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5' RACE
is available for generating the missing 5' end of a desired full-length gene.
(This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993)).
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA
presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specif c to a known sequence of the gene of interest, is used to PCR amplify the 5' portion of the desired full length gene which may then be sequenced and used to generate the full length gene. ~ This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used ~is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA
oligonucleotide using T4 RNA. ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis -reaction can then be used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide~ and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the relevant gene.
[58] The present invention also relates to vectors or plasmids which include such DNA sequences, as well as the use of the DNA sequences. The material deposited with the ATCC (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and receiving ATCC designation numbers TS-1, TS-2, AC-1, and AC-2; and/or as set forth, for example, in Table 1A, Table 6, or Table 7) is a mixture of cDNA clones derived from a variety of human tissue and cloned in either a plasmid vector or a phage vector, as described, for example, in Table 7. These deposits are referred to as "the deposits" herein.
The tissues from which some of the clones were derived are listed in Table 7, and the vector in which the corresponding cDNA is contained is also indicated in Table 7. The deposited material includes cDNA clones corresponding to SEQ ID NO:X described, for example, in Table 1A (Clone ID NO:Z). A clone which'is isolatable from the ATCC Deposits by use of a sequence listed as SEQ ID NO:X, may include the entire~coding region of a human gene or in other cases such clone may include a substantial portion of the coding region of a human gene. Furthermore, although the sequence listing may in some instances list only a portion of the DNA sequence in a clone included in the ATCC Deposits, it is well within the ability of one skilled in the art to sequence the DNA included in a clone contained in the ATCC Deposits by use of a sequence (or portion thereof) described in, for example Tables lAor 2 by procedures hereinafter further described, and others apparent to those skilled in the art.
[59] ~ Also provided in Table 7 is the name of the vector which contains the cDNA
clone. Each vector is routinely used in the art. The following additional information is provided for convenience.
[60] Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap XR
(U.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988);
Aping-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17: 9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies .5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037. pBS
contains an ampicillin resistance gene and pBK contains a neomycin resistance gene.
Phagemid pBS
may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK
may be excised from the Zap Express vector. Both phagemids may be transformed into E.
coli strain XL-1 Blue, also available from Stratagene.
[61] Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897.
AlfSport vectors contain an ampicillin resistance gene and may be transformed into E.
coli strain DHlOB, also available from Life Technologies. See, for instance, Gruber, C.
E., et al., Focus.15:59- (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR~'2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, GA 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, Clark, J:
M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., BiolTechnology 9: (I991).
[62] The present invention also relates to the genes corresponding to SEQ ID
NO:X, SEQ ID NO:Y, and/or the deposited clone (Clone ID NO:Z). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
[63] Also provided in the present invention are allelic variants, orthologs, and/or species homologs: Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, andlor species homologs of genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA contained in Clone ID NO:Z, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or.
primers from the sequences provided herein and screening a.suitable nucleic acid source for allelic variants and/or the desired homologue.
[64] The polypeptides of the ,invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
[65] The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
[66] The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art; such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.
[67] The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA
sequence contained in Clone ID NO:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ
ID NO:Y, a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or the polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1 B.
Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1 B are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of, the, complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA contained in Clone ID NO:Z.
[68] Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in Table 1 B column 6, or any combination thereof.
Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in Table 1 B column 6, or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column S). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1 B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC
ID NO:A (see Table 1 B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[69] Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten; or more of the complementary strands) of the sequences delineated in column 6 of Table 1 B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequence~~~lineated in~ column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is di~i rent from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1 B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1 B which correspond to the same Clone ID NO:Z (see Table 1 B, column 1) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[70J Further, representative examples of polynucleotides of the invention.
comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof.
Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in column 6 of Table 1B
which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X
(see Table 1B, column 2) and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2) and have a~
nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID
NO:X (see Table 1B, column 2) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (See Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[71] Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of Table 1B column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s). of the sequences delineated in the same row of Table 1B
column 6, or any combination thereof. In preferred embodiments, the polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in the same row of Table 1B column 6, wherein sequentially delineated sequences in the table (i.e.
corresponding to those exons located closest to each other) are directly contiguous in a 5' to 3' orientation. In further embodiments, above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the .
above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC TD
NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[72] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B, and the polynucleotide sequence of SEQ ID
NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[73J In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1 B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ ID NO:X
(e.g., as defined in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments, the delineated sequences) and polynucleotide sequence of SEQ ID NO:X correspond to the same Clone ID NO:Z. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[74] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, orie, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of column 6 of Table 1 B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments, the delineated sequences) and polynucleotide sequence of SEQ ID NO:X correspond to the same row of column 6 of Table 1B.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[75] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of the sequence of SEQ ID NO:X axe directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides andlor nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[76] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1 B and the 5' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies. that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[77) In specific embodiments, polynucleotides 'of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[78] In specific embodiments, ~olynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B axe directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides, are also encompassed by the invention.
[79] . In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 are directly contiguous.. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[80] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column,6 corresponding to the same Glone ID NO:Z (see Table 1B, column 1) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 lower stringency conditions, are also encompassed by the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[81] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one sequence in column 6 corresponding to the same contig sequence identifer SEQ ID
NO:X (see Table 1B, column 2) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization .
conditions or alternatively, under lower stringency, conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described .polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention. ~ .
[82] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table .1 B and the S' 10 polynucleotides of another sequence in column 6 corresponding to the same row are directly contiguous. In preferred embodiments, the 3' 10 polynucleotides of one of the sequences delineated in column ~6 of Table 1B is directly contiguous with the S' 10 polynucleotides of the next sequential exon delineated in Table IB, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polyriucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[83] . Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Accordingly, for each contig sequence (SEQ ID NO:X) listed in the fourth column of Table 1 A, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 and the final nucleotide minus I S of SEQ
ID NO:X, b is an integer of I S to the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14: More specifically, preferably excluded are one or more polynucleotides, comprising a nucleotide sequence described by the general formula of a-b, where a and b are integers as defined in columns 4 and 5, respectively, of Table 3. In specific embodiments, the polynucleotides of the invention do not consist of at least one, two, three, four, five, ten, or more of the specific polynucleotide sequences referenced by the Genbank Accession No. as disclosed in column 6 of Table 3 (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequences) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).
In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.
SEQ -ID EST Disclaimer lone ID NO: ontig Range NO: Z X ID: of a ccession #'s Range of b HE8UL90 12 942749 1 - 77S 15 - 789 D80247, D80022, D51060, D80195, D58283, D80043, D8039I, D59787, D80196, D59467, D80522, D57483, 014389, 014331, D80439, D802S3, . D80302, D59889, D80166, D59619, D80210, D80164, D80240, D59859, D59502, 014014, AA305409, D51423, D51799, D59275, D80038, D80227, D80366, D51022, D81026, D81030, 006015, D80212, D80219, D80269, D80268, D80188, D80248, D50979, D50995, D80024, D59927, 015076, AA514186, AA305578, D80133, D59610, D80193, D80045, ' D80157, AA514188, T11417, AW360811, D80378, D51103, 014429, AW177440, D80251, D51759, 075259, D80241, AW178893, 003092, D59653, H67866, T03269, AW377671, AW375405, D4S260, H67854, AA809122, AI525923, AW366296, AW 178906, AW360844, AW3608I7, C0S695, AW179328, 014227, T48593, D60010, AW37S406, AW378534, AW179332, AW377672, AW179023, AW178905, D59373, AW177731, AW378528, AW178762, AW179019, F13647, AW378532, D80014, D51221, T03116, AWI77501, 014344, AW177511, AW352170, D58246, AW360834, D80258, D81111, . - AI525917, AW179020, D59S03, D59317, AW 179024;
D80064, ' AW377676, AW352171, D80168, AW378533, 014973, AW178907, AW178908, 233452, AWI77505, AW178980, D59474, D51250, AW177733, AI535686, AW360841, AW352120, AI525920, AI525227, AI557774, AA514184, AW 178775, 014046, AW367950, AW178909, AW177456, 014407, 014957, AW179004, DS9551, D58101, AW 179329, AW 176467, AW178986, AW178914, AW178774, AW178754, AW179018, AW3521~8, AW352117, AI52S242, D60214, AI525235, AI557751, AW179009, AW179012, 016955, AW178911, AW378543, AW378525, ~
AW378540, AW177722, AW352163, AWI77734, AI52S912, T02974, AA285331, H67858, AI525925, AI525215, AW378539, AW177728, D59695, 221582, D45273, D80949, D59627, D51213, AW378542, 005763, F13796, AI525222, 004682, AW178781, ' AW360855, 014077, AI525237, . AI910186, 014298, D51053, AI905856, T02868, AW369651, 230160, 013958, AI525928, D80314, AA305720, D51231, D31458, D50981, N66429, AI525228, AI525216, AW177508, AW177497, T03048, AI525238, AI535961, AI535959, AF154671, .
A62300, AF058696, AR018138, AB002449, A82595, AR060385, A84916, A62298, AB028859, AJ132110, AR008278, I50126, I50132, I50128, I50133, AR054175, AR016514, I14842, X67155, AR060138, A45456, Y17188, A94995, D26022, A26615, AR052274, Y12724, A25909, AR066488, Y17187, Y09669, A43192, A43190, AR038669, A67220, D89785, A78862, D346I4, AR008443, AR066487, A30438, A63261, AR008277, AR008281, AR062872, A70867, D88547, AR016691, AR016690, U46128, D50010, AR008408, A64136, A68321, X82626, I79511, X68127, D13509, AR025207, AR060133, AF123263, X72378, and AR032065.
HETKR83 13 9632741 - 114115 - 1155 AW195777, AW269932, AI829559, AI571060, AI083491, AA905071, AW118125, AI049799, AI376671, N90902, W27632, AI273588, AI890622, AI393483, AA040604, . ~ W38638, ~W37154, W22119, AA.904910, N92239, AT194027;
W27681, W27896, AW367713, 014616, 002576, 014877, RS5809, AA897696, AA364393, AA298658, AA017680, W23093, W27851, . H83295, W22553, D81988, 855894, AW086128, W27371, W27944, D60284, W23268, AA040705, AF122922, AF122923, and AC044883.
_ HFIZB56 14 9556181 - 666 I S - 680 AA298425.
HFKHD91 15 9512591 - 644 15. - 658 AA422028, W79191, and AB023193.
HFKKE19 16 9474181 - 276 15 - 290 D87469, AL031597, AL157901, and AL356389.
HFOXL77 17 9106981 - 602 15 - 616 W22070, AB033063, and AR065869.
HHENW06 18 9713101 - 12571S - 1271 AW387854, 860430, H15208, AA454508, AA194058, H06926, T66357, AA159059, F06846, W40388, AA453713, AA160862, F08019, AA704013, AL117551, . L40459, and AR012385.
HKGDI91 19 9272221 - 433 15 - 447 AI952995, AI884982, AW071872, AW297176, AW131657, AI871511, AI568544, AI887085, AI214075, AW295595, AI356220, AW297665, AW006191, AI423713, AI383368, AW294321, AI363919, AF084576, ACOI 1500, and ACOI
1500.
HLCMP75 20 9447221 - 949 15 - 963 AA430329, H19258, H26693, AA853085, AW361580, AA747653, AA595199, AA730621, AI339553, AI041943, H12557, AI284951, 855547, and AI652283.
HLHCR16 21 910123I - 379015 - 3804 AA402528, AI379350, AA7I6107, AI123557, AI127175, AA234106, AA234698, AI039768, N77999, AI580137, AA424560, AA419490, AI334141, 871349, AI224976, AI417798, AI080508, N58410, AI8I8475, AA424657, N91089, AA399612, AI144265, AA3991.37, . AI498363, AA410986, AA235306, AA399148, AW292497, AI249102, W86869, AA115407, H81257, AI077499, H00194, H45499, 801206, AW104245, AA234880, AA367417, AA852175, F05822, AA853077, AA852176, 882875, H81245, AA298421, 871350, 822096, H81585, AI985171, . T49265, H81591, AA115408, 864037, T54283, AI492930, AI492932, 882876, 881695, 833935, 834138, 825630, H81595, 832025, 881696, T49264, 895688, 801319, 822040, 221579, 833043, 238741, AA514393, T97662, T34993, AA705441, D79105, 832078, H45500, AA707178, 830943, H81576, H81250, AI032721, AL079279, and I76197.
HLKAB61 22 9480021 - 284 15 - 298 W70120, W99328, AI139986, AA135576, AA399431, N45043, AA226160, AA143555, AW024960, . AA190958, and M93661.
HPTZB93 23 9718421 - 836 15 - 850 AT279486, AW206040, AW138281, AI817720, AW205987, AW206016, AI694554, AA862263, AI860959, AI29is729, AI299747, AI216051, AW340960, AI703067, AI885693, AW341220, AI681397, AI802146, AI066735, AA923300, AI127856, AI279331, AI298540, AI912139, AI630777, AW140104, AI002211, AI634502, AI268259, AI796940, AI129532, AI299414, AI914342, AI692842, AA938376, AW 137441, AW207254, AI216530, AI268955, AI459037, AW136174, AI693815, AI299132, AI912208, AI301061, AI299900, AI298698, AI702631, AI298009, AI871768, AI810454, AI689870, AW016202, AI458645, AI222004, AI689871, AW102711, AI702711, AI689859, AI804311, AI732920, AI732919, AA995350, N68345, AI351290, AA825171, AA962534, AI476744, AA878309, AA974790, AA934499, AA528135, AI702851, N94016, AI187311, AW003096, AI670694, AI223259, AW139377, AA916697, AI791238, AA829735, AA885627, AI791237, AA910380, AW003320, AA911255, AA987456, AW024083, AA971615, AA916296, AA953807, and AA128824.
HRDBE43 24 894862 1 - 186815 - 1882 AI160324, AA420500, AW263972, AI206613, AI417744, AI953056, AA778217, AI863306, AI804393, AI432712, AI587218, AI218649, AI339726, AA780366, AI825578, H70209, AA470738, AI669249, H70546, AI611674, AI792038, 808140, T82124, AI791228, AI820643, AA847657, AI342131, AI342141, AA989292, AI631664, AI697858, AI697880, AI278251, and 884867.
HSSKD85 25 908141 1 - 113615 - 1150 AW024960, AI479960, AI052585, AI139986, AA135576, AW137104, AW205456, N30762, AI631818, AI187156, AA830014, AI452434, AA634216, AW088488, AA398256, AI859053, AI088449, AI159823, AA564062, AI494095, AA158516, AI311717, AA811798, AA226160, . AI873741, N25872, AA146624, AA761003, AA190958, AI066392, AA535733, AI278434, W92242, AA225625, N40580, H94758, AI382438, AI796467, W92243, . 859937, H94384, H94363, AI969940, W70120, AW338143, W99328, AA143555, AA399431, AA190865, N45043, AI718153, -' AI245054, AI906964, AI760352, AA135514, AA653523, AI708811, AA034276, W69970, AA156400, M93661, D32210, and U57368.
HTEOF80 26 847224 1 - 506 15 - 520 AF186084, and AL117610.
HTNBMOI 27 910705 1 - 638 15 - 652 AA298841, and 228624.
HTSHM38 28 972248 1 - 604 15 - 618 AI907172, C14389, C15076, D59467, D80164, D59787, D81026, D59610, D51799, D58283, C14331, D80022, D81030, D80043, D80241, D80038, D59502, D59859, D80227, D80166, D80195, D51423, D59619, D80210, D80391, D59275, D80240, D80253, D80212, D59927, D80196, D80188, D50995, D80219, D80269, C14429, D57483, D80366, D50979, DS9889, D80193, D80251, C14014, D52291, AW177440, AA305409, D80378, D80024, D51022, D80045, AA305578, AW378532, D8~522, T03269, AW178893, D51060, C75259, T11417, AA514188, AW 179328, D80248, AW369651, ' D51250, AW352158, D80134, r D58253, C14077, AW178762, AW1,78775, AW1~77501, AW177511, C14407, D80268, AA514186, D80133, AW176467, AW360811, F13647, AW377671, AW352117, C05695, AW375405, D80132, AW378540, AI910186, AI557751, AW366296, AW360844, AW360817, AW375406, AW352170, AW378534, AW179332, AW377672, AW179023, AW178905,~D51213, AI905856, D80247, D80302, AW352171, D80014, D80439, AW377676, AW178906, AW177505, AW177731, AW178907, AW179019, AW179024, D59373, C06015, 221582, AW179020, AW360841, AW178909, AW177456, AW179329, AW178980, AW177733, AW352174, AW378528, AW178908, AW178754, AW179018, AWT79012, AW360834, D81111, D51103, AW179004, D59627, ' T02974, AW178914, AA285331, AW378525, D80258, D51097, AW367967, C14227, D80157, AW177722, AW177728, ' , ~ ' AW179009, D51759, AW178774, AW178911, AW378543, AW352163, D58101, D58246, ' AI557774, AW178983;
D59503, ' AW352120, AW178781, T48593, AI535850, AW177723, D59653, AW177508, D45260, D80168, C14975, H67854, C03092, AW378533, H67866, AW367950, AI525920, AW177497, AA809122, C14344, D80064, AW178986, AI525923, D80228, D59551, AW177734, T03116, AI525917, D45273, D59317, D80949, D51079, C14973, D51231, D60010, D51221, ' D59474, AA514184, AI535686, C14046, AI535961, T03048, ' C14957, D60214, AI525227, 233452, AI525235, AW378539, C16955, AI525242, AI525912, , AW378542, AI525925, AI525215, AI525237, AA305720, C05763, D59695, AI525222, H67858, C14298, AW360855, A62300, AR018138, AJ132110, A84916, A62298, AF058696, A25909, X67155, Y17188, D26022, AR008278, A67220, D89785, A78862~ D34614, AB028859, D88547, X82626, Y12724,'A82595, AR025207, A94995, AR060385, ' ~ AB002449, AR008443, AB012117, I50133, AR066482, I50126, I50132, I50128, X68127, A85396, A4417I, ' AR066488, AR016514, A85477, AR060138, A45456, I19525, A26615, AR052274, A86792, U87250, X93549, AR054175, " Y09669, A43192, A43190, AR038669, AR066490, AR066487, A30438, I18367, D88507, I14842, D50010, Y17187, A63261, AR008277, AR008281, A70867, " AR008408, AR062872, AR01.6691, AR016690, U46128, D13509, AF135125, A64136, A68321, AR060133, 179511, AB023656, U79457, AF123263, AB033111, AR032065, X93535, AR008382, and 242460.
HUSXE73 29 953246 1 - 129615 - 1310 AW138763, AI968244, AI671228, AI146849, AI650986, AA974891, AI935406, AI375I39, AI632343, AI580312, AI190358, AI823383, AA758662, AW166381, AI816934, AI3621'70, AI307616, AI33951 I, AI092493, AI193719, AA676785, r AA701414, AI375073, AI090245, AI077483, AW003931, N70081, AI307365, AI991601, AI967935, AI990350, AI637874, AI825545, m~
AI621021, W67234, AI186726, AW206481, N30322, AW140070, AW338117, AA031644, AI095704, H00954, AI859068, AW136394, AW263085, 839467, AI984849, AW338430, AI219050, AI334231, 862632, AI241351, AI355851, AI334036, AA449686, AI336416, H72039, AI735518, AA699736, 839468, F26300, 830863, AII30689, W67345, AI620138, AA358091, N74688, AA704504, AI524317, AI183860, AI801924, 874316, AI933476, AI933484, AA448958, H16951, N56653, 862685, AI634964, AA031725, H72038, T19026, AI433512, H00953, 831133, AW192226, AW235028, AI888621, AW190428, AI805638, AW029072, AI539153, AI628292, AI921082, AI379711, AW029606, AW188491, AI583533, AW002174, AI091468, AI598113, AI636719, AI358455, AI620093, , AI566507, AI498579, AW168723, AI811192, AI207454, AW088899, AI366549, AL046463, AI866608, AI874410, AI611743, AW083804, AW118332,.AA830821, AI696626, AI589993, AI365256, AW085786, AI805769, AW26S004, AI677797, AI364788, AI648567, AW089801, AI636619, AI866786, AW051107, AI866082, N74355, AI282651, AW129271, AI863397, AI310155, AI952920, AI536557, AW172723, AA579232, AW403717, AI539771, AW131954, AA420722, AI919345, . AI805688, AI251830, AI565125, AI862324, AA807352, AW168373, 840432, AIS39632, AI470701, AI738867, AI312428, AI434242, AI371'228, AI801605, AW080080, AI610429, AW168425, AI432736, ' AI307736, AI473598, AI499986, AI273839; AA928539, AI872064, AI568870, AI868831, AI869750, W33163, AI874151, AI950664, AI436429, AW087901, AI470293, AI570966, AI867042, AW082040, AI929108, AI537837, AI573026, AI699862, AI859464, AI242646, , W46547, AI445430, AI249877, AI862144, AI689420, AI879693, AI249946, AW130863, AI922577, AA848053, AW059713, AW068845, AI345677, AI274769, AI554218, AW192375, AA572758, AI872051, AI375730, AW 161892, AI800152, AW 191844, AI917055, AW 151750, AI702301, AW088134, AI500146, AI699255, AI570384, AI633477, AI680498, AA176980, AI453413, AI370390, AL047344, AI499512, AW162194, AI889147, AI678411, AI636445, AI494201, AI561299, AA693347, N71180, AI686823, AI539071, AI537307, AI674838, AA761557, AT344935, AI886124, .
AI476077, N75771, AI885974, AL036718, AI872074, AI560010, AI537617, AI311892, AI623682, AF18611I, AR059958, AL137556, AL122121, AL136842, AL133093, AL122111, AL080127, A08910, A08909, A08908, I41145, E15324, AJ242859, AF113676, A08916, AL133645, I00734, A18777, AR019470, E00617, E00717, E00778, AL035458, I89947,148978, A08913, I89931, AL133077, r ~ . AF090896, I49625, A08912, AR038854, AB019565, AF093119, AL133104, X62580, I26207, AL137527, AF000145, AL080060, X52128, AL080158, AF051325, E08631, D44497, AC002467, X53587, AF119337, A90832, AF097996, X70685, AL133075, AL080137, AL133031, AL122123, AL137300, AF012536, L13297, X92070, A23630, AL133081, AL117585, AF004162, U72620, AL122050, AL137281, AL137648, U96683, AL133S68, AF085809, AL133080, AL122098, AF125949, AL137273, X93495, AF081197;
AF081195, I89934, I89944;
M86826, AL133067, I09360, I33392, AL049466, I68732, AL137665, 568736, AL080086, AF003737, AF110329, 272491, AF106827, L30117, U55017, AF017152, AF090886, AL133014, A12297, AL133072, X65873, AJ0064I7, E15569, AL049465, AL137429, E03348, AF031903, AL137557, ALI37258, U68233, I92592, E07108, AF158248, AL122118, AL110222, U91329, S76508, AL137479, X72889, AF113691, AL137463, S61953, AL122049, AF118064, AF118070, AJ000937, AL133098, AJ238278, D89079, AL133557, I30339, I30334, Y09972, v U49434, U80742, AL137705, AL133113, AF026816, AL117432, X81464, AF111112, AF162270, U00763, AL137283, AL049938, AL049283, U67958, AL049382, I42402, AFI 11851, AF210052, I17767, AF159148, AL137538, AL137529, AL117457, 577771, AL096744, AJ003118, AL050146, AF106862, AF067?90, Y10655, Y10080, E02253, Y10936, AR000496, U39656, AR038969, A49I39, 237987, U00686, AF040751, AL050108, S79832, AF022363, M92439, AL110218, A93016, AF113013, L04849, .
A08907, AF078844, AF091084, AL137526, X87582, E05822, AF132676, AF061836, AL117583, X67688, X84990, E06788, E06790, E06789, 109499, A45787, I66342, AL137294, AL050138, AL122110, AL137574, AL050277, E04233, AL 110196, AL049314, A~079763, A07647, AL137712, U68387, AL110225, AL117394, AF069506, AL050393, U42766, AL133565, AL133606, X63574, AF061573, AF057300, AF057299, U88966, AF 142672, A21103, AL
110197, AF028823, AF100931, AF113689, AF126247, L19437, Y11587, AL137478, AL080159, AL137640, AL133640, AB007812, AF061795, Y14314, AF151685, AL133016, AL117440, AF061981, U78525, AL080148, AF030513, and I48979.
HWAAE95 30 789051 1 - 15 - 489 29.8743.
HWH 825 31 947020 1 - 15 - 565 AC020661, and AC020661.
HGBG022 32 11249101 - 15 - 1468 AI927400, AI678696, 1454 AA947645, AW295277, AA884167, AW296908, AA947188, AI984289, T28629, 211502, X80208, and X80209.
HCECQ23 33 938398 1 - 15 - 810 AI480182, AI500178, 796 AI873131, AA322958, AA322718, AI936088, . 885125, 890888, H05353, AA338672, H51247, 835934, AW139057, H05303, 849451, 842549, H29245, AI654790, AI638508, 851648, AW149807, 844423, AI458144, AI419465, F02105, AI499775, 240525, F09051, 885080, H51217, AI569283, D29763, D64009, and D64010.
HFXBI19 34 1136133I - 15 - 2218 AI796164, AF034611, 2204 and AF022247.
HMVCP64 36 11761521 - I S - 1422 AI215040, AW013830, 1408 AA425778, N30265, N41988, AA629285, AI867286, AI252688, AI251390, AI254897, AI252025, AI252700, AI254872, AA426241, and 297832.
HSXBV89 37 971821 1 - 15 - 652 890750, 887845, 825677, AJ245820, AJ245821, and AJ245822.
HTXAAI 38 11727351 - 1.5 - 2477 AA814365, AA648502, S 2463 H11953, 820002, AA7461 I7, H06599, AA374468, and W60570.
HWHQR10 39 915008 1 - 15 - 326 AC004235, AC004235, 312 AC004235, and AC004235.
HMZAD58 40 10021331 - 1'S - 2734 N26584, W94986, N38905, AI075815, AI367921, AI184158, AA830019, AI033601, N54995, AA830021, N27197, AA918808, AI539580, AI273730, AI262545, AA587088, AA779942, AA262747, AA676908, AA287348, AA730411, W86602, H16056, AA303482, AW 179318, W91912, AA670033, AA705754, AW300038, AA887595, AA703588, AI219099, N46479, AA312749, AA903850, AA495756, AA351345, AA887468, AA287308;
AW451932, AW197479, AI890907, AI538850, AI887308, AA761557, AL039132, AI873638, AI590686, AL036638, AI242931, AI627988, AI689702, AA848053, AW022699, AI922550, AI697372, AI440263, AI249946, AI801325;
AI683072, AI335426, AI348777, AI863382, AI818574, AL079960, AI281867, AI446538, AI567612, AI590423, AL041150, AA911767, AL038445, AI288050, AL037454, AL045626, . AI619426, AI354998, AI858827, AA502794, AI973152, AA908294, AI683395, AW161579, AI866090, AI082740, AW265004, AI619607, AI559632, AI698391, AI679506, AL048427, AW198090, AI868740, AW161156, AI680388, AI280747, AI344935, AI161279, AI537677, AI494201, AL047100, AI491852, AW051088, AA641818, AI873644, AI470293, AW023859, AI540458, AI916419, AI470648, AL119863, AI961286, AW167385, AI536685, AI570884, AI498067, AI538116, AI950664, AI624529, AI687166, AI9277S5, H89138, AI554343, AI241923, AI677824, AI473799, AI670009, AW243886, AL036631, AI913082, AI648408, AA225339, AI696612, AW059828, AI637748, AI932458, AL036772, AL036396, AAOOI397, AI872I84, AI872423, AL036980, AW029611, AW079336, AI969655, AI540674, AI251221, AW022682, AA568405, AI863321, AI174394, AI817552, AW020095, F37471, AI623941, AI500706, AI648508, AI469674, AI538342, AI345745, AI473536, AI887430, AW057937, AI500077, AI631216, AW149878, AW131428, AL046618, AI345347, AL079963, AW302924, AI538218, AI765469, AI499263, AI953562, AW192712, AI539687, AA572758, AI766348, AI868204, AI890507, AI537261, AI863191, AI269696, AI432040, AI918449, AI620284, AI263331, AI242248, AI801523, AI358701, AI445992, AI568138, AI445990, AI624293, , AW080746, AL036673, AI868931, AI783504, AI784230, AW 149925, AI568114, AI434741, AI419650, AI866770, AI343059, AI433157, AW172723, AW131999, AI702073, AI538764, AI554821, AW129271, AI612750, AW028416, AI283760, AI349933, AL039086, AI281757, AL038605, D63482, AFI24491, ' AF112366, ALI 10296, AL137574, 237987, I48978, AF146568, AL1335S7, AL133606, I89947, A08913, A08912, AL122093, AR013797, AF1I I 112, AF113694, AL137271, A089I6, A65341, A08910, AL122049, A08909, Y10080, A08908, AR029490, AL117578, AL133016, AF106657, E02221, AR038854, E08631, E05822, X83508, I89931, L19437, I48979, I41145,149625, AL050172, A93350, AF000301, AL023657, AL050I08, AF057300, AF057299, A18777, AL049283, AF153205, AF090900, ALI22110, Y16645, Y09972, AF113689, AR034830, AL050024, Y14314, 561953, AF 113019, AF090934, X79812, I33392, E02349, AF026I24, AL110225, S68736,'AL137521, AF097996, AF106862, AL133665, AF177401, AF090896, AL050155, S76508, AL133067, Y11254, AL137550, AL122100, AL137558, AL137548, AJ012755, I96214, AF113699, 282022, AF079763, AL117460, AL080158, AF008439, AL,120280, I68732, AL137476, . AL137463, A08911, X82434, AL110197, AF113691, E01614, 1m E13364, X5587, AL080074, I89934, I89944, AF118070, X70685, AL117585, AL050116,~U78525, AL137294, AL137292, I00734, AR020905, I26207, AF017437, AL049300, 272491, AL1374S9, AF017152, A23630, E00617, E00717, E00778, AL117394, AL050393, D83032, AF003737, A08907, E02253, AL117440, S78214, AF139986, AF137367, U35846, AL133560, I80064, AF090903, AR038969, U95114, A77033, A77035, AL049430, ' E06743, AL049452, AJ238278, U87620, AF125948, Y07905, AL117435, X65873, X96540, E12747, AL050277, AL080086, S36676, AL137557, AF067728, AF087943, AR000496, AL133645, AL133640, U39656, AL080154, AF111849, E07108, AL137533, U68387, AF185576, AF090901, AL133113, AL050092, AF032666, AJ005690, AF061943, AL080124, AL137429, AF028823, AF100931, AL050149, L31396, A08915, AL080I37, AF030513, AF079765, L3I397, X72889, A58524, A58523, 575997, AL133093, AL137478, AL080159, AF051325, X80340, A90832, U58996, ALI22098, ALI37529, AF061795, AF151685, AL096744, AL096720, AF06198I, A52563, AL080148, AL133010, U42766, AL 117432, AL 122121, AB019565, AF162270, AL133104, ' AL049466, AL122050, X62580, I03321, AL049314, AL133081, . M30514, D16301, AB007812, ' U68233, I92592, AF158248, . E15569, AL133565, AJ006417, U91329, U00763, E04233, AF065135, AL137560, AL117416, AL117583, AF183393, AL122045, . AL110222, AL080060, AF119337, .
I09360, X87582, Y11587, , AL110196, AJ000937, AL133080, AL049382~ AF118090, AF106827, . I42402, and AL137648.
HWDAE40 41 947007 1 - 216615 - 2180 AF150174, AI417513, AI698235, 856970, AA471187, AC008917, AC0089I7, and AC016605.
HADFCS1 42 10021421 - 392415 - 3938 AA203426, AI300I88, AW139439, AI379134, AA815253, AI568548, AW 134634, AA484158, N50870, AA687654, AW072618, N48188, AI304847, AA464588, N62844, N53335, AA043385, W80785, AI248256, AI206713, AI719303, AI630905, N77751, AI339657, AA932565, AA115602, 244436, . AW193024, AA525284, AI281971, AI298245, 854464, AI041687, 849127, AI091286, AI918287, AI887073, 240366, 806527, AA995384, M85459, AI125279, N26208, H97245, AA115137, W80892, AA043384, AA331941, AA523243, AW149086, AA902505, H02196, C21424, and AL008628.
HAWAM69 43 9431041 - 190115 - 1915 AA430300, AA541688, AA776700, AW385785, AA679037, AA573270, _ AA126614, AL045796, AA682186, AI268236, AI963606, AW192904, AI926591, AI924827, AI922590, AI032288, AI375804, AA705I72, AW081541, AA6945i4, AII30883, N25288, AA931725, AI800450, AI270687, AI366906, AW058362, AI683319, AA436891, W69578, AI597744, AI446542, 859176, AW453004, AI911821, AA687634, AI095665; AI130013, W69579, AA722782, AI191864, AI587015, AA398533, AA676733, AI476374, AA115447, AA554327, AA759328, AW242281, AA042956, AI139766, AA135916, AA886732, AA664356, AA358590, H71919, AI565897, AW304844, AA916086, AA618576, AA363371, 244808, AA430199, AI370031, AA320329, AA393105, AW452852, AA135927, AI004I40, AA135926, AA042816;
H44791, T35731, AI865731, AA813424, 842647, 827785, T32691, AI934183, AA115446, AI857286, AW008428, AI631988, AA678468, AW075384, H44790, AI569918, AI918635, AA603858, AA601518, AI745618, H42641, AI445766, 827874, AI939990, AA677131, AW364938, AI569374, AW029062, C01947, and AA732827.
HBGMG39 44 9714141 - 471 15 - 485 AA024454, AA024670, , AI458409, AI740930, AI742565, AI743686, AI066465, AI168481, AI379125, AI569972, AW069135, AI497641, AW192429, AW084071, AW339039, AW102701, AI061450, AI693756, AI991329, AW003414, AI342244, AI870883, AW007899, AI609020, AI453165, AI248142, AI129686, AI992036, AI761292, AI623708, AI864435, AW151858, AI362058, AW044270, AI378430, AW008808, AI479128, AL036585, AI923881, AI963067, AI740972, . AL045227, AI352617, AI334039, AI452903, AI700412, AI475537, AI469546, AI187911, AI066744, AI190677, AI812069, AI081231, AI050026, AI890929, AA551905, . AI080156, AI554840, AW073739, AI926062, AA577674,_AW190499, AW250073, AI524714, AI884727, AW168902, AI096904, AI369151, AI858574, AI422058, AA304774, AI890721, AI052823, AI683215, AI087154, AA682804, AW080685, AW004991, AA912478, AW079985, AA054275, AA461453, AI668789, AA970861, AA010790, AW085591, AI819302, AI217784,AI017023, AI584163, AI627434, AA709077, AA984939, AA446048, AI564659, AI000961, AI095374, AI581899, AA532435, AA864689, AW087352, AW057507, AW132118, AA744639, N53410, AA969103, AA918799, AA862362, AI672825, AA779825, AA931463, AI193638, AW 191953, N20167, AI569968, AW150032, AA612750, AI619904, AA429899, W02113, AA969104, W48745, AA827905, AW190663, AA780072, N50640, AA533847, W32574, AI640737, AI587533, AW182184, AI933617, AW073566, AI469425, AL041483, AA765028, AI952283, AI961508, AI095341, AA937508, N48059, AA228075, AI869852, ~
. N78603, AA485535, AA506502, AA877348, AA252354, AAOlOIOI, _ AA011219, T23065,'AW444850, AI917215, AA506948, AW297832, AW204879, N53257, AI917614, AI167689, AI937775, AI858564, AW043696, AW168023, AI263865, AI885655, AA450098, AWI39189, AA883859, E12258, AF153686, E12259, E12260, and AL390719.
HCEHD66 45 959160 1 - 15 - 1325 AI968437, AI824971, . 1311 AW104052, AI762197, AI598138, AI088543, AI492390, AI827280, AA058923, AW007187, AW135225, AI391466, AI808139, AA534403, AW028554, AI369729, AA460467, AA135928, AW006062, AW138526, AA507443, AI479413, AI400940, AW 137272, AW381735, AA135929, AA085774, W811S3, AA918755, C03738, 885039, AI472852, AI937792, AI867512, AA599118, N62215, . AI864402, H41491, N62216, D44882, H14329, W30972, H40979, AA4.63408, AI744140, H40980, N62166, AA319197, AI766568, N62223, AF186409, AF020184, L27421, L27420, and AC006241.
HCESP56 46 8276711 - 500 15 - 514 AW247740, AW247029,-AW204207, W39269, AA325536, 814422, W52568, Y16752, AL022170, and 265186.
HCHAT01 47 8672091 - 154215 - 1556 817167, and AB014576.
HCHMW40 48 9515181 - 870 15 - 884 AI732539, AI791495, AI791325, ' AA709067, AW082062, AI791964, . AI732667, AA505923, AI909857, AI909862, AA601601, AW057561, and AI909853.
HCUEV29 49 8160651 - 491 15 - SOS AW410192, AI570209, AA583494, AW087991, AW337550, T30350, T24722, AW246233, and ' AL031283.
HDQID90 50 8319761 - 953 15 - 967 AA767219; AI809238.,.AI219470, AA767092, AA114887, T71487, AA464762, AA504439, 225261, N87679; D57415, AA278335, AW300598, W46278, AA669095, D56990, D54675, AI797687, AI948608, AA909071, AW236181, AI718165, AB033082, AF132480, AF132479, and AC002350.
HE8PY29 51 8878621 - 741 15 - 755 AI27I550, AI753504, AA809220, AW081079, W78099, AW386283, AI264068, AI219556, AW082138, AA455733, AI382746, AA548778, AA431230, AF0921.37, AF100751, ' AF040252, AC009948, AC009948, ' and AC009948.
HE8QZ34 52 9522831 - 107015 - 1084 835313, AA210809, W28575, ' AA112I26, 886156, AA286.753, AW405566, AA385668, AA384297, H25863, H50786, 825032, 246079, . AA334931, AA490204, and AW367213. ' HE9TD31 54 8158451 - 941 15 - 955 AI475682, AI439613, AA815076, ' AB033082, AF132480, and AF 132479.
HELHB88 55 8119351 - S54 15 - S68 AI750406, AI580905, AA024853, AF114488, AF114487, AF064243, .
AP000116, AF064244, AP000049, AP000311, AF132672, AF127798, AF132478, and AF132481.
HEOPP67 56 8276301 - 436 15 - 450 AA641653, 299396, AF181972, and AF 181973.
HGBDG55 57 8158581 - 522 15 - 536 AA368408, and AL360268.
HHFOC79 58 9354061 - 1024_ AI569931, AA450162, 15 - 1038 AA405198, ' H26214, H14443, N28528, 873380, AW015358, 848456, AI750978, AA358230, 219130, AA359395, AA744173, H26831, and AC008745.
HHGAE47 59 9221941 - 705 15 - 719 AW025529, AW026010, AA657904, AA662803, AA886335, AA158820, AI475932, AW050607, AI885090, AI056120, AI244837, AA485S66, AI375435, AA922036, AA878578, AA643750, AI056614, AW449834, AW 197722, AI393408, AA485405, AI560410, AA161103, AI749095, AI72093I, AW058170, AI446208, N57590, AI268967, AI832600, AI913781, AA910277, N52768, AI277003, AI914599, AI192693, N57604, AI673692, AW050712, AA631339, N52783, and AI919380:
HKAOV71 60 8276791 - 743 15 - 757 AF123303, and AF004161.
HKGDE58 61 945039I - 925 15 - 939 T75535, AA287162, AA448686, ' 813858, H12041, 246111, T94956, F07044, H05358, AA053290, ' AA454139, F05720, F05717, AL122084, and AL049611.
HMCGL45 62 9221951 - 114115 - 1155 AW025529, AI475932, AW026010, ' AA886335, AA662803, AI056120, . ~ AWOS0607, AI885090, AI375435, AW449834, AA161103, AI244837, . AA878578, AA922036, AW197722, AI056614, AI393408, AW058170, AA643750, AI560410, AI749095, AI720931, AI446208, N52768, AI913781, AI277003, AI268967, AA910277, AI914599, AI192693, . ' N52783, AW050712, AA48S405, AI673692, AA631339, AA657904, AA485566, AA158820,~AI619710, AI560351, AI919380, N57590, .
AI832600, N57604, T25136, AW198090, AI499963, AW023338, AI638644, AI890214, AI538850, N75779, AI633125, AI686817, AI499570, AI500061, AI860027, AI473536, AI925164, AW163834, AI690813, AW162194, AA641818, AI684244, AI469505, AI376425, AI802542, AI673363, AI670009, AI498067, AW082532, AI433I57, AI886055, AW080700, AI702073, AI884318, AW128834, AI633198, AI289310, AI620056, AI590043, AW103928, AI890907, AW152182, AI589428, AI961S89, AI679550, AI961414, AI698391, AI479292, ' AI701097, AI570861, AI147292, AW169604, AL045413, AW081383, AI345688, AI245008, AI539800, AI950729, AI927233, AL079799, AI491775, AI872423, AI538980, AI288050, AI635634, AI440239, AA805434, AW161579, AW151893, AW148363, AI866465, AI538564, AI571439, AW083374, AL041150, AI973152, AI445611, AL047100, AI538116, AI687362, AI281757, AI241923, AA580663, AI440399, AI095003, AW022808, AI540674, AI954475, AI499890, AI471282, AI648494, AI927755, AI621341, AI445829, AI432030, AI915291, AI932503, AI341690, AI613038, AI866040, AW073865, AI699823, AW190194, AI55961.9, AL037582, AL037602, AI627988, AI685005, AL039086, AI348901, 841605, AI932794, AI345415, AI636588;
AW163554, AI572096, AI612852, AW050998, AI580436, AI627893, AI568138, AA587590, AI869377, AL046466, AL118781, AI819545, AI270183, AI912434, AI567513, AI524179, AA830709, AW051088, I48978, I48979, AL137550, AF061573, A15345, I89947, I68732, A65340, AL117460, A77033, A77035, X63-162, AF153205, AR029490, AL023657, D16301, AL117587, U78525, AL050366, E12747, AF067728, AF115392, AF090900, AR038854, A76335, ' E02349, AF102578, I33392, A58524, A58523, A08910, A08909, A6S341, AJ005690, AL080159, A08908, 282022, A58545~
AL137294, AL080074, AL137533, A52563, AL137479, U373S9, X70685, A93350, AF047716, , A03736,.AL,122100, A08913, U49908, AL122110, X82434, AF183393, AF094480, 237987, AL050393, A08912, Y10080, S54890, A08911, A08907, AL133049, AF113690, AF100931, AL137271, U35846, AL096744, 213966, Y14314, AR034821, S76508, AF026816, AL136884, AF215669, AL049347, AR068753, AL050149, AF177401, AF039138, AF039137, AF146568, AL117435, X72889, X53587, AL050155, AL133112, AL133637, AF031147, US5017, X67688, AF090903, AL137480, D83032,. AL050277, AF097996, AF200464, AL117457, AL133665, AL080163, 297214, AL133623, AR020905, AF017437, AL122049, AF113699, ALI10158, AF158248, AL137656, AL133010, A21103, AF 111849, AL
110221, AF061981, AL080148, X81464, L04849, A18777, S36676, AL137530, I25049, E01614, E13364, AF115410, AC002471, AC005374, S75997, I89931, AF145233, AL049339, A07588, AF087943, U95114, AL080110, AL080139, AL137529, L04504, S77771, AL117392,I49625, AF107847, AL117416, AL137459, Y09972, AF058921, I09499, - . AL137476, AL137537, AF114170, AL137526, AF090943, AL133558, E05822, I33391, Y11254, AJ000937, AL133080, AL122104, AF141289, U58996, AL133075, AL050116, S53987, AL137488, U72621, AF030513; AL133113, AL133072, .
- AF032666, AJ012755, X83508, AF061943, A17115, A18079, A08916, AF106945, I89934;
AL133067, U83980, Y10823, . U90884, AL117585, X66862, X66871, AL050024, 582852, AF176651, X84990, I03321, U96683, AL137256, AF180525, A12297, AL137521, AF106862, AF057300, AF057299, AL110225, AL110218, AR011880, A18788, AF113694, AB016226, A08456, A86558, A76337, AF118092, E 12806, AF026124, AF061795, AF15I685, U73682, I32738, AF090901, AL122093, AL050092, AL050138, and AL137292.
HMSOL52 63 921126 1 - 15 - 1304 AI911515, AI360955, 1290 AW028045, < - AI796049, AI609712, AW195544, AI184337, AI470056, AI361065, N34939, AI017177, AI038779, AI440241, AI651451, AA789292, AA854683, AI765258, AI702748, and AA384884.
HMTBB 64 950884 1 - 15 - 514 AA582539, AI963340, 17 500 AI097093, - AI460219, AA286856, AI761614, AI149781, AI032670, AI819154, AI636161, AI089302, AI811219, .
H12042, T95010, AA836993, AW271462, H05308, 837000, AI001803, AA904906~
AA743196, - AI015200, AA453607, F05000, AA578803, AI241466, AI033193, AA037601, AA330970, F03322, F01968, T75492, N47542, AW183219, AI288171, AA651907, AA054759, F01965, AL122084,~
and AL049611.
HOEET48 65 963290 1 - 15 - 1480 AI797684, AW239200, 1466 AA456267, _ AI478733, AI751749,AI990902, AA427646, AI379565, AI970534, W95460, AA788855, AA405402, AW068453, AW294114, AI751750, AA594137, AA947297, AW177719, AI057073, AA427487, AI341112, AA232452, AA041304, AW068711, H73236, AA041328, W95567, AW 167569, AA853047, AI652166, ' W02069, H74.164, 834003, AI341381, AW176526, AA580289, D30965, D31 I76, AA367502, and AR035969. ' HOUHL51 66 815891 1 - 15 - 647 AA431822, AA037543, 633 AI656610, AA431419, AA974280, AA815270, AA037457, AI341790, AW295199, AI651702, AW292290, AA583011, AI208605, AI419858, AA620408, AA417333, AA4I7321, W28051, AA251183, AI917695, W28536, and _AI024754.
HSIA078 67 889498 1 - 15 - 829 AA377072, AA298640, 815 AA453038, AC002302, AC012317, AC012185, and AC002302.
HSOBC04 68 927280 1 - 15 - 1220 AA115298, AI741325, 1206 ~ AI688227, AI819333, AA452504, AI9256.64, AI742595, AI174,530, AA115338, ' AI567500, AA563582, AA461615, AI142563, AA807844, N94422, AI095261, AA569395, W58424, ' AA687480, AA479551, AA582573, AI081428, AA779677, AI280806, N24393, AA988617, AI863187, AA834079,. AW302361, AI362861, AW273442, AA150123, AA553678, AI752480, AI312661, WS2661, AI298150, AA463418, W72509, AA024450, W72139, AI037968, W79868, AI028169, AA477651, H39596, W02690, AI198327, AI952450, AA926794, AI087245, W74236, AW004736, AI334346, AI870989, H98040, AI689546, AI332748, W76066, AAI50031, 840403, AI349417, AA595996, . W80872, H99144, AW166280, W52767, AA496878, H2,5985, AI357863, 855375, AA363023, AW 104147, AA378409, AI979074, AI376184, AI687489,T32290, N26307, H97338, N95244, W77880, AI917258, W2S604, AI536791, AA024802, AA577352, AA328156, AA359865, AA367475, AA461442, AA358275, W80763, T09474, AA987427, AI611160, AA888165, AA595303, AI9I8172, W30769, AI201782, AA187662, W21074, AA4119S5, AA935961, AA090719, AA4119S6, AA451977, AI371307, AW074526, N79974, AI635472, N39751, AI612934, AA478489, AA102215, AI802295, AI750502, AA496836, AL133116, L07063, and AC012192.
HTEIL07 69 953803 1 - 15 -_459 H55431, and AL031843.
HTEKS20 70 846714 1 - IS - 1077 AI936596, AA868353, 1063 AI797296, AA725553, AI221970, AA429551, AA428462, AA629305, AA629047, AA431190, AI073397, AW235895, AI123443, AI808267, AA609412, AI914363, AA953895, AI214385, AA431516, AA911681, AA781953, AI825106, AA298758, AI215028, AA909534, AA723768, D10393, 563991, AL137023, and AL137023.
HTEON29 71 815852 1 - 15 - 992 AW004028, AI968030, 978 AW237673, AA432290, AW138422, AA112090, AA428635, AI143780, AI143791, and AA861634.
HTLEN77 72 772363 1 - 15 - 424 T89583.
HTTEK47 73 573649=1 - 15 - 410 AA400005, AW205244, 396 AW 134723, and AI689951.
HTTJW49 74 948107 1 - 15 - 662 AA199865, AI769428, 648 AI061340, AW268880, AA707168, AI970984, AI884812, AW444872, AI479954, AI356088; AI701720, AI765045, AA722812, AW236544, AA410516, AI267987, AA005114, AI298592, AI865503, AI633370, AA878382, AW389168, AF118838, Y17571, AF164632, AF164526, AF164527;
AC004458, AF164528, and AF 164525.
HUJCT05 75 929264 1 - 15 - 639 AI124874, and AC003962.
HWBEG18 76 909798 1 - 15 - 954 AA906863, AW408789, 940 AW452373, .
AA352977, AA732349, AI269653, - AI492098, arid AB020653.
HWLFG75 77 916563 1 - 15 - 1269 AI356559, AW163067, 1255 AA443325, AW005140, C18386, 815375, 817389, 860462, AA442531, H16941, AA740299, AA025666, AA443338, R42I16, 860229, 842625, AW444512, AW450707, AW157098, AA724594, AA978110, AI810652, AA927875, AI924004, H16834, AI886594, AI376913, AA609873, AW173645, AA578062, AA578362, AA467933, AI147260, 852646, AI672253, and AI347103.
HWNCY05 78 928789 1 - I5 - 1380 AA210942, W40569, AW025860, .
D63226, AA362635, AA334307, AA211707, AI613267, and AC006928.
Code Descri tion Tissue Or an Cell DiseaseVector Line AR022 a_Heart a_Heart AR023 a_Liver a_Liver AR024 a_mammar gland a_mamma oland AR025 a_Prostate ~ a_Prostate AR026 a_smail intestinea_small intestine AR027 a_Stomach a_Stomach AR028 Blood B cells Blood B cells AR029 Blood B cells Blood B cells activated activated AR030 Blood B cells Blood B cells resting resting AR031 Blood T cells Blood T cells activated activated AR032 Blood T cells Blood T cells resting i:estin~
AR033 brain brain AR034 breast breast AR035 breast cancer breast cancer AR036 Cell Line CAOV3Cell Line AR037 cell line PA-1 cell line AR038 cell line transfomnedcell line transformed AR039 colon colon AR040 colon (9808co65R)colon (9808co65R) AR041 colon (9809co15)colon (9809co15) AR042 colon cancer colon cancer AR043 colon cancer colon cancer (9808co64R) (9$08co64R) AR044 colon cancer colon cancer 9809co14 9809co14 AR045 corn clone 5 corn clone AR046 corn clone 6 corn clone _ AR047 corn clone2 corn clone2 AR048 com clone3 corn clone3 AR049 Corn Clone4 Corn Clone4 AR050 Donor II B CellsDonor II
24hrs B Cells 24hrs AR05I Donor II B CellsDonor II
72hrs B Cells 72hrs AR052 Donor II B-CellsDonor II
24 hrs. B-Cells ' hrs.
AR053 Donor II B-CellsDonor II
72hrs B-Cells 72hrs AR054 Donor II RestingDonor II,Resting.
B Cells B .
Cells ' AR055 Heart Heart AR056 Human Lung (clonetech)Human Lung (clonetech) AR057 Human Mammary Human Mammary ' (clontech) (clontech) AR058 Human Thymus Human Thymus (ctonetech) (clonetech) AR059 Jurkat (unstimulated)Jurkat (unstimulated) _ AR060 . Kidne Kidne AR061 Liver Liver AR062 Liver(Clontech)Liver(Clontech) AR063 Lymphocytes Lymphocytes chronic chronic I
1 m hoc tic m hoc tic leukaemia leukaemia AR064 Lymphocytes Lymphocytes diffuse large B cell lymphomadiffuse large B cell 1 m homa AR065 Lymphocytes Lymphocytes follicular 1 m homa follicular I m homa AR066 normal breast normal breast AR067 Normal Ovarian Normal Ovarian (4004901) (4004901) AR068 Normal Ovary Normal Ovary AR069 Normal Ovary Normal Ovary AR070 Normal Ovary Normal Ovary AR071 Ovarian Cancer Ovarian Cancer AR072 Ovarian Cancer Ovarian Cancer (97026001 ) (97026001 ) AR073 Ovarian Cancer Ovarian Cancer (97076029) (97076029) .
AR074 Ovarian Cancer Ovarian Cancer (98046011 ) (9804601 I ) AR075 Ovarian Cancer Ovarian Cancer (98066019) (98066019) AR076 Ovarian Cancer Ovarian Cancer (980760 I7) (98076017) AR077 Ovarian Cancer Ovarian Cancer (98096001 ) (98096001 ) AR078 ovarian cancer ovarian cancer ' 15799 AR079 Ovarian Cancer Ovarian Cancer AR080 Ovarian Cancer Ovarian Cancer AR081 Ovarian Cancer Ovarian Cancer AR082 ovarian cancer ovarian cancer AR083 Ovarian Cancer Ovarian Cancer AR084 Ovarian Cancer Ovarian Cancer AR085 Ovarian Cancer Ovarian Cancer AR086 ovarian cancer ovarian cancer AR087 Ovarian Cancer Ovarian Cancer AR088 Ovarian cancer Ovarian cancer 3rd C00 3rd AR089 Prostate Prostate AR090 Prostate (clonetech)Prostate (clonetech) AR091 rostate cancer rostate cancer AR092 prostate cancerprostate #15176 cancer #15176 AR093 prostate cancerprostate #15509 cancer #15509 AR094 prostate cancerprostate #15673 cancer #15673 AR095 Smalf IntestineSmall Intestine (Clontech) (Clontech) AR096 Spleen Spleen AR097 Thymus T cells Thymus T
activated cells activated AR098 Thymus T cells Thymus T
resting cells resting AR099 Tonsil Tonsil AR100 Tonsil geminal Tonsil geminal center centroblast center centroblast AR101 Tonsil germinalTonsil germinal centerB
cell center B
cell R102 Tonsil 1 m h Tonsil I
A node m h pode _ Tonsil memory Tonsil memory, AR103 B cell B
cell AR104 Whole Brain Whole Brain AR105 Xeno~raft ES-2 Xeno raft ~ ES-2 AR106 Xeno raft S'W626W626 Xeno~raft S
H0008 Whole 6 Week _ Uni-ZAP
Old .Embryo ~ XR
H0009 Human Fetal - Uni-ZAP
Brain XR
H0012 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney Kidney XR
H0013 Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old Emb o Emb o XR
r H0014 Human Gall BladderHuman Gall Gall Uni-ZAP
Bladder Bladder XR
HOOIS Human Gall Bladder,Human Gall Gall Uni-ZAP
Bladder Bladder fraction II XR
H0017 Human Greater Human Greaterperitoneum Uni-ZAP
Omentum Omentum XR
H0024 Human Fetal Human Fetal Lung Uni-ZAP
Lung III Lung XR
H0028 Human Old'Ovar ar Ovar Bluescri Human Old , t Ov H0030 Human Placenta _ Uni-ZAP
XR
H0031 Human Placenta Human PlacentaPlacenta Uni-ZAP
XR
H0036 Human Adult Human Adult Small Uni-ZAP
Small Small Int.
Intestine Intestine XR
H0038 Human Testes Human TestesTestis Uni-ZAP.
XR
H0039 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor Tumor XR
H0040 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor Tumor XR
H0041 Human Fetal Human Fetal . Bone , Uni-ZAP
Bone Bone XR
H0042 Human Adult Human Adult Lung Uni-ZAP
Pulmonary Pulmonar XR
H0046 Human EndometrialHuman EndometrialUterus diseaseUni-ZAP
Tumor Tumor ' XR
H0048 Human Pineal Human Pineal _ Uni-ZAP
Gland Gland XR
H0050 Human Fetal Human Fetal Heart Uni-ZAP
Heart Heart XR
H0051 Human HippocampusHuman Brain Uni-ZAP
.
Hi ocam us XR
H0052 Human CerebellumHuman CerebellumBrain Uni-ZAP
XR
H0056 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein, Endo. remake Vein Endothelialvein XR
Cells H0057 Human Fetal ~ ~ ~ ~ Uni-ZAP
Spleen XR
HOOS9 Human Uteiine Human UterineUterus, diseaseLambda Cancer Cancer ZAP 1I
H0063 Human Thymus Human ThymusThymus Uni-ZAP
XR
H0069 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells T-Celts Line XR
H007I Human Infant Human InfantAdrenal Uni-ZAP
Adrenal Gland Adrenal Glandland XR
H0081 Human Fetal Human Fetal Skin Uni-ZAP
Epithelium Skin (Skin) XR
H0083 HUMAN JURKAT Jurkat Cells Uni-ZAP
MEMBRANE BOUND XR
POLYSOMES
H0086 Human epithelioidEpithelioid Sk Muscle diseaseUni-ZAP
sarcoma Sarcoma, XR
muscle H0087 Human Th mus Human Th Bluescri mus t H0090 Human T-Cell T-Cell LymphomaT-Cell diseaseUni-ZAP
Lymphoma XR
HO100 Human Whole Human Whole Embryo Uni-ZAP
Six Week Six Old Emb o Week Old XR
Embr o HO101 Human 7 Weeks Human Whole Embryo Lambda Old 7 Embryo, subtractedWeek Old ZAP II
Embryo HOIOS Human Fetal Human Fetal Heart pBluescript Heart, Heart subtracted H0116 Human Thymus Human ThymusThymus pBluescript Tumor, subtracted Tumor H0122 Human Adult Human SkeletalSk Muscle Uni-ZAP
Skeletal -Muscle Muscle XR
HOI23 Human Fetal Human Fetal Brain Uni-ZAP
Dura Mater Dura Mater XR
H0124 Human Human Sk Muscle diseaseUni-ZAP
Rhabdom osarcomaRhabdom osarcoma XR
H0125 Cem cells cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line treated Treated Cem, XR
Jurkat, Ra'i, and Su t H0130 LNCAP untreatedLNCAP Cell ProstateCell Uni-ZAP
Line Line XR
H0131 LNCAP + o.3nM LNCAP Cell ProstateCell Uni-ZAP
81881 Line Line XR
H0134 Raji Cells, CyclohexamideBlood Cell Uni-ZAP
cyclohexamide Line treated Treated Cem, ' XR
Jurkat, Ra'i, and ' Su t H013S Human Synovial Human Synovial_ Uni-ZAP
Sarcoma Synovium Sarcoma XR
H0141 Activated T-Cells,Activated Blood Cell Uni-ZAP
12 hrs. T-Cells Line XR
H0144 Nine Week Old 9 Wk Old Embryo Uni-ZAP
Early Early -Sta a Human Sta~e Human XR
H0149 7 Week Old EarlyHuman Whole Embryo Uni-ZAP
Stage 7 Human, subtractedWeek Old XR
Embr o HO1S0 Human EpididymusEpididymis Testis Uni-ZAP
XR
H0156 Human Adrenal Human AdrenalAdrenal diseaseUni-ZAP
Gland Tumor Gland Tumor Gland XR
H0163 Human Synovium Human SynoviumSynovium Uni-ZAP
XR
H0169 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer, Stage C fractionCancer, stage - XR
C
H0170 12 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old Human Earl Sta~e XR
Human H0171 12 Week Old Twelve Week Emb o Uni-ZAP
Earl Stage Old Human, II Earl Sta~e XR .
Human H0179 Human NeutrophilHuman NeutrophilBlood Cell Uni-ZAP
Line XR
H0181 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast Cancer Breast Cancer XR
H0188 Human Normal Human NormalBreast Uni-ZAP
Breast Breast _ XR
H0192 Cem Cells, cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line treated, subtraTreated Cem, ~ XR
Jurkat, Ra'i, and _ Su t H0194 Human Cerebellum,Human CerebellumBrain . pBluescript subtracted H0205 Human Colon Human Colon Colon pBluescript Cancer, differential Cancer H0208 Early Stage Human Fetal Lung pBluescript Human Lung, Lung subtracted H0213 Human Pituitary,Human Pituitary Uni-ZAP
subtracted , XR
' H0214 Raji cells, CyclohexamideBlood Cell pBluescript cyclohexamide Line treated, subtractedTreated Cem, Jurkat, Ra'i, and Su t H0231 Human Colon, Human Colon Bluescri subtraction t H0244 Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old Emb o, subtractedEmbr o XR
H0251 Human ChondrosarcomaHuman Cartilage diseaseUni-ZAP
Chondrosarcoma XR
H0252 Human OsteosarcomaHuman Bone diseaseUni-ZAP
Osteosarcoma XR
H0253 Human adult Human Adult Testis Uni-ZAP
testis, large Testis inserts XR
H0255 breast lymph Breast LymphLymph Lambda node CDNA Node Node libr ZAP II
H0261 H: cerebellum, Human CerebellumBrain Uni-ZAP
Enzyme , subtracted XR
H0264 human tonsils Human TonsilTonsil ~ Uni-ZAP
XR
H0265 Activated T-CellT-Cells Blood Cell Uni-ZAP
Line ( l2hs)/Thiouridine _ XR
labelledEco H0266 Human MicrovascularHMEC Vein Cell Lambda Line Endothelial ZAP II
Cells, Tract.
A
H0267 Human MicrovascularHMEC Vein Cell Lambda Line Endothelial ZAP II
Cells, fract.
B
H0270 HPAS (human Human PancreasPancreas Uni-ZAP
pancreas, subtracted) XR
H0271 Human Neutrophil,Human NeutrophilBlood Cell Uni-ZAP
- Line Activated Activated XR
H0284 Human OB MG63 Human Bone Cell Uni-ZAP
control Line fraction I Osteoblastoma XR
MG63 cell line H0286 Human OB MG63 Human Bone Cell Uni-ZAP
treated Line ( 10 nM E2) Osteoblastoma XR
fraction I
MG63 cell line H0288 Human OB HOS Human Bone Cell Uni-ZAP
control Line fraction I Osteobiastoma XR
HOS
cell line H0290 Human OB HOS Human Bone Cell Uni-ZAP
treated Line ( 1 nM E2) fractionOsteoblastoma XR
I HOS
cell line H0294 Amniotic Cells Amniotic PlacentaCell Uni-ZAP
- TNF Cells - Line induced TNF induced XR
H0295 Amniotic Cells Amniotic PlacentaCell Uni-ZAP
- Primary Cells - Line I Culture Prima Culture XR
H0309 Human Chronic Synovium, Synovium diseaseUni-ZAP
Synovitis Chronic Synovitis/ XR
Osteoarthritis H0316 HUMAN STOMACH Human StomachStomach Uni-ZAP
XR
H0318 HUMAN B CELL Human B CellLymph diseaseUni-ZAP
Node LYMPHOMA L m homa H0327 human corpus Human CorpusBrain Uni-ZAP
colosum Callosum XR
H0328 human ovarian Ovarian CancerOvary diseaseUni-ZAP
cancer _ XR
H0329 DermatofibrosarcomaDermatofibrosarcomSkin diseaseUni-ZAP
Protuberance a Protuberans XR
H0331 Hepatocellular HepatocellularLiver diseaseLambda Tumor .
Tumor ' ZAP II
H0333 HemangiopericytomaHemangiopericytomBlood diseaseLambda vessel a ZAP II
H0334 Kidney cancer Kidney CancerKidney diseaseUni-ZAP
XR
H0341 Bone Marrow Bone Marrow Bone Cell Uni-ZAP
Cell Line Cell Marrow Line (RS4; I 1 j Line R_S4;11 XR
H0352 wilm"s tumor Wilm"s Tumor diseaseUni-ZAP_ XR
H0355 Human Liver Human Liver, , pCMVSport normal Adult 1 H0369 H. Atrophic Atrophic Uni-ZAP
Endometrium Endometrium XR
and myometrium H0370 H. Lymph node Lymph node . diseaseUni-ZAP
breast with Cancer Met. Breast XR
Cancer H0386 Leukocyte and Human LeukocytesBlood Cell pCMVSport Lung; 4 , Line screens I
H0391 H. Meniin~ima, Human MeninQimabrain S ortl H0392 H. Menin~ima, Human MeninQimabrain S ortl MI
H0393 Fetal Liver, Human Fetal Liver Bluescri subtraction Liver t II
H0402 CD34 depleted CD34 DepletedCord ZAP Express Buffy Coat Blood (Cord Blood), Buffy Coat re-excision (Cord Blood) H0409 H. Striatum Human Brain,Brain pBluescript Depression, subtracted Striatum Dl: ression H0411 H Female Bladder,Human FemaleBladder pSportl Adult Adult Bladder H0412 Human umbilicalHUVE Cells 'UmbilicalCell pSportl vein Line endothelial vein cells, IL-4 induced H0413 Human UmbilicalHUVE Cells UmbilicalCell pSportl Vein Line Endothelial vein Cells, uninduced H0416 Human Neutrophils,Human NeutrophilBlood Cell pBluescript - Line Activated, re-excisionActivated H0418 Human Pituitary,Human Pituitary pBluescript subtracted VII
H0421 Human Bone Marrow,Bone Marrow pBluescript re-excision H0423 T-Cell PHA 24 T-Cells Blood Cell pSportl hrs Line H0424 Human Pituitar Human Pituita Bluescri ,~subt IX t H0427 Human Adipose Human A_ pSportl dipose, left hi 1i oma H0428 Human Ovary _ Ovary pSportl Human Ovary Tumor H0434 Human Brain, Human Brain, Bluescri striatum, t re-excision Striatum H0435 Ovarian Tumor Ovarian Tumor,_ Ovary pCMVSport OV3S0721 2.0 H0436 Resting T-Cell T-Cells Blood Cell pSportl.
Library,II Line H0438 H. Whole Brain Human Whole ZAP Express #2, re- Brain excision #2 H0441 H. Kidney Cortex,Kidney cortexKidney _ pBluescript subtracted H0445 Spleen, Chronichuman Spleen,Spleen diseasepSportl CLL
1 m hoc tic leukemia _ H0448 Salivary gland,Human SalivarySalivary Lambda subtracted Gland gland ZAP II
H0455 H. Striatum Human Brain,Brain pBluescript Depression, subt Striatum De ression H0457 Human Eosino Human Eosino S ortl hils hils H0483 Breast Cancer Breast Cancer pSportl cell line, Cell MDA 36 line, MDA
H0484 Breast Cancer Breast Cancer pSportl Cell line, Cell angiogenic line, Angiogenic, , H0486 Hodgkin"s LymphomaHodgkin"s diseasepCMVSport II
L m homa 2.0 II
H0488 Human Tonsils, Human Tonsils ~ pCMVSport Lib 2 2.0 H0494 Keratinocyte Keratinocyte pCMVSport , 2.0 H0497 HEL cell line HEL cell HEL pSportl-line 92.1.7 H0505 Human Astroc Human Astroc S ortl to to H0506 Ulcerative ColitisColon Colon S ortl H0509 Liver, HepatomaHuman Liver,Liver diseasepCMVSport He atoma, 3.0 atient 8 H0510 Human Liver, Human Liver,Liver pCMVSport normal normal, Patient ' 3.0 # 8 H0518 pBMC stimulatedpBMC stimulated pCMVSport w/ poly I/C with of IIC 3.0 H0519 NTERA2, controlNTERA2, pCMVSport Teratocarcinoma 3.0 cell line H0520-NTERA2 + retinoicNTERA2, ' pSportl acid, 14 days Teratocarcinoma cell line H0521 Primary DendriticPrimary Dendritic pCMVSport Cells, lib 1 cells 3.0 H0522 Primary DendriticPrimary Dendritic , pCMVSport cells,frac 2 cells 3.0 H0529 Myoloid ProgenitorTF-1 Cell pCMVSport Cell Line;
Line Myoloid progenitor 3.0 ' cell line H0538 Merkel Cells Merkel cellsL m h S octl node H0539 Pancreas Islet Pancreas Pancreas diseasepSportl Cell Tumor Islet Cell Tumour H0542 T Cell helper Helper T pCMVSport I cell 3.0 H0543 T cell helper HelperT cell pCMVSport II
3.0 H0544 Human endometrialHuman endometrial pCMVSport stromal cells stromal cells 3.0 H0545 Human endometrialHuman endometrial ' pCMVSport stromal cells-treatedstromal cells-treated 3.0 with ro~esterone with roe H0546 Human endometrialHuman endometrial CMVS
ort stromal cells-treatedstromal cells-treated~ 3.0 avith estradiol ~ with estra -H0547 NTERA2 teratocarcinomaNTERA2, pSportl cell line+retinoicTeratocarcinoma acid ( 14 da s) cell line H0549 H. Epididiymus,Human Uni-ZAP
caput &
corpus Epididiymus, XR
captit and co us H0550 H. Epididiymus,Human Uni-ZAP
cauda E ididiymus, XR
cauda H0551 Human Thymus Human Thymus pCMVSport Stromal Cells Stromal Cells 3.0 H0553 Human Placenta Human Placenta pCMVSport 3.0 H0555 Rejected Kidney,Human Rejected. Kidney diseasepCMVSport lib 4 Kidne 3.0 H0556 Activated T- T-Cells Blood Cell Uni-ZAP
Line cell( 12h)/Thiouridine-re- XR
excision H0559 PMA 4H, re- HL-60 Cells,Blood Cell Uni-ZAP
HL-60 PMA Line , stimulated XR
excision 4H
H0560 KMH2 , KMH2 pCMVSport 3.0 H0561 L428 L428 pCMVSport 3.0 H0562 Human Fetal Human Fetal pCMVSport Brain, Brain normalized c5-11-26 2.0 H0572 Human Fetal Human Fetal pCMVSport Brain, Brain normalized AC5002 2.0 H0574 Hepatocellular ' HepatocellularLiver , diseaseLambda Tumor; re- .
excision Tumor ZAP II
H0575 Human Adult Human Adult Lung Uni-ZAP
Pulmonar ;re-excisionPulmonar XR
H0576 Resting T-Cell;T-Cells Blood Cell ~ Lambda re- Line ZAP II
excision H0579 Pericardium Pericardium Heart S ortl ' H0580 Dendritic cells,Pooled dendritic pCMVSport pooled cells 3.0 H0581 Human Bone Marrow,Human Bone Bone Marrow pCMVSport treated Marrow 3.0 H0583 B Cell lymphomaB Cell LymphomaB Cell diseasepCMVSport . ~
3.0 H0586 Healing groin healing groingroin diseasepCMVSport wound, 6.5 hours post incisionwound, 6.5 f 3.0 hours ost incision H0587 Healing groin Groin-2!19/97groin diseasepCMVSport wound; 7.5 hours ost incision 3.0 .
H0590 Human adult Human Adult Small .
small Small Int. Uni-ZAP
intestine,re-excisionIntestine XR
H0591 Human T-cell T-CeII LymphomaT-Cell diseaseUni-ZAP
lymphoma;re-excision XR
H0592 Healing groin HGs wound diseasepCMVSport wound- healing zero hr post-incisionproject; , 3.0 abdomen (control) H0593 Olfactory Olfactory pCMVSport epithelium epithelium;nasalcavityfrom roof - 3.0 of left nasal cacit H0594 Human Lung Cancer;re-Human Lung Lung diseaseLambda Cancer ZAP II
excision H0595 Stomach cancer Stomach Cancer diseaseUni-ZAP
= , (human);re-excision5383A (human) XR
H0596 Human Colon Human Colon Colon ' Lambda Cancer;re-excision ' _ Cancer ZAP II
HOS98 Human Stomach;re-Human StomachStomach Uni-ZAP
excision ' XR
HOS99 Human Adult Human Adult Heart . Uni-ZAP
Heart;re- Heart excision XR
H0600 Healing AbdomenAbdomen diseasepCMVSport wound;70&90 3.0 min post incision _ H0601 Healing AbdomenAbdomen diseasepCMVSport Wound;l5 days 3.0 post incision H0606 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast Cancer;re-excisionBreast Cancer XR
H0610 H. Leukocytes, H.Leukocytes pCMVSport normalized cot 1 SA
H0616 Human Testes, Human TestesTestis Uni-ZAP
Reexcision XR
H0617 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast-Cancer ReexcisionBreast Cancer XR
H0618 Human Adult Human Adult Testis , Uni-ZAP
Testes, Testis ' Lame Inserts, XR
Reexcision H0619 Fetal Heart Human Fetal Heart Uni-ZAP
Heart XR .
HOf20 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney; Kidney Reexcision XR
H0622 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor;
Reexcision Tumor ' XR
H0623 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein;
Reexcision Vein Endothelialvein XR
Cells H0624 12 Week Early Twelve Week Embryo Uni-ZAP
Stage Old Human II; ReexcisionEarly Stage XR
Human H062S Ku 812F BasohilsLineKu 812F Baso S ortl hits H0626 Saos2 Cells; 5aos2 Cell pSportl Untreated Line;
Untreated H0627 Saos2 Cells; Saos2 Cell pSportl Vitamin D3 Line;
Treated Vitamin D3 Treated H0628 Human Pre-DifferentiatedHuman Pre- Uni-ZAP
Adipocytes Differentiated ' XR
Adipocytes .
H0631 Saos2, DexamethosomeSaos2 Cell pSportl Line;
Treated Dexamethosome Treated H0632 Hepatocellular HepatocellularLiver Lambda Tumor;re-excision Tumor ZAP II
H0633 Lung Carcinoma TNEalpha diseasepSportl A549 activated TNFalpha activatedAS49--Lung Carcinoma H0634 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor, re-excision Tumor XR
H063S Human ActivatedActivated Blood. Cell Uni-ZAP
T-Cells, T-Cells Line re-excision XR
H0638 CD40 activated CD40 activated pSportl monocyte dendridic cellsmonocyte dendridic cells H0641 LPS activated LPS activated , pSportl derived ' dendritic cellsmonocyte d derive dendritic cells H0644 Human Placenta Human PlacentaPlacenta Uni-ZAP
(re- .
excision) XR
H064S Fetal Heart, human Fetal Heart . Uni-ZAP
re-excision Heart XR
i H0646Lung, Cancer Metastatic pSportl (4005313 A3): 'Invasive squamous Poorly cell lung Differentiated carcinoma, Lung poorly di Adenocarcinoma, H064$ Ovary, Cancer: Papillary diseasepSportl (4004562 Cstic B6) Papillary neoplasm , Serous of low Cystic Neoplasm,malignant Low potentia Ivlali~nant Pot H0649 Lung, Normal: Normal Lung pSportl (4005313 Bl) H0650 B-Cells B-Cells pCMVSport 3.0 H06S Ovary, Normal: Normal Ovary pSporti (9805C040R) H06S2 Lung, Normal: Normal Lung pSportl (4005313 B1) H0653 Stromal Cells Stromal Cells S ortl H0656 B-cells (unstimulated)B-cells pSportl (unstimulated) H0657 B-cells (stimulated)B-cells (stimulated) S ortI
H0658 Ovary, Cancer 9809C332- Ovary diseasepSportl Poorly &
(9809C332): differentiateFallopian Poorly differentiated Tubes adenocarcinoma H0659 Ovary, Cancer Grade II ' Ovary ' , diseasepSportl - ' Papillary (15395A1F): Carcinoma, Grade II Ovary Papillary Carcinoma' H0660 Ovary, Cancer: Poorly differentiated diseasepSportl (15799A1F)Poorlycarcinoma, ovary differentiated carcinoma H0661 Breast, Cancer:Breast cancer diseasepSportl (4004943 AS) H0662 Breast, Normal:Normal BreastBreast pSportl -(400552282) #4005522(B2) H0664 Breast, Cancer:Breast CancerBreast diseasep5portl (9806C012Rj H0665 Stromal cells Stromal cells 5 ortl 3.88 3.88 H0667 Stromal cells(HBM3.18)Stromal cell(HBM pSportl 3.18) H0668 stromal cell stromal cell pSportl clone 2.5 clone 2.5 H0670 Ovary, Cancer(4004650Ovarian Cancer- pSportl A3): Well-Differentiated4004650A3. ' .
Micropapillary Serous Carcinoma H0672 Ovary, Cancer: Ovarian Ovary ' pSportl (4004576 Ag) Cancer(4004576A8) H0673 Human Prostate Human ProstateProstate Uni-ZAP
Cancer, Stage B2; re-excisionCancer, stage XR
H0674 Human Prostate Human ProstateProstate Uni-ZAP
Cancer, . Stage C; re-excissionCancer, stage XR
C
H0675 Colon, Cancer: Colon Cancer pCMVSport (9808C064R) 9808C064R 3.0 H0677 TNFR degenerateB-Cells PCRII
olio H0682 Serous Papillaryserous papillary pCMVSport Adenocarcinoma adenocarcinoma 3.0 (9606G304SPA3B) H0683 Ovarian Serous Serous papillary pCMVSport Papillary Adenocarcinoma adenocarcinoma, 3.0 stage 3C
(9804601 H0684 Serous PapillaryOvarian Cancer-Ovaries pCMVSport Adenocarcinoma 98106606 3.0 H0686 Adenocarcinoma Adenocarcinoma pCMV
of of Sport Ovary, Human Ovary, Human 3.0 Cell Line Cell Line, # SW-626 H0687 Human normal Human normalOvary pCMVSport ova (#9610G21S)ova (#9610G21S) 3.0 H0688 Human Ovarian Human Ovarian pCMVSport Cancer(#98076017)cancer(#9807G017), 3.0 mRNA from Maura Ru H0689 Ovarian Cancer Ovarian Cancer, pCMVSport #98066019 3.0 H0690 Ovarian Cancer,Ovarian Cancer, pCMVSport #
97026001 #97026001 - 3.0 H0693 Normal ProstateNormal Prostate pCMVSport #ODQ3958EN Tissue # 3.0 H069S mononucleocytesmononucleocytes pCMVSport from patient from patient 3.0 at Shad Grove Hos it S0001 Brain frontal Brain frontalBrain Lambda cortex cortex ZAP II
50002 Monocyte activatedMonocyte-activatedblood CeILLine Uni-ZAP
XR
S0003 Human OsteoclastomaOsteoclastomabone diseaseUni-ZAP
XR
SOOOS Heart Heart-left Heart CDNA
ventricle 50007 Early Stage Human Fetal Uni-ZAP
Human Brain Brain XR' S0010 Human Amygdala Amygdala Uni-ZAP
' XR
50011 STROMAL - Osteoclastomabone diseaseUni-ZAP
.
OSTEOCLAS'TOMA XR
50022 Human OsteoclastomaOsteoclastoma Uni-ZAP
Stromal Cells Stromal Cells XR
-unamplified S0026 Stromal cell stromal cellBone Cell Uni-ZAP
TF274 marrow Line XR
S0027 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
serum Line treated arter XR
S0028 Smooth muscle,controlSmooth musclePulmanaryCell Uni-ZAP
Line arter XR
50029 brain stem Brain stem brain Uni-ZAP
XR
50030 Brain pons Brain Pons Brain Uni-ZAP
XR
S0031 Spinal cord Spinal cord spinal Uni-ZAP
cord XR
S0032 Smooth muscle-ILbSmooth musclePulmanaryCell Uni-ZAP
Line induced arter XR
50036 Human SubstantiaHuman Substantia Uni-ZAP
Nigra Ni ~ra XR
S0037 Smooth muscle, Smooth muscle.PulmanaryCell Uni-ZAP
IL16 Line induced arter XR
50038 Human Whole Human Whole ZAP Express Brain #2 - Brain Oli o dT > 1.SKb#2 50040 Adipocytes , Human Adipocytes , Uni-ZAP
from Osteoclastoma XR
S004S Endothelial Endothelial endothelialCell ~ Uni-ZAP
cells-control cell Line cell-lun XR
S0046 Endothelial-inducedEndothelial endothelialCell Uni-ZAP
cell Line cell-lung XR
50049 Human Brain, Human Brain, Uni-ZAP
Striatum Striatum ~ XR
S0051 Human Human diseaseUni-ZAP
Hypothalmus,SchizophrenHypothalamus,, . XR
is Schizo hrenia 50052 neutrophils human neutrophilsblood Cell Uni-ZAP
control Line XR
S0053 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS Line induced induced XR
50112 Hypothalamus Brain Uni-ZAP
XR
S0114 Anergic T-cell Anergic T-cell Cell Uni-ZAP
Line XR
50116 Bone marrow Bone marrow Bone marrow Uni-ZAP
XR
S0126 Osteoblasts Osteoblasts Knee Cell Uni-ZAP
Line XR' 50132 Epithelial-TNFaAirway Epithelial Uni-ZAP
and INF
induced XR
50134 Apoptotic T-cellapoptotic Cell Uni-ZAP
cells Line XR
50144 Macrophage (GM-CSFMacrophage Uni-ZAP
(GM-treated) CSF treated) ~ XR
S0146 prostate-editedprostate Prostate Uni-ZAP
BPH
XR
SO150 LNCAP prostate LNCAP Cell Prostate Cell Uni-ZAP
cell line Line Line XR
50152 PC3 Prostate PC3 prostate Uni-ZAP
celi line cell line XR
S0174 Prostate-BPH Human Prostate pBluescript subtracted II
BPH
S0182 Human B Cell Human B- Uni-ZAP
8866 Cell 8866 XR
50192 Synovial FibroblastsSynovial pSportl Fibroblasts (control ) S0194 S novial h oxiaS novial S ortl Fibroblasts 50196 Synovial IL-1/TNFSynovial pSportl Fibroblasts , stimulated S0206 Smooth Muscle- Smooth musclePulmanaryCell pBluescript HASTE Line normalized arter S0210 MessanQial cell,MessanQial S ortl frac 2 cell 50212 Bone Marrow Bone Marrow pSportl Stromal Cell, untreatedStromal Cell ,untreated S0216 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS ~ Line induced induced ~ XR
S0222 H. Frontal H. Brain, Brain diseaseUni-ZAP
Frontal cortex,epileptic;re-Cortex, Epileptic XR
excision S0242 Synovial FibroblastsSynovial p5portl Fibroblasts (I11/TNF), subt 50250 Human OsteoblastsHuman OsteoblastsFemur diseasepCMVSport II
2.0 S0260 Spinal Cord, Spinal cord spinal Uni-ZAP
re-excision cord XR
50276 Synovial hypoxia-RSFSynovial Synovial pSportl fobroblasts subtracted (rheumatoid)tissue S0278 H Macrophage Macrophage Uni-ZAP
(GM-CSF (GM-treated),re-excisionCSF treated) XR
S0280 Human Adipose Human Adipose Uni-ZAP
Tissue, re-excision Tissue XR
50282 Brain Frontal Brain frontalBrain Lambda Cortex, re- cortex ZAP II
excision 50294 Larynx tumor Larynx tumorLarynx,vocal diseasepSportl cord 50300 Frontal lobe,dementia;re-Frontal LobeBrain Uni-ZAP
excision . dementia/Alzheimer' XR
's S0312 Human Human diseasepSportl osteoarthritic;fractionosteoarthritic' ' II
cartilage S0316 Human Normal Human Normal p5portl Cartilage,FractionCartilage I
S0328 Palate carcinomaPalate carcinomaUvula diseaseSor_tl _ S0330 Palate normal Palate normalUvula S ortl S0332 Pha nx carcinomaPhar nx carcinomaH o har S ortl nx S0338 Human OsteoarthriticHuman diseasepSportl Cartilage Fractionosteoarthritic III
cartilage S0340 Human OsteoarthriticHuman diseasepSportl Cartilage Fractionosteoarthritic IV
cartilage ' S0342 Adipocytes;re-excisionHuman Adipocytes Uni-ZAP
from Osteoclastoma XR
50344 Macrophage-oxLDL;macrophage- blood Cell - Uni-ZAP
re- Line excision oxidized , XR
LDL
treated 50346 Human Amygdala;re-Amygdala Uni-ZAP
excision XR
S0354 Colon Normal Colon NormalColon S ortl II
50356 Colon CarcinomaColon CarcinomaColon diseaseS ortl 50358 Colon Normal Colon NormalColon ' _S o_rtl III
S0360 Colon Tumor Colon Tumor Colon disease_S ortl II
S0364 Human Quadrice Quadrice pSportl s s muscle S0366 Human Soleus Soleus Muscle S ortl S0370 Lar nx carcinomaLar nx carcinoma diseaseS ortl II
S0374 Normal colon Normal colon S ortl S0376 Colon Tumor Colon Tumor diseaseS ortl S0378 Pancreas normalPancreas pSportl PCA4 Normal No PCA4 No S0380 Pancreas Tumor Pancreas diseasepSportl PCA4 Tu Tumor PCA4 Tu S0386 Human Whole Whole brain Brain ZAP Express Brain, re-excision ' S0388 Human Human . diseaseUni-ZAP
Hypothalamus,schizophreHypothalamus, XR
re-excision Schizo hrenia .
nia, 50390 _ Smooth musclePulmanaryCell Uni-ZAP
Smooth muscle, Line control;
re-excision arter XR
~
50392 Salivary Gland Salivary pSportl , gland;
normal 50400 Brain; normal Brain; normal S ortl 50404 Rectum normal Rectum, normal S ortl 50406 Rectum tumour Rectum tumour S ortl S0408 Colon, normal Colon, normal 5 ortl S0410 Colon,tumo Colon, tumour S ortl ur 50418 _ CHME Cell pCMVSport CHME Cell Line;treatedLine;
hrs treated 3.0 S0420 CHME Cell . CHME Cell pSportl line, .
Line,untreated untreatetd ' S0422 Mo7e Cell Line Mo7e Cell pCMVSport GM-CSF Line treated (lng/ml)GM-CSF treated 3.0 ( 1 n~/ml) S0424 TF-1 Cell Line TF-1 Cell pSportl GM-CSF Lane Treated GM-CSF Treated 50426 Monocyte activated;Monocyte-activatedblood, Cell Uni-ZAP
re- Line excision XR
50428 Neutrophils human neutrophilsblood Cell Uni-ZAP
control; re- Line ' excision XR
50434 Stomach Normal Stomach Normal diseaseS ortl 50436 StomachTumour Stomach Tumour diseaseS ortl ~
S0438 Liver Normal Liver Normal p5portl MetSNo MetSNo S0440 Liver Tumour Liver Tumour S ortl Met 5 Tu S0442 Colon Normal Colon Normal S ortl 50444 Colon Tumor Colon Tumour diseaseS ortl S0456 Ton~ue Normal Ton~ue Normal S ortl S0458 Thyroid Normal Thyroid normal pSportl (SDCA2 No) S0460 Th roid Tumour Th roid Tumour S ortl S0468 Ea.h .926 cell Ea.h .926 S ortl line cell Tina S0472 Lun MesotheliumPYBT S ortl S0474 Human blood Platelets Blood Other platelets .
platelets S3012 Smooth Muscle Smooth musclePulmanaryCell ~ pBluescript Serum Line Treated, Norm arter ' S3014 Smooth muscle, Smooth musclePulmanaryCell pBluescript serum Line induced,re-exc arter 56016 H. Frontal Cortex,H. Brain, Brain diseaseUni-ZAP
Frontal E ile tic Cortex, E ~ XR
ile tic S6022 H. Adipose TissueHuman Adipose Uni-ZAP
Tissue XR
S6024 Alzheimers, Alzheimer"s/SpongyBrain diseaseUni-ZAP
spongy chan a chance XR
S6026 Frontal Lobe, Frontal LobeBrain Uni-ZAP
Dementia dementialAlzheimer' XR
's S6028 Human Manic Human Manic Brain diseaseUni-ZAP
Depression Tissue de ression XR
tissue T0003 Human Fetal Human Fetal pBluescript Lung Lung SK-.
T0004 Human White Human White pBluescript Fat Fat SK-T0006 Human Pineal Human Pinneal pBluescript Gland Gland SK-T0010 Human Infant Human Infant Other Brain Brain T0039 HSA 172 Cells Human HSA ~ pBluescript 172 cell line SK-T0040 HSC172 cells SA172 Cells pBluescript SK-T0041 JurkatT-cell JurkatT-cell pBluescript G1 phase SK-T0048 Human Aortic Human Aortic' pBluescript .
- Endothelium Endothilium ~ SK-T0049 Aorta endothelialAorta endothelial pBluescript cells +
TNF-a cells SK-T0060 Human White Human White pBluescript Adipose Fat SK-T0067 Human Thyroid Human Thyroid pBluescript SK-T0068 NormalOvary, Normal Ovary, pBluescript Premeno ausal Premeno ausal SK-T0069 Human Uterus, Human Uterus, pBluescript ~ normal normal SK-T0082 Human Adult Human Adult pBluescript Retina Retina SK-.
T0109 ~ Human (HCC) Bluescript ~ cell line liver (mouse) SK-metastasis, remake TO Human colon pBtuescript I carcinoma IO
(HCC) cell line, SK-remake L0004 ClonTech HL
1065a L0005 Clontech human aorta of A+ mRNA (#6572) L0021 Human adult (K.Okubo) L0022 Human adult .
lung 3"
directed MboI
cDNA
L0103 DKFZ ham 1 am ~dala L0109 Human brain brain cDNA
L0142 Human placenta placenta cDNA
(TFu'iwara) L0143 Human placenta placenta polyA+
(TFu'iwara) _ Human fetal brain L0157 brain (TFu'iwara) L0163 Human heart . heart cDNA
(YNakamura) .
L0351 Infant brain, , BA, M13-Bento Soares derived L0352 Normalized infant BA, M
brain, 13-Bento Soares derived L0362 Stratagene ovarian Bluescript cancer (#937219) SK-_ L0364 NCI_CGAP_GC5 germ cell Bluescript tumor SK-L0366 Stratagene schizoschizophrenic Bluescript brain brain S11 ' S-I1 frontal SK-lobe L0367 NCI CGAP Schl Schwannoma Bluescript _ _ tumor SK- .' L0368 NCI_CGAP_SS synovial Bluescript 1 sarcoma SK-L0369 NCI CGAP_AA1 adrenal adenomaadrenal Bluescript' -' gland SK-L0370 Johnston frontalpooled frontalbrain Bluescript cortex lobe SK-L0372 NCI_CGAP_Col2 colon tumor colon Btuescript SK-L0374 NCI CGAP_Co2 tumor colon Bluescript SK-L0375 NCI CGAP_Kid6 kidney tumorkidney Bluescript -SK-L0378 NCI_CGAP_Lul lung tumor lung Bluescript SK-L0381 NCI_CGAP_HN4 ' squamous pharynx Bluescript cell carcinoma SK-L0383 NCI_CGAP_Pr24 invasivetumor(cellprostate Bluescript line) ' SK-L0384 NCI_CGAP_Pr23 prostate prostate Bluescript tumor ~
SK-L0438 normalized infanttotal brain brain lafmid brain BA
cDNA
L0439 Soares infant whole~brain Lafmid brain 1NIB BA
L0455 Human retina retina eye lambda cDNA gtl0 randomly primed sublibrary L0456 Human retina retina eye lambda cDNA gtl0 Tsp509I-cleaved subtibrary L0471 Human fetal Lambda heart, Lambda ZAP Ex ~ ZAP Ex ress ress L0480 Stratagene cat#937212 Lambda (1992) ZAP, p$luescript SK(-) L0~183Human pancreatic Lambda islet ZAPII
L0485 STRATAGENE Humanskeletal leg muscle Lambda muscle skeletal muscle ZAPII
cDNA
library, cat.
#936215.
L0517 NCI_CGAP_Prl AMP10 L0518 NCI_CGAP Pr2 AMP10 ~
L0519 NCI CGAP_Pr3 - AMP10 L0520 NCI_CGAP_Alvl alveolar pAMPlO
rhabdomyosarcoma L0521 NCI_CGAP_Ervl Ewin~"s sarcoma AMP10 L0522 NCI CGAP_Kidl kidne AMP10 L0526 NCI_CGAP_Prl2 metastatic pAMPlO
prostate bonelesion L0527 NCI_CGAP_Ov2 ovary pAMPlO
L0533 NCI_CGAP_HSC1 stem cells bone AMP10 marrow L0540 NCI_CGAP_PrlO invasive prostate pAMPlO
prostate tumor L0542 NCI_CGAP_PrII normal prostaticprostate pAMPlO
a ithelial cells LOS46 NCI_CGAP_Prl8 stroma rostate AMPIO
L0564 Jia bone marrowbone man-orv Bluescri stroma stroma t LOS65 Normal Human Bone . Hip pBluescript Trabecular Bone ' Cells ~
L0590 Stratagene fibroblast pBluescript ' (#937212) SK-L0591 Stratagene HeLa pBluescript cell s3 L0592 Stratagene hNT pBluescript neuron (#937233) SK-L0593 Stratagene pBluescript neuroepithelium SK-(#937231) L0595 Stratagene NT2 neuroepithelialbrain pBluescript neuronal cells ~
recursor 937230 SK-L0596 Stratagene colon colon pBluescript (#937204) ~ SK-L0597 Stratagene corneal cornea pBluescript stroma (#937222) ' SK-L0598 Morton Fetal cochlea ear pBluescript Cochlea SK-L0599 Stratagene lung lung p131uescript (#937210) SK-L0600 Weizmann Olfactoryolfactory nose ~ p$luescript epithelium E ithelium ' SK-L0601 Stratagene pancreas pancreas pBluescript (#937208) SK-L0602 Pancreatic Isletpancreatic pancreas~ pBluescript islet SK-L0603 Stratagene placenta placenta~ pBluescript (#937225) SK-L0604 Stratagene musclemuscle skeletal pBluescript muscle SK-L0605 Stratagene fetalfetal spleenspleen pBluescript spleen (#937205) SK-L0608 Stratagene lunglung carcinomalung NCI-H69 pBluescript carcinoma 937218 ~SK-I L0617Chromosome 22 pBluescriptII
exon KS+
L0622 HM1 pcDNAII
(Invitro~en) L0623 HM3 pectoral pcDNAII
muscle (after mastectom (Invitro~en) ) L0629 NCI_CGAP_Mel3 metastatic bowel pCMV-(skin melanoma rima SPORT4 to bowel ) L0631 NCI_CGAP_Br7 breast pCMV-L0635 NCI_CGAP_PNS1 dorsal root peripheral pCMV-ganglion nervous SPORT4 s stem L0636 NCI CGAP_Pitl four pooled brain pCMV-pituitary adenomas SPORT6 L0637 NCI_CGAP_Brn53 three pooledbrain pCMV-menin~iomas SPORT6 L0638 NCI_CGAP_Brn35 tumor, 5 brain pCMV-pooled (see descri lion) SPORT6 L0639 NCI_CGAP_Brn52 tumor, 5 brain pCMV-pooled (see descri lion) SPORT6 L0640 NCI_CGAP_Brl8 four pooled breast pCMV-high-grade tumors, SPORT6 includin two rima L0641 NCI_CGAP_Col7 juvenile colon pCMV-granulosa tumor ~ SPORT6 L0642 NCI_CGAP_CoI8 moderately colon pCMV-differentiated SPORT6 adenocarcinoma L0643 NCI_CGAP_Col9 moderately colon pCMV-differentiated' SPORT6 adenocarcinoma L0644 NCI_CGAP_Co20 moderately colon pCMV-differentiated SPORT6 adenocarcinoma L0646 NCI_CGAP_Col4 moderately- colon pCMV-differentiated SPORT6 adenocarcinoma L0647 NCI_CGAP_Sar4 five pooled connective pCMV-sarcomas, tissue SPORT6 including myxoid hposarcoma L06~18~NCI_CGAP Eso2 squamous esophagus pCMV-cell carcinoma SPORT6 L0649 NCI_CGAP_GLJ1 2 pooled genitourinary pCMV-' high-grade transitionaltract SPORT6 cell tumors L0650 NCI_CGAP_Kidl3 2 pooled kidney pCMV-, Wilms"
tumors, one SPORT6 primary and one metast L0651 NCI_CGAP Kid8 renal cell kidney pCMV-tumor L0653 CGAP_Lu28 two pooled lung , pCMV-NCI
_ squamous SPORT6 cell carcinomas L0654 NCI CGAP_Lu31 lung, pCMV-cell line L0655 CGAP_Lyml2 lymphoma, lymph pCMV-NCI node _ follicular SPORT6 mixed small and large cell L0656 CGAP_Ov38 normal epitheliumovary pCMV-NCI
_ SPORT6 L0657 CGAP_Ov23 tumor, S ovary ' pCMV-NCI pooled (see _ description) SPORT6 L0658 NCI_CGAP_Ov35 .tumor, 5 ovary pCMV-- pooled (see descri tion) SPORT6 L0659 NCI_CGAP_Panl . adenocarcinomapancreas pCMV-L0662 NCI_CGAP_Gas4 poorly differentiatedstomach pCMV-adenocarcinoma SPORT6 with signet r L0663 CGAP Ut2 moderately- uterus pCMV-NCI
_ differentiated SPORT6 endometrial adenocarcino L0664 CGAP_Ut3 poorly-differentiateduterus pCMV-NCI
_ endometrial SPORT6 adenocarcinoma, L0665 CGAP_Ut4 serous papillaryuterus pCMV-NCI
_ carcinoma, SPORT6 high Qrade, 2 ooled t L0666 CGAP_Utl well-differentiateduterus pCMV-NCI
_ endometrial SPORT6 adenocarcinoma, L0667 NCI_CGAP_CML1 myeloid cells,whole pCMV-18 blood pooled CML SPORT6 cases, BCRIABL rearra L0717 Gessler Wilms SPORTI
tumor L073I Soares_pregnant_uterus_' uterus pT7T3-Pac NbHPU
L0738 Human colorectal pT7T3D
cancer L0740 Soares melanocytemelanocyte pT7T3D
2NbHM (Pharmacia) with a modified of linker L0741 Soares adult - brain pTTT3D
brain N2b4HB55Y (Pharmacia) with a modified of linker L0742 Soares adult brain pTTT3D
brain N2b5HB55Y (Pharmacia) with a modified polyiinker L0743 Soares breast breast pTTT3D
2NbHBst (Pharmacia) - with a modified of linker L0744 Soares breast breast pTTT3D
3NbHBst (Pharmacia) with a . ~ modified of linker L0745 Soares retina retina eye pTTT3D
N2b4HR
(Pharmacia) with a modified of linker L0746 Soares retina retina eye pTTT3D
N2b5HR
(Pharmacia) with a modified of linker L0747 Soares fetal_heart heart T7T3D
NbHH
19W (Pharmacia) with a modified of linker L0748 Snares fetal Liver pTTT3D
liver spleen and 1NFLS Spleen (Pharmacia) with a modified of linker L0749 Soares_fetal Liver pTTT3D
liver_spleen and 1NFLS_S 1 Spleen (Pharmacia) with a modified of linker L0750 Snares fetal lung pTTT3D
lung_NbHLI
9W (Pharmacia) with a modified ' ~ of linker L07S1 Snares ovary ovarian tumorovary pTTT3D
tumor NbHOT ~ (Pharmacia) with a modified of linker L0752 Soares_parathyroidparathyroid parathyroid pTTT3D
tumor tumor NbHPA gland (Pharmacia) - with a modified polylinker .
L0753 5oares_pineal_gland_N3H pineal pTTT3D
gland pG (Pharmacia) with a modified of linker L0754 Snares placenta placenta pTTT3D
Nb2HP
(Pharmacia) with a modified of linker L0755 Soares_placenta_8to9wee~ placenta pTTT3D
ks_2NbHP8to9W (Pharmacia) with a modified polylinker L0756 Soares_multiplemultiple pTTT3D
sclerosis sclerosis 2NbHMSP lesions (Pharmacia) - with a . modified polylinker V_TYPE
L0757 Soares_senescent_fibroblasenescent pTTT3D
fibroblast sts NbHSF - (Pham~acia) - ' with a modified polylinker V_TYPE
L0758 Soares_testis pTTT3D-Pac NHT
(Pharmacia) with a modified of linker L0759 Soares_total_fetus_Nb2H pT7T3D-Pac F8 9w (Pharmacia) - with a ' modified - of linker L0761 NCI_CGAP_CLL1 B-cell, chronic pT7T3D-Pac lymphotic (Pharmacia) leukemia with a modified of linker L0762 NCI_CGAP_Brl.l breast pT7T3D-Pac (Pharmacia) with a modified of linker L0763 NCI_CGAP_Br2 breast pT7T3D-Pac (Pharmacia) with a modified of linker L0764 NCI_CGAP_Co3 colon pT7T3D-Pac (Pharmacia) vYith a modified of linker L0766 NCI_CGAP_GCB1 germinal ~ pT7T3D-Pac centerB
cell (Pharmacia) with a modified of linker LD767 NCI_CGAP_GC3 pooled germ pTTT3D-Pac cell tumors (Pharmacia) with a modified ' polylinker L0768 NCI_CGAP_GC4 pooled germ pT7T3D-Pac cell tumors . (Pharmacia) with a modified of linker L0769 NC1 CGAP_Brn25 anaplastic brain pT7T3D-Pac _ oligodendroglioma Pharmacia) ( with a modified of linker L0770 NCI_CGAP_Brn23 glioblastomabrain pTTT3D-Pac ~ .
(pooled) (Pharmacia) ' with a modified polylinker L0771 NCI_CGAP_Co8 adenocarcinomacolon pTTT3D-Pac (Pharmacia) with a modified of linker L0772 NCT_CGAP_CoIO colon tumor colon pT7T3D-Pac RER+ .
(Pharmacia) with a modified of linker L0773 NCI_CGAP_Co9 colon tumor colon pT7T3D-Pac RER+
(Pharmacia) with a modified polylinker L0774 NCI CGAP_Kid3 kidney pT7T3D-Pac (Pharmacia) with a modified of linker L0775 NCI_CGAP_KidS 2 pooled kidney pTTT3D-Pac tumors (clear cell (Pharmacia) type) with a modified of linker L0776 NCI_CGAP_Lu5 carcinoid lung pTTT3D-Pac (Pharmacia) with a modified of linker L0777 Soares_NhHMPu_SPooled humanmixed pTTT3D-Pac 1 (see melanocyte, below) (Pharmacia) fetal heart, and with pregnant a modified of linker L0778 Barstead pancreas pancreas pTTT3D-Pac HPLRB 1 (Pharmacia) . with a modified ' of linker L0779 Soares NFL T_GBC_S pooled pTTT3D-Pac -(Pharmacia) with a modified polylinker L0780 Soares NSF_F8_9W_OT pooled pTTT3D-Pac PA P_S1 (Pharmacia) with a modified of linker L0782 NCI CGAP Pr21 normal prostateprostate pTTT3D-Pac - (Pharmacia) with a modified of linker .
L0783 NCI CGAP_Pr22 normal prostateprostate pTTT3D-Pac ..
- (Pharmacia) with a - modified polylinker L0785 Barstead spleen spleen pTTT3D-Pac (Pharmacia) with a modified _ of linker L0786 Soares NbHFB whole pTTT3D-Pac brain - ~ (Pharmacia) with a modified of linker L0788 NCI_CGAP_Sub2 . pTTT3D-Pac (Pharmacia) with a modified of linker L0789 NCI_CGAP_Sub3 pTTT3D-Pac (Pharmacia) with a modified of linker L0790 NCI_CGAP_Sub4 pT7T3D-Pac ~
(Pharmacia) with a modified of linker L0791 NCI_CGAP~SubS pTTT3D-Pac (Phannacia) with a modified oI linker L0792 NCI_CGAP_Sub6 pT7T3D-Pac (Pharmacia) with a modified of linker L0793 NCI_CGAP_Sub7 ~ pTTT3D-Pac (Pharmacia) with a modified polylinker L0794 CGAP_GC6 pooled germ pT7T3D-Pac NCI cell _ tumors . (Pharmacia) with a modified of linker L0800 NCI CGAP Col6 colon tumor,colon pTTT3D-Pac RER+
- (Pharmacia) with a modified of linker L0803 NCI CGAP_Kid kidney pTTT3D-Pac - (Pharmacia) with a modified polylinker L0804 NCI_CGAP_Kidl2 2 pooled kidney pT7T3D-Pac tumors (clear cell (Pharmacia) type) with a modified of linker L0805 NCI CGAP_Lu24 carcinoid lung pT7T3D-Pac - ( . Pharmacia) with a modified of linker L0806 CGAP_Lul9 squamous lung pT7T3D-Pac NCI cell ~
_ carcinoma, (Pharmacia) poorly differentiated with (4 a modified ' ' of linker L0807 CGAP_Ov I8 fibrotheoma ovary pTTT3D-Pac NCI
_ (Pharmacia) with a modified of linker L0809 NCI CGAP Pr28 prostate pT7T3D-Pac - ~
(Pharmacia) with a modified of linker OMIM ~ Description Reference 120160 Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420, 120160 Osteo orosis, idio athic, 166710 120160 Ehlers-Danlos s ndrome, a VIIA2, 130060 120160 Marfan syndrome, aty ical 126650 Chloride diarrhea, con enital, Finnish e, 214700 126650 Colon cancer 129900 EEC s ndrome-1 147450 Am tro hic lateral sclerosis, due to SOD I deficienc , 105400 154276 Mali nant h erthermia susce tibili 3 173360 Thrombo hilia due to excessive lasmino en activator inhibitor 173360 Hemorrha is diathesis due to PAI1 deficienc 176261 Jervell and Lan e-Nielsen syndrome, 220400 183600 S lit hand/foot malformation, a 1 187680 6-merca to urine sensitivi 253270 Multi 1e carbox lase deficienc , biotin-res onsive 300000 O itz G s ndrome, a I
300066 . Deafness, X-linked 6, sensorineural 300077 Mental retardation, X-linked 29 300310 A aroma lobulinemia, a 2, X-linked 301220 Partin ton s ndrome II
302350 Nance-Horan s ndrome 304050 Aicardi s ndrome 3041 I0 Craniofrontonasal d s lasia 306100 Gonadal d s enesis, XY female a 309530 Mental retardation, X-linked I, non-dysmo hic 309585 Mental retardation, X-linked, syndromic-6, with gynecomastia and obesit ~
312040 N s ndrome, 310465 601 Platelet disorder, familial, with associated m 399 eloid mali nanc _ Refsum disease, infantile, 266510 602136 Zellwe er s ndrome-1, 214100 _ Adrenoleukod stro h , neonatal, 202370 602447 Coronary artery disease, susce tibilit to PolynZrcleotide and Polypeptide Variants [84] The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide sequences encoding the polypeptide of SEQ. ID NO:Y, the nucleotide sequence of SEQ ID
NO:X
encoding the polypeptide sequence as defined in column 7 of Table 1A, nucleotide sequences encoding the polypeptide as defined in column 7 of Table 1A, the nucleotide sequence as defined in columns 8 and 9 of Table 2, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9, of Table 2, the nucleotide sequence as defined in column 6, of Table 1B, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in column 6 of Table 1B, the cDNA sequence contained in Clone ID NO:Z, and7or nucleotide sequences encoding the polypeptide encoded by the cDNA sequence contained in Clone ID NO:Z. .
[85] The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y, the polypeptide sequence as defined in column 7 of Table 1A, a polypeptide sequence encoded by the polynucleotide .sequence. in SEQ ID NO:X, a polypeptide sequence encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as ,defined in column 6 of Table 1B, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO.:X, and/or a polypeptide sequence encoded by the cDNA
sequence contained in Clone ID NO:Z.
[86j "Variant" refers to a polynucleotide. or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are ovexall closely similar, arid, in many regions, identical to the polynucleotide or polypeptide of the present.invention.
[87] . Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting o~ (a) a nucleotide sequence described in SEQ ID NO:X
or contained in the cDNA sequence of Clone ID NO:Z; (b) a nucleotide sequence in SEQ
ID NO:X or the cDNA in Clone ID NO:Z which encodes the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid_sequence encoded by the cDNA in Clone ID
NO:Z; (c) a nucleotide sequence in SEQ ID NO:X or the cDNA in Clone ID NO:Z
which encodes a mature polypeptide; (d) a nucleotide sequence in SEQ ID NO:X or the cDNA
sequence of Clone ID NO:Z, which encodes a biologically active fragment of a polypeptide;
(e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of Clone ~ ID
NO:Z, which encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence encoding a polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDN.A in Clone ID NO:Z; (g) a nucleotide sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID
NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (h) a nucleotide sequence encoding a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (i) a nucleotide sequence encoding an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.
j88] The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (fj, (g), (h), (i), or (j) above, the nucleotide coding sequence in SEQ
ID NO:X or the complementary strand thereto, the nucleotide coding sequence of the cDNA
contained in Clone ID NO:Z or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding a,polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide.
sequence encoded by the complexrient of the polynucleotide sequence in SEQ ID NO:X, a nucleotide sequence encoding the. polypeptide encoded by the cDNA contained in Clone ID
NO:Z, the nucleotide coding sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, the nucleotide coding sequence in SEQ ID
NO:B as defined in column 6 of Table IB or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto, the nucleotide sequence in SEQ ID NO:X encoding the polypeptide sequence as defined in column 7 of Table 1A or the complementary strand thereto, nucleotide sequences encoding the polypeptide as defined in column 7 of Table 1A or the complementary strand thereto, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides and nucleic acids.
j89] In a preferred embodiment, the invention encompasses nucleic acid molecules .
which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent hybridization conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (fj, (g), (h), or (i), above, as are polypeptides encoded by these polynucleotides. In- another preferred embodiment, ~polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions, or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[90] In another embodiment, the invention provides a purified protein comprising, or alternatively consisting of, a polypeptide having an amino acid' sequence selected from the group consisting of: (a) the complete amino acid.sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (b) the amino acid sequence of a mature form of a polypeptide having the amino acid sequence of SEQ ID
NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (c) the amino acid sequence of a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; and (d) the amino acid sequence of an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded ~by the cDNA in Clone ID NO:Z. .
[91] The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the amino acid sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid sequence .
encoded by the cDNA contained in Clone ID NO:Z, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B, the amino acid sequence as defined in column 7 of Table 1A, an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are also provided (e.g., those fragments described herein). Further proteins encoded by polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these amino acid sequences under stringent hybridization, conditions or alternatively, under lower astringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins.
(92] ~By a nucleic acid having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to ~5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referred to irx Table 1A or 2 as the ORF (open reading frame), or any fragment specified as described herein.
(93] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention.can be determined conventionally using known~computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). .
In a sequence alignment the query and subject sequences are both DNA
sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, 153 .
k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=S, Gap Size Penalty O.OS, Window Size=S00 or the length of ' the subject nucleotide sequence, whichever is shorter.
[94) If the subject sequence is shorter than the query sequence because of S' or 3' deletions, not because of internal deletions, a manual correction must be made to the results.
This is because the FASTDB program does not account for S' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the S' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are S' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the present invention. Onlybases outside the S' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the~percent identity score.
[95] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the S' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment'of the first 10 bases at S' end. The 10 unpaixed bases represent 10% of the sequence (number of bases at the S' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the S'. or 3' of the subject sequence which are not matched/aligned with the query. In this case.the percent identity calculated by FASTDB is not manually corrected. Once again, only bases S' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
[96] By a polypeptide having an amino acid sequence at least, for example, 9S~%
"identical" to a query amino acid sequence of the present invention, it is intended. that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 9S% identical to a query amino acid sequence, up to S% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur .
at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
(97) As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 9S%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a polypeptide referred to in Table 1A (e.g., the amino acid sequence identified in column 6) or Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the polynucleotide sequence defined in columns 8 and 9 of Table 2) or a fragment thereof, the amino acid sequence of the polypeptide encoded by the polynucleotide sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or a fragment thereof, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment thereof, or the amino acid sequence of the polypeptide encoded by cDNA contained in Clone ID
NO:Z, or a fragment thereof, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-24S (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM
0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group' Length=0, Cutoff Score=I, Window Size=sequence length, Gap Penalty=S, Gap Size Penalty=0.05, Window Size=S00 or the length of the subject amino acid sequence, whichever is shorter.
[98) If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results.
This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global~percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity, calculated by the above FASTDB
program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and Ci-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N-and C- terminal residues of the subject sequence..
[99] For example, a 90 amino acid residue subject sequence is aligned with a residue query sequence to determine percent identity. The deletion occurs at the N-terminus of'the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matchedltotal number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence., This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment, which are not matched/aligned with the query sequnce axe manually corrected for. No other manual corrections are to made for the purposes of the present invention.
[100] The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
Moreover, polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, I-5, or 1-2 amino. acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced fox a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
(101] Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.
(Genes II, Lewin, B., ed., John Wiley & Sons, New York (I985)). These allelic variants can vary at either the polynucleotide and/or polypeptide' level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. -[102] . Using known methods of protein engineering and recombinant DNA
technology, variants may be generated to impxove or alter the characteristics of the polypepfides .of the present invention. For instance, one or more amino acids can be deleted fxom the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem.,268: 2984-2988 (1993)) xeported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).) .
[103] Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring. protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1 a. They used random mutagenesis to generate over 3,500 individual IL-1 a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity]." In fact, only 23 unique amino acid sequences, out of more than 3;500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
[104] ~ Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide . results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N-or C-terminal residues of a protein retains such immunogeriic activities can readily be determined by routine methods described herein and otherwise known in the art.
[105] Thus, the invention further includes polypeptide variants which show a functional activity (e.g., biological activity) of the polypeptides of the invention.
Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
[106] The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95°,%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide having the amino acid sequence of an N
and/or C
terminal deletion), irrespective of whether they encode a polypeptide having functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having functional activity include, inter alias (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the gene, as described~in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA
expression in specific tissues (e.g., normal or diseased tissues); and (4) in situ hybridization '(e.g., histochemistry) for detecting mRNA expression ~in specific tissues (e.g., normal or diseased tissues).
[107] Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, which do, in..fact, encode a polypeptide having functional activity. By a polypeptide having "functional activity" is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein of the invention. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an anti-polypeptide of the invention antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for' a polypeptide of the invention.
[108] The functional activity of the polypeptides, and fragments, variants and derivatives of the invention, can be assayed by various methods.
[109] For example, in one embodiment where one is assaying for the ability to bind or compete with a full-length polypeptide of the present invention for binding to an anti-polypetide antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western. blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
[110] In another embodiment, where a ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of a ~polypeptide o~ the present invention to bind to a substrates) of the polypeptide of the invention can be routinely assayed using techniques known in the art.
[111] In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the present invention and fragments, variants and derivatives thereof to elicit polypeptide related biological activity (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.
[112] Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acid sequence of the cDNA contained in Clone ID NO:Z, the nucleic acid sequence referred to in Table 1A (SEQ ID NO:X), the nucleic acid sequence disclosed in Table 2 (e.g,. the nucleic acid sequence delineated in columns 8 and 9) or fragments thereof, will encode polypeptides "having functional activity." In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
[113] For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., "Deciphering the Message in Protein Sequences:
' Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
[114] The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast; the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
[115] The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. . See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
[116] As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the. protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and 11e; replacement of the hydroxyl residues Ser and Thr; replacement, of the acidic residues Asp and Glu;
replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitutions with one or more of the amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) fusion of the polypeptide with additional amino acids, such as, for example, an 1gG Fc fusion region peptide, serum albumin (preferably human serum albumin) or.a fragment thereof, or leader or secretory sequence, or a sequence facilitating purification, or (v) fusion of the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)).
Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
[117J For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance- due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Bobbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
[118] A further embodiment of the invention relates to polypeptides which comprise the amino acid sequence of a polypeptide having an amino acid sequence which contains at least one amino acid substitution, but not more than~50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions from a polypeptide sequence disclosed herein. Of course ~ it is highly preferable fox a polypeptide to have an amino acid sequence which comprises the ~arnino acid sequence of a polypeptide of SEQ ID NO:Y, an~ amino acid sequence encoded by SEQ
ID NO:X, an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2, an amino acid sequence encoded by the complement of SEQ
ID NO:X, and/or an amino acid sequence encoded by cDNA contained in Clone ID
NO:Z
which contains, in order of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
[119] In specific embodiments, .the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of a reference amino acid sequence selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof (e.g., the mature form and/or other fragments described herein); (b) the amino acid sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the complement of SEQ ID NO:X or fragments thereof; (d) the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns, 8 and 9 of Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA contained in Clone ID
NO:Z or fragments thereof; wherein the fragments or variants have 1-5, 5-10, 5-25, 5-S0, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Polynucleotide aid Polypeptide Fragments .
(120] The present invention is also directed to polynucleotide fragments of the polynucleotides (nucleic acids) of the invention. In the present invention, a "polynucleotide fragment" refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in Clone ID NO:Z or the 'complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide encoded by the cDNA
contained in Clone ID NO:Z or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ
ID NO:X as defined in columns 8 and'9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence in SEQ ID NO:X or the complementary strand thereto; is a polynucleotide sequence encoding a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence encoding a portion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide sequence encoding a portion of a polypeptide encoded by the complement of the polynucleotide sequence in SEQ ID NO:X; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:B as defined in column 6 of Table 1B or the complementary strand thereto; or is a portion of the polynucleotide sequence of SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto.
[121] The polynucleotide fragments of the invention are preferably at least about 15 nt, and more-preferably at least about 20 nt, still more preferably at least about 30 nt, arid even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at Least about 150 nt in length. A fragment "at least 20 nt in length,'-' for example, is intended to include 20 or more contiguous bases from the, cDNA sequence contained in Clone ID
NO:Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto. In this context "about" includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at.both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 160, I70, 180, 190, 200, 250, 500, 600, 1000, or 2000 nucleotides in length ) are also encompassed by the invention.
[I22] ~ Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number I-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 45I-500, 50I-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1400, 1401-1450, 1451-1500, 1501-1550, 1551-'1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551.-3600, 3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400,'5401-5450, 5451-5500,.
5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 58b1-5850, 5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, .6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of SEQ ID NO:X, .or the complementary strand thereto. In this context "about"
includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein.
Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or. alternatively, under lower stringency ~
conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[123] ' Further representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 175I-1800, I80I-1850, 1851-1900, 1.901-1950, 1951-2000, 2001-2050, 2051-2I00;
2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of the cDNA sequence contained in Clone ID NO:Z, or the complementary strand thereto.
In this context "about" includes the particularly recited range or a range larger or smaller by several (5, 4,'3, 2, or 1) nucleotides, at either terminus or at both termini.
Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity).
More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[124] Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist .of, a nucleic acid sequence comprising one, two, three, four, five,' six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence delineated in Table 1B column 6. Additional, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described' polynucleotide fragments of the invention in combination with a polynucleotide sequence that is the complementary strand ,of a sequence delineated in column 6 of Table 1 B. In further embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID
NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated Table 1B, column 6, and have a nucleic acid sequence~which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally;
fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[12S] In additional specif c embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1 B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[126] In additional specific embodiments; polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ
ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are .also encompassed by the invention.
[127] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in the same row of column 6 of Table 1B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defned in Table 1A or 1B) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[128] In additional specific embodiments, polynucleotides of the invention comprise, :or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of the sequence,of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also.
encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention. ' [129] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein) are directly contiguous Nucleic acids which hybridise to the complement of these 20 contiguous polynucleot'ides under stringent hybridization conditions ox alternatively, 'under lower stringency conditions, are also encompassed by the invention. ~Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and po~lypeptides are also encompassed by the invention.
[130J In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also .
encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[I3IJ In specific embodiments, .polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 are directly contiguous. In preferred embodiments, the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1 B is directly contiguous with the 5'' 10 polynucleotides of the next sequential exon delineated in Table 1B, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[132) ~ In the present invention, a "polypeptide fragment" refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y, a portion of an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2, a portion of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID
NO:X, a portion of an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, and/or a portion of an amino acid sequence encoded by the cDNA contained in Clone ID NO:Z. Protein (polypeptide) fragments may be "free-standing," or comprised within a larger polypeptide of which~the fragment forms a part or region, most preferably as a single continuous region. Representative examples of .
polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700; 701-720, 721-740, 741-760, 76I -780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of cDNA. and SEQ ID NO: Y. In a preferred embodiment, polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140,.141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID NO:Y. Moreover, polypeptide fragments of the invention may be at least v about 10, 15, 20, 25, 30, 35, 40, 45, 50, S5, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about" includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at Goth extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
[133j Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the maj ority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether- a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such.
immunblogic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
[134] Accordingly, polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the. carboxy terminus, or both. For eXample,..any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form.
Similarly, any number of amino acids, ranging from I-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
[135] The present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptid~e_ of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1B, and/or a polypeptide, encoded by the cDNA contained in Clone ID NQ:Z). In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ID NO:Y, or the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
[136] The present invention further provides polypeptides having one or more residues from the caxboxy terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ
ID
NO:X as defined in columns 8 and 9 of Table 2, and/or a polypeptide encoded by the cDNA
contained in Clone ID NO:Z). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where n corresponds to the position of amino acid residue, in a polypeptide of the invention.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[137] In addition, 'any of the above 'described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. 'The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide disclosed as SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA~ contained in Clone ID NO:Z, and/or the complemexlt thereof, where n and m are integers as described above.
Polynucleotides encoding these polypeptides are also encompassed by the invention:
[138] Also as mentioned above, even if deletion of orie~ or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. Fox example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or~mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
[139] The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions.
Polynucleotides encoding these polypeptides are also ericoinpassed by the invention.
[140] Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for example, in Tables 1A and 2), the cDNA contained in Clone ID NO:Z, or the polynucleotide sequence as defined in column 6 of Table IB, znay be analyzed to determine certain preferred regions of the polypeptide. For example, the amino acid sequence of a polypeptide encoded by a polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ ID NO:Y
and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2) or the cDNA contained in Clone ID NO:Z may be analyzed using the default parameters bf the DNASTAR computer algorithm (DNASTAR, Inc., 1228. S. Park St., Madison, WI 53715 USA; http://www.dnastar.com~.
[I41] Polypeptide regions that may be routinely obtained using the DNASTAR
computer algorithm include, but are not limited to; Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle hydrophilic regions and hydrophobic regions;
Eisenberg alpha-and beta-amphipathic regions; Karplus-Schulz .flexible regions; Emini surface-forming regions; and Jameson-Wolf regions of high antigenic index. Among highly preferred polynucleotides of the invention in this regard are those that encode polypeptides comprising regions that combine several structural features, such as several (e.g., l, 2, 3 or 4) of the features_set out above.
[142] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from data by DNASTAR
analysis by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
[143] Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a functional activity (e.g.
biological activity) of the polypeptide sequence of which the amino acid sequence is a fragment. By a polypeptide displaying a "functional activity" is meant a polypeptide capable of one or more known functional activities associated' with a full-length protein, such as, for example, biological activity, antigenicity, immunogenicity, and/or multimerization, as described herein.
[144] Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or, a decreased undesirable activity.
[145] In preferred embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the antigenic fragments. of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides encoding these polypeptides are also encompassed by the invention.
j146] The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y; a polypeptide sequence encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the portion of SEQ ID NO:B
as defined imcolumn 6 of Table 1B or the complement thereto; the polypeptide sequence encoded by the cDNA contained in Clone ID NO:Z; or the polypeptide sequence encoded by a polynucleotide that hybridizes to the sequence of SEQ ID NO:X, the complement of the sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, or the cDNA sequence contained in Clone ID NO:Z
under stringent hybridization conditions or alternatively, under lower stringency hybridization as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or alternatively, under lowei stringency hybridization conditions defined supra.
(147] The term "epitopes," as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses- a polypeptide comprising an~ epitope, as well as the polynucleotide encoding this polypeptide. An "immunogenic epitope," as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983)). The term "antigenic epitope," as used herein, is defined as a portion of .a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[148] Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135.
(1985) further described in U.S. Patent No. 4,631,211.) [149] In the. present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 1~1,, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids.
Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 3S; 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of -two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
[150] Non-limiting examples of epitopes of polypeptides that card be used to generate .
antibodies of the invention include a polypeptide comprising, or alternatively consisting of, at least one, two, three, four, five, six or more of the portions) of SEQ ID
NO:Y specified in column 7 of Table 1A. These polypeptide fragments have been determined to bear antigenic epitopes of the proteins of the invention by the analysis of the Jameson-Wolf antigenic index which is included in the DNAStar suite of computer programs.
By "comprise" it is intended that a polypeptide contains at least one, two, three, four, five, six or more of the portions) of SEQ ID NO:Y shown in column 7 of Table 1A, but it may contain additional flanking residues on either the amino or carboxyl termini of the recited portion. Such additional flanking sequences are preferably sequences naturally found adjacent to the portion; i.e., contiguous sequence shown in SEQ ID NO:Y. The flanking sequence may, however, be sequences from a heterolgous polypeptide, such as from another protein described herein or from a heterologous polypeptide not described herein.
In. particular embodiments, epitope portions of a polypeptide of the invention comprise one, two, three, or more of the portions of SEQ ID NO:Y shown in column 7 of Table 1A.
[151] Similarly, immunogenic epitopes can be used, for example, to~-induce antibodies -according to methods well known in the art. See, for instance, Sutcliffe et al., supra;
Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985): Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well-as any combination .of two, three, four, five or .more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have -been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
[152] Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo ' immunization, in vitro immunization, and phage display methods. -See, e.g., Su'tcliffe et al., supra; Wilson et al., supra, and-Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer rnay be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide. ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ~.g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide ,antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
[153] As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including fragments or variants thereof), may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides.
By way of another non-limiting example, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S.
Patent No.
5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP
Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Patent 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N.- or'C-terminal end of the heterologous protein (e.g., immurioglobulin Fc ~polypeptide or human , serum albumin ~polypeptide).
Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.
[154) Such fusion proteins as those described above may facilitate purification and may increase half life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of 'the heavy or light chains of mammalian immunoglobulins. See, e.g., EP
394,827;
Traunecker et al., Nature, 331:84-86 (I988). Enhanced.delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT
Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been~found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-(1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows 'for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Fusion Proteins [155] Any polypeptide of the present invention can be used to generate fusion proteins.
For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
Moreover, because secreted proteins target cellular locations based on trafficking signals, polypeptides of the present invention which are shown to be secreted can be used as - targeting molecules once fused to other proteins.
[156] Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
[157] - In certain preferred embodiments, proteins of the invention are fusion proteins comprising an amino acid sequence that is an N , and/or C- terminal deletion of a polypeptide of the invention. In preferred embodiments, the invention is directed to a fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence.of the invention.
Polynucleotides encoding these proteins are also encompassed by the invention.
[158] Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to. final preparation of the polypeptide.
The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
[159] As one of skill in the art will appreciate that, as discussed above, polypeptides of the present invention, and epitope-bearing fragments thereof, can be combined with heterologous polypeptide sequences. For example, the ,polypeptides of the present invention may be fused with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHl, CH2, CH3, and any combination thereof, including both. entire domains and portions thereof), or albumin (including, but not limited to, native or recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. For example, EP-A-O 464 S~3 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy. and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP-A
0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the, Fc portion may hinder therapy -and diagnosis if the fusion protein is used as an antigen for immunizations.
In drug discovery, for example,' human proteins, such as hIL-S, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-S. See, D. Bennett et al., J. Molecular Recognition 8:52-S8 (1995); K. Johanson et al., J. Biol.
Chem. 270:9459-9471 (1995).
[160] Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a polypeptide which'facilitates purification of the fused polypeptide. In ' .preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, .Inc., 9259 Eton Avenue, Chatsworth, CA, 91.311), among others, many of which are commercially available: As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. .Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)).
[161] Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;
5,830,721; 5,834,252;
and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997);
Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);
and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each of these patents and publications are hereby incoiporated ~ by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In. another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, . , etc. of one or more heterologous molecules.
[162] Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
Recombinant and Synthetic Production of Polyp~tides of the Invention [163] The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective.
In the latter case, viral propagation generally will occur only in complementing host cells.
[164] The polynucleotides of the invention may be joined ~to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vecfor, is a virus, it may be packaged in vitro using an appropriate packaging cell line and then txansduced into host cells.
[165] The polynucleotide insert should be operatively linked ~ to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, imthe transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
[166] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, 6418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal ,cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178));
insect cells such as Drosophila S2 and Spodoptera S~ cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
[167] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNHl6a, pNHlBA, p~1H46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG
available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
Preferred expression vectors for~use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S 1 ~ pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogeri, Carlbad, CA). Other suitable vectors will be readily apparent to the skilled artisan.
[168] Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative.
Glutamine synthase expression systems can also functiom in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine~ synthase expression system and components thereof are detailed in PCT publications: W087/04462;
W086/05807;
W089/01036; W089/10404; and W091/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using a GS expression system in marine myeloma cells is described in Bebbington et al., Bioltechr~ology 10:169(1992) and in Biblia and Robinson Biotech~ol.
Prog. 11:1 (1995) which are herein incorporated by reference.
[169] The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic~cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
[170] Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector. .
[171] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, ~e.g., US Patent Number 5,641,670, issued June 24, 1997; International Publication Number WO 96/29411;
International Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad.
Sci. USA
86:8932-8935 (1989); and Zijlstra et al., Nature 32:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
[172] Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or canon exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
[173] Polypeptides of the present invention can also be recovered from:
products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal. process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
[174] In one embodiment, the yeast Pichia pastoris is used to express polypeptides of the invention in a eukaryotic system. Pichia pasto~is is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using OZ. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for 02. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOXI) is highly active. In the presence of methanol, alcohol oxidase produced from the ADXI gene comprises up to approximately 30% of the total soluble protein in Pichia pastor~is. See Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al., Yeast 5:167-77 (1989); Tschopp, J.F., et al:, Nucl. Acids Res. 15:3859-76 (1987).
Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the A OXl regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
[175] In one example, the plasmid vector pPIC9K is used to express DNA
encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J.
Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOXl promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
[176) Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDI, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in the art would readily appreciate, as Long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
[177] . In another embodiment,. high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol. ' [178] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucl~eotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polyriucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent No.
5,641,670, issued June 24, 1997; International Publication No. WO 96/29411', published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:.435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
[179] In addition, polypeptides of the invention can. be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman' & Co., N.Y., and Hunkapiller et al., Nature, 310:105-('1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, .if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common .amino acid°s, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic .acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, .
norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary).
[180] The invention encompasses polypeptides of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V 8 protease, NaBH4; acetylation, formylation, oxidation, reduction;
metabolic synthesis in the presence of tunicamycin; etc.
[181] Additional post-translational modifications encompassed by the .
invention include, for example, e.g., N-linked or 0-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
[182] Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples ~of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine (1211, i23I,.izsl, i3il), carbon (14C), sulfur (3sS), tritium (3H), indium (111In, llzln, 113mIn, llsmln), technetium (99Tc,99mTc), thallium (2olTi), gallium (68Ga, 67Ga), palladium (lospd), molybdenum (991VIo), xenon (133Xe), fluorine (18F), ls3Sm,, 177Lu, ls9Gd, 149Pm~ la°La~ 17s~' 166Hp~ 9oY~ 47SC~ ls6Re~ ls8Re~ 142Pr~ lose and 97Ru.
[183] In specific embodiments, a polypeptide of the present invention or, fragment or variant thereof is attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 177Lu, 9oY, 166Ho, and ls3Sm, to polypeptides.
In a preferred 'embodiment, the radiometal ion associated with the macrocyclic chelators is 111In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is 9°Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via a 186 _ linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art - see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
[184] As mentioned, the proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide.
Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et aL, Meth.
Enzymol.
182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[185] Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at-random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
[186] The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used,- depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).-For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,S00, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
[187j As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in IJ.S. Patent No.
5,643,575;
Morpurgo et al., Appl. Biochem. Biotech~zol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioco~jug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
[188] The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to-those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having ar free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol. molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
[189] As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g.., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
[190] One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated 'moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modif cation may be accomplished by reductive alkylation which exploits differential reactivity of-different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
[191] _ As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev.
Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S.
Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98132466, the disclosures of each of which are incorporated herein by reference. _ [192] One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the, modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (CISOzCH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[193) Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Patent No. 5,612,460, the entire disclosure of which is incozporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to, the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described' in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of.the invention.
[194] The number of polyethylene glycol moieties attached to each protein bf the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, S, 6, 7, 8, 9, 10, 12, 1 S, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-S, 4-6, S-7, 6-8, 7-9, 8-10, 9-LI, 10-I2, 11-13, I2-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule.
Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Theca. Drug Carrier Sys. 9:249-304 (1992).
[195] The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are .not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC".) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
[196] The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly; the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trirt~ers, or at least tetramers.
[197] Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer refers to a multimer containing only polypeptides corresponding to a protein of the invention (e.g., the amino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID
NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or an amino acid sequence encoded by cDNA contained in Clone ID
NO:Z (including fragments, variants, splice variants, and fusion proteins, corresponding to these as described herein)). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence.
In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specif c embodiments, the multimer of the invention is a homodimer (e.g., containing two polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing three polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
[198] As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
[199] Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked by, for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, lieteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ
ID NO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or encoded by the cDNA contained in Clone ID NO:Z). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein. In one example, covalent associations are~between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a Fc fusion protein of the invention (as described herein). In another specific.example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO:
WO
98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
[200] Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
[201] Trimeric polypeptides of the invention may offer.the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S.
patent application Ser. No. 08/446,922, hereby incorporated by reference.
Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides 'of the invention.
[202] In another example, proteins of the invention are associated by interactions between Flag~ polypeptide sequence contained in fusion proteins of the invention containing Flag~ polypeptide sequence. In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in .
Flag~ fusion proteins of the invention and anti-Flag~ antibody.
[203] The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques, known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-Iinks between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US
Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is~ herein incorporated by reference' in its entirety).
[204] Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, .
which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., ,US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). ' Antibodies [205] Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of the invention (e.g., a polypeptide or fragment or variant of the.
amino acid sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in Clone ID
No:Z, and/or an epitope, of the present invention) as determined by immunoassays well known in the art for assaying specific antibody-antigen binding. Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of.the above. The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The irnmunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the immunoglobulin molecules of the invention are IgGl . In other preferred embodiments, the immunoglobulin molecules of the invention are IgG4.
[206) Most preferably the antibodies are human antigen-binding antibody fragments of the present invention.and include, but are not limited to, Fab, Fab' and.F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable regions) alone or in combination with the' entirety or a portion of the following: hinge region, CH1; CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable regions) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibodies include antibodies having the amino acid sequence'of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.~
J
[207] The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92105793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5;601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[208] Antibodies of the present invention may be described or,specif ed in terms of the epitope(s) or portions) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portions) may be specified as described herein, e.g., by N-terminal and C-terminal positions, or by size in contiguous amino acid residues, or listed in the Tables and Figures. Preferred epitopes of the invention include the predicted epitopes shown in column 7 of Table 1A, as well as polynucleotides that encode these epitopes. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the .
same.
(209] Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind'any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, Less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, Iess than 55%, and Less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combinations) of 2, 3, 4, 5,. or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affnities include those with a dissociation constant or Kd less than 5 X 10'2 M, 10'2 M, 5 X 10'3 M, 10'3 M, 5 X 10' 4 M, 10'4 M, 5 X 10'5 M, 10'5 M, 5 X 10'6 M, 10'6M, 5 X 10'7 M, 107 M, 5 X
10'8 M, 1 b'8 M, 5X10'9M,10'9M,5X10'1°M,10'1°M,5X10'11M,10'11M,SX10''2M,10''2 M,SX
10'I3M, 10'13M,SX 10'14 M, 10'14M,SX10'lSM,orlO'15M.
[210J The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
[211] Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation.. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. ' For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for examphe, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the.activity in absence of the antibody.
[212J . The invention also features receptor-specific antibodies which .both prevent ligand binding and receptor activation as well as antibodies that recognize the.receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are. neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the Iigand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT
publication WO
96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et aL, Cancer Res. 58(15):3209-3214 (I998); Yoon et aL; J.
Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998);
Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997);
Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
[213] Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have utility in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
incorporated by reference herein in its entirety.
[~14] As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be rscombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules-useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT
publications WO 92/08495; WO 91114438; WO 89112624; U.S. Patent No. 5,314,995;
and EP 396,387; the disclosures of which are incorporated herein by reference in their entireties.
[215] The antibodies of the invention include derivatives that are modifed, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, arnidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by~ known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic. synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical amino acids. ~ ' [216] The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc.
to induce the production of sera containing polyclonal antibodies specific for the antigen.
Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG, (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
[217] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-(Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
[218] Methods for producing and screening for specif c antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and' cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
(219] Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
(220] Another well known method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr Virus (EBV). Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference. The source of B cells for transformation is commonly human peripheral blood, but B cells for. transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues.
Tissues axe generally made into single cell suspensions prior to EBV
transformation.
Additionally, steps maybe taken to either physically remove or inactivate T
cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
[221J In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast micxoscopy, transformed cells may appear large, clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV
lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV
transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SILO-007, RPMI 8226, and KR-4). Thus, the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
[222] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) ox pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
[223] For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected ox identified with antigen, e.g., using labeled antigen or antigen bound-or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.
Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994);
Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994);
PCT application No. PCTlGB91/01134; PCT publications WO 90/02809; WO 91/10737;
WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.
Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
[224] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
[225] Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos.
5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., ~ by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al:, PNAS 91:969-973 (1994)), and chain shuffling (0.S.
Patent No. 5,565,332).
[226j Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111;
and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO
96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
[227] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma_ technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int.
Rev.
Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92101047; WO 96/34096;
WO
96/33735; European Patent No. 0 598 877; U.S. Patent Nos. 5,413,923;
5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771;
5,939,598;
6,075,181; and 6,114,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, CA) arid Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
[228] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Biotechnology 12:899-903 (1988)).
[229] Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic". polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona, FASEB J.
7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby block its biological activity. Alternatively, antibodies which bind to and enhance polypeptide multimerization and/or binding, and/or receptor/ligand multimerization, binding and/or signaling can be used to generate anti-idiotypes that function as agonists of a polypeptide of the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens as agonists of the polypeptides of the invention or its ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its.
ligand(s)/receptor(s), and thereby promote or enhance its biological activity.
j230] Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen et al., Hum. Gene Ther. 5:595-601 (1994);
Marasco, W.A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev.
Microbiol.
51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999);
Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999);
Zhu et al., J. Immunol. Methods 231:207-222 (1999); and references cited therein. .
Poly~ueleotides Encoding Antibodies [231] The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ
ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or to a polypeptide encoded by the cDNA contained in Clone ID
NO:Z.
[232] The polynucleotides. may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
[233] Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR
amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody.
Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
[234] Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
[235] In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA
techniques, one or more of the CDRs may be inserted within framework regions, e..g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
[236] In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable .region derived from a murine rilAb and a human immunoglobulin constant region, e.g., humanized antibodies.
[237] Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778; Bird Science 242:423- 42 (1988); Huston et al., Proc. Natl.
Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) cari be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. colt may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
Methods ofP~oducingAntibodies [238] The antibodies of the invention can be produced by any method known in the art 'for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Methods of producing antibodies include, but are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below.
[239] Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody.molecule may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an . antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an~antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a-promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S.
Patent No. 5,122,464) and the variable domain of the antibody may be cloned- into such a vector for expression of the entire heavy or light chain.
[240] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
[241] , A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule: For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 ( 1990)).
[242] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX
vectors may also be used to express . foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin,or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
[243] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
(244] . In mammalian host cells, a number of viral-based expression systems 'may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)).
Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences.
Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons, can be of a variety of origins, both natural and synthetic. The efficiency of expression may -be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods, in Enzymol. 153:51-544 (1987)).
[245] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e:g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
[246] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which~contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of. the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasniid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and ,grow to form foci which in turn can be cloned and' expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
[247] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxaxithine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad.
Sci. USA
48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes:
dhfr, which confers resistance _to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.
32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.
Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).
Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY
(1990); and in Chapters 12 and' 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981); which are incorporated by reference herein in their entireties.
[248] The expression levels of an .antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplif canon for the expression of cloned genes in mammalian cells in DNA
cloning, Vol.3. ~ (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Grouse et al., Mol. Cell. Biol. 3:257 (1983)).
[249] Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drubs methinninP c»inhnximinP pr mPthntraxatP
reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suplliers, including, for example Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using a GS expression system in marine myeloma cells is described in Bebbington et al., Bioltechnology 10:169(1992) and in Biblia and Robinson Biotechnol.
Prog. 1 1:l (1995) which are incorporated in their entirities by reference herein.
[250] The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986);
Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
[251] Once an antibody molecule of the invention has been produced by an,animal, chemically,synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
[252] The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does ' not necessarily need to be direct, but may occur through linker sequences. , The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232;,EP
439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981;
Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
[253] The present invention fizrther includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other thar~
the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain' or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053;
5,447,8SI;
5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 9-1/06570;
Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:1OS3S-1OS39 (1991); Zheng et al., J.
Immunol. 1S4:SS90-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA
89:11337-11341 (1992) (said references incorporated by reference in their entireties).
[254] As discussed, sZrpra, the polypeptides corresponding to a polypeptide,_ polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to ~
facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See EP 394,827; and Traunecker et 214:
al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. See, for example, Fountoulakis et al., J.
Biochem. 270:3958-3964 (1995). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. See, for example, EP A 232,262. Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).
[255] Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al,, Cell 37:767 (1984)) and the "flag" tag.
[256] The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part' of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferi~n, and aequorin; and examples of suitable radioactive material include 125I, 131I, 11 lIn or 99Tc.
[257] Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for,example, 213Bi. A
cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol~, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin~ etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, Iidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, rrielphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthrac~clines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
[258] The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent .or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, 13-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator,.an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as~
for example, lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
[259] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
[260] Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985)? Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.
(Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", I-mmunol. Rev. 62:119-58 (1982).
[261] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety. ' [262] An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factors) and/or cytokine(s) can be used as a therapeutic.
Immunophenotyping [263] The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. Translation products of the gene of the present invention may be useful as cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Patent 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
[264] These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and "non-self' cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
Assays Fot~ Antibody Binding.
[265] The antibodies of the invention may be assayed for imrnunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). .Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
[266] Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hburs) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds., (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.16.1.
[267] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non=fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g:, an anti-human antibody) conjugated to .an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane . in wash buffer, and detecting the presence of the antigen., One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York, section 10.8.1.
[268] ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, (1994), Current Protocols. in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 11.2.1.
[269] The binding affinity of an antibody to an antigen and the off rate of an antibody=
antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
[270] Antibodies of the invention may be characterized using immunocytochemisty methods on cells (e.g., mammalian .cells, such as CHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art. Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.
Therapeutic Uses , [271] The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
[272] In a specific and preferred embodiment, the present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more diseases, disorders, or conditions, including but not limited to: neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions., and/or as described elsewhere herein.
Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g., antibodies directed to the full length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a polypeptide of the invention (such as, for example, a predicted linear epitope shown in column 7 of Table 1A; or a conformational epitope, including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
[273] A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g.
as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary sleill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
[274] The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as; e.g., IL-2, IL-3 and IL-7), for example, which serve o increase the number or activity of effector cells which interact with the antibodies.
[275] The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
[276] It is preferred to use high affinity and/or potent i~ vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof.
Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10'2 M, 10'2 M, S X 10'3 M, 10'3 M, S X 10-4 M, 10'4 M, S X 10'' M, 10'5 M, S X 10'6 M, 10'6 M, S X
10'7 M, 10'7 M, S X 10-8 M, 10'$ M, S X 10-9 M, 10'9 M, S X 10'1° M, 10-1° M, S X 10-11 M, 10-11 M, S X 10-12 M, 10-12 M, S X 10'13 M, 10' 13 M, S X 10'14 M, 10-14 M, S
X 10'15 M, and lp-ls M.
222.
Gene Therapy [277] In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an'expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
[278] Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
[279) For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-SOS (1993); Wu and Wu, Biotherapy 3:87-9S (1991); Tolstoshev, Ann.
Rev. Pharmacol. Toxicol. 32:573-S96 (1993); Mulligan, Science 260:926-932 (1993); and Moigan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH
11 (S):1 S S-21 S ( 1993 ). Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY ( 1990).
[280] In a preferred embodiment, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In-another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 ( 1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
(281] Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
[282] In a specific embodiment, the nucleic acid sequences are directly administered ih vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; W092/20316; W093/1418$, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Roller and Smithies, Proc. Natl.
Acad. Sci.
USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
[283] In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
[284] Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia.
Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. I~ozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991);
Rosenfeld et al., Cell 68:143- 155 (1992); Mastrangeli et al., J. Clin.
Invest. 91:225-234 (1993); PCT Publication W094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995).
In a preferred embodiment, adenovirus vectors are used.
[285] Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).
[286] Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells axe then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
[287] ~ In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.
217:599-618 (1993); Gohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac.
Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide.for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
[288] The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
[289) Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
[290] In a preferred err~bodiment, the cell used for gene therapy is autologous to the patient.
[291] In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered i~
vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used.
Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992);
Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc.
61:771 (1986)).
[292] In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by the presence or absence of an appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity [293] The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
Ther-~apeuticlProphylactic Administration and Composition [294] The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably a polypeptide or antibody of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that Iimit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
[295] Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above;
additional appropriate formulations and routes of administration can be selected ,from among those described herein below.
[296] Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
[297] In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or b'y means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
[298] In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Larger, Science 249:1527-1533 (1990);
Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 3S3- 36S (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) [299] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Larger, supra;
Sefton, CRC Crit. Ref. Biomed. Erg. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Larger and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);
Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). ' [300] Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
[301] In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector 'and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.
USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
[302] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium_stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
[303] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[304] The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[305] The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention 'of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
[306] For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to mg/kg of the patient's body weight. Generally, human antibodies have a longer half life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
[307] The invention also provides a pharmaceutical pack or kit comprising one or more containers f lled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. - .
Diagnosis and Imaging [308] Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
[309] The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the 1 presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
[310] Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J.
Cell . Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (I25I, 12II), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[3I1] One facet of the invention is the detection and diagnosis of a disease or. disorder s associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds' to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
[312] It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S.W.
Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments."
(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.
A.
Rhodes, eds., Masson Publishing Inc. (1982)).
[313] Depending on several variables, including the type of label used and'the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or to 10 days.
[314] In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
[315] Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used.
Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
[316] In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S.
Patent No. 5,441,050). Imanother embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
Kits [317] The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specif cally immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound;
an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
[318] ~ In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recbmbinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
[319] In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
[320] In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody.
The detecting means of the kit may include a second, labeled monoclonal antibody.
Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
[321] In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
[322] The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
[323] Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
Uses of the Polynucleotides [324] Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
[325] The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), axe presently available. Each sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome, thus each polynucleotide of the present invention can routinely be used as a chromosome marker using techniques known in the art. Table 1A, column 9 provides the chromosome location of some of the polynucleotides of the invention. .
[326] Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably at least 15 by (e.g., 15-25 bp) from the sequences shown in SEQ ID
NO:X.
Primers can optionally be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR
screening of somatic cell hybrids containing individual human chromosomes.
Only those hybrids containing the human gene corresponding to ~SEQ ID NO:X will yield an amplified fragment.
[327] Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, preselection by hybridization to construct chromosome specific-cDNA
libraries, and computer mapping techniques (See, e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is hereby incorporated by reference in its entirety).
[328] Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread.
This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 by are preferred. For a review of this technique, see Verma et al., "Human Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York (1988).
[329] For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes).
[330] Thus, the present invention also provides a method for chromosomal localization which involves (a) preparing PCR primers from the polynucleotide sequences in Table 1A
and/or Table 2 and SEQ ID NO:X and (b) screening somatic cell hybrids containing individual chromosomes.
[331] The polynucleotides of the present ~ invention would likewise be useful for radiation hybrid mapping, HAPPY mapping, and long range restriction mapping.
For a review of these techniques and others known in the art, see, e.g. Dear, "Genome Mapping:
A Practical Approach," IRL Press at Oxford University Press, London (1997);
Aydin, J.
Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry 3:483-492 (1998);
Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol.
62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999) each of which is hereby incorporated by reference in its entirety.
[332] Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis.
Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library)). Column 10 of Table 1A provides an OMIM reference identification number of diseases associated with the cytologic band disclosed in column 9 of Table 1A, as determined using techniques described herein and by reference to Table 5.
Assuming I
megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
[333] Thus, once coinheritance is established, differences in a polynucleotide of the invention and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene .
from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.
[334] Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using the polynucleotides of the invention. Ariy of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker. Diagnostic and prognostic methods, kits and reagents encompassed by the present invention are briefly described below and more thoroughly elsewhere herein (see e.g., the sections labeled "Antibodies", "Diagnostic Assays", and "Methods for Detecting Diseases").
[335] Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder. Additional non-limiting examples of diagnostic methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., Example 12).
[336] In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general embodiment, the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the invention, where each probe has one strand containing a 31'mer-end internal to the region. In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification.
[337] Where a diagnosis of a related disorder, including, for example, diagnosis of a tumor, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
[338] ~ By "measuring the expression Ievel of polynucleotides of the invention" is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA
level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the related disorder or being determined by averaging levels from a population of individuals not having a related disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
[339] By "biological sample" is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains polypeptide of the present invention or the corresponding mRNA. As indicated, biological samples include body fluids (such as semen, lymph, vaginal pool, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
[340] The methods) provided above may preferably be applied in a diagnostic method andlor kits in which polynucleotides and/or polypeptides of the invention are attached to a solid support. In one exemplary method, the support may be a "gene chip" or a "biological chip" as described in US Patents 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the invention attached may be used to identify polymorphisms between the isolated polynucleotide sequences of the invention, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, such as for example, in neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, digestive disorders, metabolic disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. Such a method is described in US Patents 5,858,659 and 5,856,104. The US Patents referenced supra are hereby incorporated by reference in their entirety herein.
[341] The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides of the invention are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA
analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by Nielsen et al., Science 254, 1497 (1991); and Egholm et al., Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than.DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA
hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (Tm) by 8°-20° C, vs. 4°-16° C for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
[342] The compounds of the present invention have uses which, include, but are not limited to, detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to:
acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.;
and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
[343j Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., "The Etiology of Acute Leukemia:
Molecular Genetics and Viral Oncology," in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types. (Gelmann et al., supra) Indeed, the, human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra) [344] For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated. (International Publication Number WO
91/15580). However, it has been shown that exposure of HL-60 cells to a DNA
construct that is complementary to the 5' end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells.
(International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci.
85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the present invention's usefulness is not be limited to treatment, prevention, and/or prognosis of proliferative disorders of cells and tissues of hematopoietic origin, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.
[345] In addition to the foregoing, a polynucleotide of the present invention can be used to control gene expression through triple helix formation or through antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem.
56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC
Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney_ et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding .of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix - see Lee et al., Nucl.
Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense - Okano, J.
Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC
Press, Boca Raton, FL (1988)). Triple helix formation optimally results in a shut-off of RNA
transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. The oligonucleotide described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of polypeptide of the present invention antigens. Both techniques are effective in model systems, and the information disclosed hereim can be used to design antisense or triple helix polynucleotides in an effort to treat disease, and in particular;
for the treatment of proliferative diseases and/or conditions. Non-limiting antisense and triple helix methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the section labeled "Antisense and Ribozyme (Antagonists)").
[346] Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner.
Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled "Gene Therapy Methods", and Examples 16, 17 and 18).
[347] The polynucleotides axe also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel.
This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.
[348] The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA
sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
[349] Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood,, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can be amplified using PCR'. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992)). Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA
corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.
(350] There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention, specific to tissues, including but not limited to those shown in Table 1A. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen 'tissue cultures for contamination. Additional non-limiting examples of such uses are further described herein.
[351] The polynuc~eotides of the present invention are also useful as hybridization probes for differential identification of the tissue(s)~or cell types) present in a biological sample. Similarly, polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissues) (e.g., immunohistochemistry assays) or cell types) (e.g., immunocytochemistry assays). In addition, for a number of disorders of the above tissues or cells, significantly higher or lower levels of gene expression of the polynucleotides/polypeptides of the present invention may be detected in certain tissues.(e.g., tissues expressing polypeptides and/or polynucleotides of the present invention, for eXample, those disclosed in column 8 of Table 1A, and/or cancerous and/or wounded tissues) or~bodily fluids (e.g., semen, lymph, vaginal pool, serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
[352] Thus, the invention provides a diagnostic method of a disorder, which involves:
(a) assaying gene expression level in cells or body fluid of an individual;
(b) comparing the gene expression level with a standard gene expression level, whereby an increase or decrease in the assayed gene expression level compared to the standard expression level is indicative of a disorder.
[353] In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip" or other support, to raise anti-DNA antibodies using DNA
immunization techniques; and as an antigen to elicit an immune response.
Uses of the Polypeptides [354] Each of the polypeptides identified herein can be used in numerous ways.
The following description should be considered exemplary and utilizes known techniques.
[355] Polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissues) (e.g., immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et al., J.
Histochem. Cytochem. 29:577-580 (1981)) or cell' types) (e.g., immunocytochemistry assays). ' [356] Antibodies can be, used to assay levels of polypeptides encoded by polynucleotides. of the invention in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J.
Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (131h lzsh lzsh izil)~ c~.bon (14C), sulfur (3sS), tritium (3H), indium (1 tsmln~ 113mIn, llzln, 111In), and technetium (99Tc, 99mTc), thallium (z°1Ti), gallium (68Ga, 67Ga), palladium (losPd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), is3Sm~ ~77Lu~ is9Gd~ ia9Pm~ i4oLa~ i7syb~ 166Ho~ goy~ a7Sc~ ia6Re~ lasRe~
i4zPr~ ios~~ 97Ru;
luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[357] In addition to assaying levels of polypeptide of the present invention in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
[358] A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for ~ example, ~31I, llzIn, 99m~Z~~~ (lslh i2sl~ lz3h izil)~ carbon (14C), sulfur (3sS), tritium (3H), indium (11'"'In, 113"'In, llzln, 111In), and technetium (99Tc, ~99mTc), thallium (zo.lTi), gallium (6~Ga, 67Ga), palladium (lo3pd), molybdenum (~9Mo), xenon (l3sXe), fluorine (18F, ls3Sm, 177Lu, is9Gd, la9Pm, laoLa, ms~,b~ 166H~~ 9oY.~ a.7Sc~ is6Re~ issRe, i4zPr~ lose 97Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which express the polypeptide encoded by a polynucleotide of the invention. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc.
(1982)).
[359] In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g., polypeptides encoded by polynucleotides of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell.
In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
[360] In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention in association with toxins or cytotoxic prodrugs.
[361] By "toxin" is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. "Toxin" also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, Zi3Bi, or other radioisotopes such as, for example, lo3Pd, 133Xe, i3ih 68Ge,'7Co, 6'Zn, 85Sr, 32P, 3'S, Soy, ls3Sm, ls3Gd, 169Yb, ~ICr, 54Mn, 75Se, 113Sn, 9oyttrium, 117Tin, lg6Rhenium, 166Holmium, arid 188Rhenium;
luminescent labels, such as luminol;~ and fluorescent labels, such as fluorescein and rhodamine, and biotin. In a specific embodiment, the invention,provides a method for the ' specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 9°Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope lllln. In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 1311.
[362] Techniques known in the art may be applied to label polypeptides of the invention (including antibodies). Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. PatentNos_ 5,756,065;
5,714,631;5,696,239;
5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560;
and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety).
[363] Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying,Vthe expression level of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
[364] Moreover, polypeptides of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
[365] Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease (as described supra, and elsewhere herein). For example, administration of an antibody directed to a polypeptide of the present invention can bind, and/or neutralize the polypeptide, and/or reduce overproduction of the polypeptide.
Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
[366] At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS=PAGE gels or.on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the biological activities described herein.
Diagnostic Assays [367] The compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described herein under the section heading "Biological Activities".
[368] For a number of disorders; substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding the polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression levels) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
[369] The present invention is also useful. as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
[370] In certain embodiments, a, polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognose diseases and/or disorders associated with the tissues) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[371] By "assaying the expression level of the gene encoding the polypeptide"
is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or, the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA
level in a second biological sample). Preferably, the polypeptide expression level or mRNA
level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
[372] By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof] or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mamriials are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
[373] Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S 1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the -ligase chain reaction (RT-LCR).
[374J The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of polypeptides of the invention compared to normal control tissue samples may be used to detect the presence of tumors. Assay techniques that can be used to determine levels of a polypeptide, such as a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying polypeptide levels in a biological sample can occur. using any art-known method.
[375J Assaying polypeptide levels in a biological sample can occur using antibody-based techniques. For example, polypeptide expression in tissues can be studied ' with classical immunohistological methods (Jalkanen et al., J. Cell. Biol.
101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (lzsl, izil), carbon (14C), sulfur (3sS), tritium (3H), indium (llzln), and technetium (99mTC), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[376] The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of inteest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.
[377] For example, antibodies, or fragments of antibodies, such as those described herein, may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
[378] In a preferred embodiment, antibodies, or fragments of antibodies directed to any one or all of the predicted epitope domains of the polypeptides of the invention (shown in column 7 of Table 1A) may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
[379] In an additional preferred embodiment, antibodies, or fragments of antibodies directed to a conformational epitope of a polypeptide of the invention may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection. .
[380] The antibodies (or fragments thereof), and/or polypeptides of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The antibody (or fragment thereof) or polypeptide is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the gene product, or conserved variants or peptide fragments, or polypeptide binding, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection. _ [381] Immunoassays and non-immunoassays for gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detestably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
[382] The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detestably labeled antibody or detectable polypeptide of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.
(383] By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylaxnides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc.
Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
[384] The binding activity of a given lot of antibody or antigen polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
[385] In addition to assaying polypeptide levels or polynucleotide levels in a biological sample obtained from an individual, polypeptide or polynucleotide can also be detected in vivo by imaging. For example, in one embodiment of the invention, polypeptides and/or antibodies of the invention are used to image diseased cells, such as neoplasms. In another embodiment, polynucleotides of the invention (e.g., polynucleotides complementary to all or a portion of an mRNA) and/or antibodies (e.g., antibodies directed to any one or a combination of the epitopes of a polypeptide of the invention, antibodies directed to a conformational epitope of a polypeptide of the invention, or antibodies directed to the full length polypeptide expressed on the cell surface of a mammalian cell) are used to image diseased or neoplastic cells.
[386] Antibody labels or markers for in vivo imaging of polypeptides of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR
and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. Where a in vivo imaging is used to detect enhanced levels of polypeptides for diagnosis in humans, it may be preferable to use human antibodies or "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using techniques described herein or otherwise known in the art. For example methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567;
Taniguchi et al., EP
171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO
8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 31=1:268 (1985).
[387] Additionally, any polypeptides of the invention whose presence can be detected, can be administered. For example, polypeptides of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.
[388] ~ A polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 13i1, nzln~ 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the antigenic protein. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc.
(1982)).
[389] With respect to antibodies, one of the ways in which an antibody of the present invention can be delectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et al., J. Clin.
Pathol. 31:507-520 (1978); Butler, J.E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotornetric, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody include, but. are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme.
Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
[390] Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect polypeptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Tiaining Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, 'but not limited to, a gamma counter, a scintillation counter, or autoradiography.
[391] It is also possible to label the antibody with a fluorescent compound.
When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
[392] The antibody can also be detectably labeled using fluorescence emitting metals such as ls2Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[393] The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
(394] Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Methods for Detecting Diseases [395] In general, a disease may be detected in a patient based on the presence of one or more proteins of the invention and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, urine, and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a disease or disorder, including cancer and/or as described elsewhere herein. In addition, such proteins may be useful for the detection of other diseases and cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding polypeptides of the invention, which is also indicative of the presence or absence of a disease or disorder, including cancer. In general, polypeptides of the invention should be present at a level that is at least three fold higher in diseased tissue than in normal tissue.
[396] There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, supra. In general, the presence or absence of a disease in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value. , [397] In a preferred embodiment, the assay involves the use of a binding agents) immobilized on a solid support to bind to and remove the polypeptide of the invention from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include polypeptides of the invention and portions thereof, or antibodies, to which the binding agent binds, as described above.
[398] The solid support may be any material known to those of skill in the art to which polypeptides of the invention may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
Alternatively, the support may be a bead or disc, such as glass fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No.
5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature.
In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support br may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for the suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 ug, and preferably about 100 ng to about 1 ug, is sufficient to immobilize an adequate amount of binding agent.
[399] Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
Gene Therapy Methods [400] Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate t~o the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of the polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, W090/11092, which is herein incorporated by reference.
[401] Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide of the present invention. Such methods are well-known in the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 ( 1993); Ferrantini, M.
et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J.
Immunology 153:
4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-(199.6); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3 : 31-3 8 ( 1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
(402] As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like).
The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
[403] In one embodiment, the polynucleotide of the present invention is delivered as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotide of the present invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
[404] The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVI~3, pBPV, pMSG and pSVL
available from Pharmacia; and pEFl/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
[405] Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter;
inducible promoters, such as the MMT promoter, the metallothionein promoter;
heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters;
viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter;
retroviral LTRs; the b-actin promoter; and human growth. hormone promoters.
The promoter also may be the native promoter for the polynucleotide of the present invention.
[406] Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
(407] The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,. cartilage, pancreas, kidney, gall bladder,. stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue.
Interstitial space bf the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising These cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
[408] For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection.
The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
[409] The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used,, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during. angioplasty by the catheter used in the procedure.
[410] The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called "gene guns". These delivery methods are known in the art.
[411] The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
[412) In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.
However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid.
Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA
(Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:607.7-6081, which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol.
Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in functional form.
[413] Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA
(1987) 84:7413-7416, which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
[414] Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092-(which is herein incorporated by reference) for a description of the synthesis of DOTAP
( 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA
liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc.
Natl. Acad. Sci.
USA 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.
[415] Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such' materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatid~l glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also, be mixed with the DOTMA and DOTAP starting materials in appropriate ratios.
Methods for making liposomes using these materials are well known in the art.
[416] For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following 261 .
day with deionized water. The sa~rrple is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size.
Other methods are known and available to those of skill in the art.
[417] The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527, which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water ar an isotonic buffer solution such as 10 mM. Tris/NaCI, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art.
Commonly used methods include Ca2+-EDTA chelafion (Papahadjopoulos et al., Biochim.
Biophys.
Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D.- and Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem.
Biophys.
Res. Commun. 76:836 (1977); Fraley et aL, Proc. NatI. Acad. Sci. USA 76:3348 (1979));
detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad.. Sci.
USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem.
255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978);
Schaefer-Ridder et al., Science 215:166 (1982)), which are herein incorporated by reference.
[418] Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10.
Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
(419J U.S. Patent No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice.
U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA
into cells and.
mammals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.
[420] In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding a polypeptide of the present invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, .avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and. mammary tumor virus.
[421] The retroviral plasrnid vector is employed to transduce packaging cell lines to form producer cell lines. Exarr~ples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 ( 1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO~ precipitation. In one alternative, the retroviral plasmid vector may-be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
(422] The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a polypeptide~ of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a polypeptide of the present invention.
[423] In certain other embodiments, cells are engineered, ex vivo or i~ vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses a polypeptide of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis.109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA
76:6606).
[424] Suitable adenoviral vectors useful in the present invention are described, for example, in I~ozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993);
Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692 (1993); and U.S. Patent No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the El region of adenovirus and constitutively express Ela and Elb, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are also useful in the present invention. .
[425] Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide ~of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E 1 a, E 1 b, E3, E4, E2a, or L 1 through L5.
[426] In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr.
Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb.
Methods for producing and using such AAVs are known in the art. See, for example, U.S.
Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377. .
[427] For example, an appropriate AAV vector for use in the present invention will include aII the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc.
Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct.
These viral particles are then used to transduce eukaryotic cells,. either ex vivo or in vivo. The transduced cells will contain: the polynucleotide construct integrated into its genome, and will express a polypeptide of the invention.
[428] , Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994;
Koller et aL, Proc. Natl. Acad. .Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), which are herein encorporated by-reference. This method involves the activation of a gene which is .present in the target cells, but .which is not normally expressed in the cells; or is expressed at a Lower level than desired.
[429] Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter.
Suitable promoters are described herein. The targeting sequence is suff ciently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5' end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to~the endogenous sequence upon homologous recombination.
[430] The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5' and 3' ends. Preferably, the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3' end of the amplified promoter. The 'amplified promoter and targeting sequences are digested and ligated together.
(431] The promoter-targeting sequence construct is delivered to the cells, either as .
naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles,' whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
[432] The promoter-targeting sequence construct is taken up by cells.
Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
[433] The polynucleotide encoding a.polypeptide of the present invention may contain a secretory signal sequence that facilitates secretion of the protein.
Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5' end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
[434] Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, . catheter infusion, biolistic injectors, particle accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e;g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. Fox example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).
[435] A preferred method of local administration is by direct injection.
Preferably, a recombinant molecule of the present invention completed with a delivery vehicle is administered by direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
[436] Another method of local administration is to contact a~polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
[437] Therapeutic compositions useful in systemic administration, include' recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.
In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising polypeptides of the invention for targeting the vehicle to a particular site.
[438] Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery: Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad.
Sci. USA
189:11277-11281, 1992, W hick is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal.
Examples of such carriers, include plastic capsules or tablets, such as those known in.the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into 'the skin.
[439] Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
(440] Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
Biolo ical Activities [441] Polynucleotides or polypeptides; or agonists or antagonists of the present invention, 'can be used in assays to test for one or more biological activities. If these polynucleotides or polypeptides, or agonists or antagonists of the present invention, do exhibit activity in a particular assay, it is likely that these molecules may be involved in the _ diseases associated with the biological activity. Thus, the polynucleotides and polypeptides, and agonists or antagonists could be used to diagnose, prognose, prevent and/or treat the associated disease.
[442] Members of the calcium-binding family of proteins are believed to be involved in biological activities associated with gene expression, cell proliferation, cell death, and secretion processes. Accordingly, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated with aberrant calcium-binding protien activity.
[443] In preferred embodiments, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders relating to blood disorders (e:g., blood coagulation disorders, fibrinolysis disorders, and/or as described under "Immune activity" and "Cardiovascular Disorders"
below) neoplastic disorders (e.g., inappropriate cell mitosis, aberrant cytokinesis, and/or as described under "Hyperproliferative Disorders" below), and neural transmission (e.g., disorders of neurotransmitter exocytosis, dysfunctions associated with neural excitation, and/or as described under "Neural Activity and Neurological Disorders" below), and digestive/endocrine disorders (e.g., hypercholesterolemia, and/or as described under "Gastrointestinal Disorders" and "Endocrine Disorders" below).
[444] In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognose diseases and/or disorders associated with the tissues) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[445] Thus, polynucleotides, translation products and antibodies of the invention are useful in the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated. with activities that include, but are not limited to, blood coagulation, exo- and ~endocytosis, cytokinesis, gene expression, neurotransmitter release, and cholesterol uptake.
[446] More generally, polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated with the following systems.
Immune Activity [447] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation,, differentiation, or mobilization (chemotaxis) of immune cells.
Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T
lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious. Moxeover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
[448] In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissues) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[449] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing,' diagnosing, and/or prognosing immunodeficiencies,. including both congenital and acquired immunodeficiencies.
Examples of B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinem.ia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA
deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with increased IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired), and transient hypogammaglobulinemia of infancy.
[450] In specific embodiments, ataxia-telangiectasia or conditions associated with ataxia-telangiectasia are treated, prevented, diagnosed, and/or prognosing using the polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof.
[451] Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion,. chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
[452] In specific embodiments, DiGeorge anomaly or conditions associated with DiGeorge anomaly are treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides of the invention, or antagonists or agonists thereof.
[453] Other immunodeficiencies that may be treated, prevented, diagnosed, andlor prognosed using polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, include, but are not limited to, chronic granulomatous disease, Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including Cl, G2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs:
[454] In a preferred embodiment, the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
[455] In a preferred embodiment polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among B cell and/or T
cell iminunodeficient individuals.
(456] The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing andlor prognosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of polynucleotides and polypeptides of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing,autoimmune disorders.
[457] Autoimmune diseases or disorders that may be treated, prevented, diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoirrimune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thromliocytopenia, idiopathic, thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.
[458] Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.
(459] Additional disorders that are likely to have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by ~glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).
[460] Additional disorders that may have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g.; by IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous, degenerative, and atrophic disorders.
(461) In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using for example, antagonists or agonists, polypeptides or polynucleotides, or antibodies of the present invention. In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
[462] In another specific preferred embodiment, systemic lupus erythematosus is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies; and/or agonists or antagonists of the present invention.
[463] In another specific preferred embodiment IgA nephropathy is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
[464] In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or. antagonists of the present invention (465] In preferred embodiments, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a iminuno,suppressive agent(s).
[466] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, prognosing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells.
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia.
Alternatively,~Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the piesent invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.
[467] Allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosed using polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof. Moreover, these molecules can be used to treat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility. -[468] Additionally, polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, may be used to treat, prevent, diagnose andlor prognose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema. In specific embodiments, polynucleotides, polypeptides;
antibodies, and/or agonists or antagonists of the present invention may be used to modulate IgE
concentrations in vitro or in vivo.
[469] ' Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or ' antagonists of the present invention have uses in the diagnosis, prognosis, prevention, and/or treatment of inflammatory conditions. For example, since polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic. inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy);
gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis;
ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative'disorders (e.g., Parkinson's disease and Alzheimer's disease); AIDS-related dementia; and prion disease);
cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional diseases, conditions, and disorders that are characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout,' trauma, pancreatitis, , sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus, diabetes mellitus, and allogenic transplant rejection).
[470j Because inflammation is a fundamental defense mechanism, inflammatory disorders can effect virtually any tissue of the body. Accordingly, polynucleotides, polypeptides, and antibodies of the invention, as well as agonists or antagonists thereof, have uses in the treatment of tissue-specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, _ osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis. .
[471] In specific embodiments, polypeptides, antibodies, .or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat organ transplant rejections and graft-versus-host disease. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.
[472] In other embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat immune complex diseases, including, but not limited to, serum sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.
[473] Polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the invention can be used to treat, detect, and/or prevent infectious agents. For example, by .
increasing the immune response, particularly increasing the proliferation activation and/or differentiation of B and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.
(474] In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen. In a specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance tumor-specific immune responses.
[475] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or~antagonists of the present invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In .specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment; the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, paxainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
[476] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
[477] In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B
streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, and Borrelia burgdorferi.
[478] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an ~adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania.
[479] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis;
for example, by preventing the recruitment and activation of mononuclear phagocytes.
f (480] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.
[481J In one embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, carriel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
[482] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B
cell responsiveness to pathogens.
(483] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an activator of T
cells.
[484] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.
[485] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to induce higher affinity antibodies.
j486] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to increase serum immunoglobulin concentrations.
[487J In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to accelerate recovery of immunocompromised individuals. ' [488] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among aged populations and/or neonates.
[489] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or,-agonists or antagonists of the present invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, .
prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
[490] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or .
agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function.
Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
[491J In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency.
Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious .
mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.
[492] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo. Moreover, in related embodiments, said enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.
[493] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to direct an individual's immune 'system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.
[494] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.
[495] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B
cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.
[496] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect.
In another.
specific embodiments polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in the pretreatment of bone marrow samples prior to transplant.
[497] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetencelimmunodeficiency such as observed among SCID patients.
[498] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present . invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.
[499] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of regulating secreted eytokines that are elicited by polypeptides of the invention.
[500] In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in one or more of the applications decribed herein, as they may apply to veterinary medicine.
[501] In another specific embodiments polypeptides, antibodies, polynucleotides and/or agonists or antagonists, of the present invention are used as a means of blocking various aspects of immune responses to foreign agents or self. Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimrnune disorders such as lupus, and arthritis, as well as immunoresponsiveness to. skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.
[502] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for preventing the B
cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erytheriiatosus and multiple sclerosis.
[503] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a inhibitor of B
and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
[504] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for chronic hypergammaglobulinemia evident in such diseases a's monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.
[505] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells,.in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes. .
[506] The polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.
[507] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit complement mediated cell lysis.
[508] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.
[509] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.
[510] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed to treat adult respiratory distress syndrome CARDS).
[511] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, agonists and antagonists of the invention may be used to stimulate the regeneration of mucosal surfaces.
[512] In a specific. embodiment, polynucleotides or polypeptides, and/or agonists thereof are used to diagnose, prognose, treat, and/or prevent a disorder characterized by primary or acquired immunodeficiency, deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, polynucleotides or polypeptides, and/or agonists thereof may be used to treat or prevent infections of the joints, bones, skin, andlor parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, andlor osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseases and disorders that may be prevented, diagnosed, prognosed, and/or treated with polynucleotides or polypeptides, and/or agonists of the present invention include,~but are not limited to, HIV
infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.
[513] In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention axe used to treat, and/or diagnose an individual having common variable immunodeficiency disease ("CVID"; also known as "acquired agammaglobulinemia" and "acquired hypogammaglobulinemia") or a subset of this disease.
[514] In a specific embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to diagnose, prognose, prevent, and/or treat cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms. Examples of cancers or neoplasms that may be prevented, diagnosed, or treated by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and disorders described in the section entitled "Hyperproliferative Disorders" elsewhere herein.
[515] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
[516] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
[517] In specific embodiments, the compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.
[518]. Antagonists of the invention include, for example, binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms of the polypeptides of the present invention (e.g., Fc fusion protein; see, e.g., Example 9). Agonists of the invention include, for example, binding or stimulatory antibodies, and soluble forms of the polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9). ,polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described herein.
[519] In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of.producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin.
molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. W098/24893, WO/9634096, WO/9633735, and . W019110741). Administration of polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention to such animals is useful for the generation of monoclonal antibodies against the polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
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Claims (24)
1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of:
(a) a polynucleotide fragment of SEQ ID NO:X or a polynucleotide fragment of the cDNA sequence contained in Clone ID NO:Z, which is hybridizable to SEQ ID
NO:X;
(b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(c) a polynucleotide encoding a polypeptide fragment of a polypeptide encoded by SEQ ID NO:X or a polypeptide fragment encoded by the cDNA sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X;
(d) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y or a polypeptide domain encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(e) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:Y or a polypeptide epitope encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(f) a polynucleotide encoding a polypeptide of SEQ ID NO:Y or the cDNA
sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X, having biological activity;
(g) a polynucleotide which is a variant of SEQ ID NO:X;
(h) a polynucleotide which is an allelic variant of SEQ ID NO:X;
(i) a polynucleotide which encodes a species homologue of the SEQ ID NO:Y;
(j) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A
residues or of only T residues.
(a) a polynucleotide fragment of SEQ ID NO:X or a polynucleotide fragment of the cDNA sequence contained in Clone ID NO:Z, which is hybridizable to SEQ ID
NO:X;
(b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(c) a polynucleotide encoding a polypeptide fragment of a polypeptide encoded by SEQ ID NO:X or a polypeptide fragment encoded by the cDNA sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X;
(d) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y or a polypeptide domain encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(e) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:Y or a polypeptide epitope encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(f) a polynucleotide encoding a polypeptide of SEQ ID NO:Y or the cDNA
sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X, having biological activity;
(g) a polynucleotide which is a variant of SEQ ID NO:X;
(h) a polynucleotide which is an allelic variant of SEQ ID NO:X;
(i) a polynucleotide which encodes a species homologue of the SEQ ID NO:Y;
(j) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A
residues or of only T residues.
2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a protein.
3. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:Y
or the polypeptide encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X.
or the polypeptide encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X.
4. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:X or the cDNA
sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X.
sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X.
5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
7. A recombinant vector comprising the isolated nucleic acid molecule of claim 1.
8. A method of making a recombinant host cell comprising the isolated nucleic acid molecule of claim 1.
9. A recombinant host cell produced by the method of claim 8.
10. The recombinant host cell of claim 9 comprising vector sequences.
11. An isolated polypeptide comprising an amino acid sequence at least 90%
identical to a sequence selected from the group consisting of:
(a) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(b) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z, having biological activity;
(c) a polypeptide domain of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(d) a polypeptide epitope of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(e) a full length protein of SEQ ID NO:Y or the encoded sequence contained in cDNA
Clone ID NO:Z;
(f) a variant of SEQ ID NO:Y;
(g) an allelic variant of SEQ ID NO:Y; or (h) a species homologue of the SEQ ID NO:Y.
identical to a sequence selected from the group consisting of:
(a) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(b) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z, having biological activity;
(c) a polypeptide domain of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(d) a polypeptide epitope of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(e) a full length protein of SEQ ID NO:Y or the encoded sequence contained in cDNA
Clone ID NO:Z;
(f) a variant of SEQ ID NO:Y;
(g) an allelic variant of SEQ ID NO:Y; or (h) a species homologue of the SEQ ID NO:Y.
12. The isolated polypeptide of claim 11, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
13. An isolated antibody that binds specifically to the isolated polypeptide of claim 11.
14. A recombinant host cell that expresses the isolated polypeptide of claim 11.
15. A method of making an isolated polypeptide comprising:
(a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.
(a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.
16. The polypeptide produced by claim 15.
17. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polynucleotide of claim 1.
18. A method of diagnosing a pathological condition or a susceptibility to a .
pathological condition in a subject comprising:
(a) determining the presence or absence of a mutation in the polynucleotide of claim 1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.
pathological condition in a subject comprising:
(a) determining the presence or absence of a mutation in the polynucleotide of claim 1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.
19. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or amount of expression of the polypeptide of claim 11 in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
(a) determining the presence or amount of expression of the polypeptide of claim 11 in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
20. A method for identifying a binding partner to the polypeptide of claim 11 comprising:
(a) contacting the polypeptide of claim 11 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.
(a) contacting the polypeptide of claim 11 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.
21. The gene corresponding to the cDNA sequence of SEQ ID NO:Y.
22. A method of identifying an activity in a biological assay, wherein the method comprises:
(a) expressing SEQ ID NO:X in a cell;
(b) isolating the supernatant;
(c) detecting an activity in a biological assay; and identifying the protein in the supernatant having the activity.
(a) expressing SEQ ID NO:X in a cell;
(b) isolating the supernatant;
(c) detecting an activity in a biological assay; and identifying the protein in the supernatant having the activity.
23. The product produced by the method of claim 20.
24. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 11.
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Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2395787A1 true CA2395787A1 (en) | 2001-08-02 |
Family
ID=27587117
Family Applications (37)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002395724A Pending CA2395724A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395885A Withdrawn CA2395885A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392398A Abandoned CA2392398A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002397839A Withdrawn CA2397839A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002393002A Withdrawn CA2393002A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395693A Pending CA2395693A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002394039A Abandoned CA2394039A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395858A Withdrawn CA2395858A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395787A Withdrawn CA2395787A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395849A Pending CA2395849A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395666A Withdrawn CA2395666A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395671A Withdrawn CA2395671A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395734A Withdrawn CA2395734A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395699A Pending CA2395699A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392757A Abandoned CA2392757A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392450A Abandoned CA2392450A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002394841A Withdrawn CA2394841A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395178A Withdrawn CA2395178A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395729A Pending CA2395729A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395403A Withdrawn CA2395403A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002397407A Withdrawn CA2397407A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002393652A Withdrawn CA2393652A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392751A Abandoned CA2392751A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392438A Abandoned CA2392438A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395816A Withdrawn CA2395816A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins and antibodies |
| CA002395827A Withdrawn CA2395827A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395872A Withdrawn CA2395872A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002393912A Withdrawn CA2393912A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395654A Withdrawn CA2395654A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002393618A Abandoned CA2393618A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395398A Withdrawn CA2395398A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002398877A Withdrawn CA2398877A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395295A Withdrawn CA2395295A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins and antibodies |
| CA002398411A Withdrawn CA2398411A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395738A Withdrawn CA2395738A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392428A Abandoned CA2392428A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392422A Abandoned CA2392422A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
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| CA002395885A Withdrawn CA2395885A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392398A Abandoned CA2392398A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002397839A Withdrawn CA2397839A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002393002A Withdrawn CA2393002A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
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| CA002395666A Withdrawn CA2395666A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395671A Withdrawn CA2395671A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395734A Withdrawn CA2395734A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395699A Pending CA2395699A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392757A Abandoned CA2392757A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392450A Abandoned CA2392450A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002394841A Withdrawn CA2394841A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395178A Withdrawn CA2395178A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395729A Pending CA2395729A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395403A Withdrawn CA2395403A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002397407A Withdrawn CA2397407A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002393652A Withdrawn CA2393652A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392751A Abandoned CA2392751A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392438A Abandoned CA2392438A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395816A Withdrawn CA2395816A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins and antibodies |
| CA002395827A Withdrawn CA2395827A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395872A Withdrawn CA2395872A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002393912A Withdrawn CA2393912A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395654A Withdrawn CA2395654A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002393618A Abandoned CA2393618A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395398A Withdrawn CA2395398A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002398877A Withdrawn CA2398877A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395295A Withdrawn CA2395295A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins and antibodies |
| CA002398411A Withdrawn CA2398411A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002395738A Withdrawn CA2395738A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392428A Abandoned CA2392428A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002392422A Abandoned CA2392422A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
Country Status (3)
| Country | Link |
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| AU (16) | AU2001241409A1 (en) |
| CA (37) | CA2395724A1 (en) |
| WO (48) | WO2001055312A2 (en) |
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