AU5816799A - Compositions and methods for the treatment of tumor - Google Patents

Description

WO 00/15666 PCT/US99/20594 5 COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR Field of the Invention The present invention relates to compositions and methods for the diagnosis and treatment of tumor. 10 Backg-round of the Invention Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin. 43:7 [1993]). Cancer is characterized by an increase in the number of abnormal, or neoplastic cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor 15 cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis). In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness. Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differentiation 20 which are a common feature of all cancers. The genomes of certain well studied tumors have been found to show decreased expression of recessive genes, usually referred to as tumor suppression genes, which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes, that act to promote malignant growth. Each of these genetic changes appears to be responsible for importing some of the traits that, in aggregate, represent the full neoplastic phenotype (Hunter, Cell, 64:1129 [1991] and Bishop, 25 Cell 64:235-248 [1991]). A well known mechanism of gene (e.g., oncogene) overexpression in cancer cells is gene amplification. This is a process where in the chromosome of the ancestral cell multiple copies of a particular gene are produced. The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome (Alitalo et al., Adv. Cancer Res., 47:235-281 30 [1 986]). It is believed that the overexpression of the gene parallels gene amplification, i.e., is proportionate to the number of copies made. Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer. For example, it has been found that the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd 35 transmembrane glycoprotein receptor (p1 85 H"; HER2) related to the epidermal growth factor receptor EGFR), is overexpressed in about 25%to 30% of human breast cancer (Slamon et al., Science 235:177-182 [1987]; Slamon et al., Science 244:707-712 [1989]). -1- WO 00/15666 PCT/US99/20594 It has been reported that gene amplification of a proto-oncogene is an event typically involved in the more malignant forms of cancer, and could act as a predictor of clinical outcome (Schwab et al., Genes Chromosomes Cancer, 1_:181-193 [1990]; Alitalo et al., supra). Thus, erbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et al., 5 [1987] and [1 989], supra; Ravdin and Chamness, Gene, .59:19-27 [1995]; and Hynes and Stern, Biochim. Biophys. Acta, 1198:165-184 [1994]), and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines (Baselga et al., Oncology, j (3 Suppl 1):43-48 [1997]). However, despite the association of erbB2 overexpression with poor prognosis, the odds of HER2-positive patients responding clinically to treatment with taxanes were 10 greater than three times those of HER2-negative patients (Ibid). A recombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (a humanized version of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or Herceptin T M ) has been clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. (Baselga et al., J. Clin. Oncol.. 14:737-744 [1996]). In light of the above, there is obvious interest in identifying novel methods and compositions which are 15 useful for diagnosing and treating tumors which are associated with gene amplification. Summary of the Invention The present invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans. The present invention is based on the identification 20 of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to be associated with the overexpression of the gene product and contribute to tumorigenesis. Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevention) of certain cancers, and may act as predictors of the prognosis of tumor treatment. In one embodiment, the present invention concerns an isolated antibody which binds to a polypeptide 25 designated herein as a PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 polypeptide. In one aspect, the isolated antibody specifically binds to a PRO87, PRO533, PRO214, PR0240, PRO211, PRO230, PRO261, PRO246 or PRO317 polypeptide. In another aspect, the antibody induces the death of a cell which expresses a PRO187, PR0533, PRO214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 polypeptide. Often, the cell that expresses the PRO 187, PR0533, PRO214, PRO240, PRO2 11, PR0230, 30 PR0261, PR0246 or PR0317 polypeptide is a tumor cell that overexpresses the polypeptide as compared to a normal cell of the same tissue type. In yet another aspect, the antibody is a monoclonal antibody, which preferably has non-human complementarity determining region (CDR) residues and human framework region (FR) residues. The antibody may be labeled and may be immobilized on a solid support. In yet another aspect, the antibody is an antibody fragment, a single-chain antibody, or a humanized antibody which binds, preferably specifically, to a 35 PRO187, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 polypeptide. In another embodiment, the invention concerns a composition of matter which comprises an antibody which binds, preferably specifically, to a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, -2- WO 00/15666 PCT/US99/20594 PR0246 or PRO317 polypeptide in admixture with a pharmaceutically acceptable carrier. In one aspect, the composition of matter comprises a therapeutically effective amount of the antibody. In another aspect, the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent. Preferably, the composition is sterile. 5 In a further embodiment, the invention concerns isolated nucleic acid molecules which encode anti PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PRO246 or anti-PRO317 antibodies, and vectors and recombinant host cells comprising such nucleic acid molecules. In a still further embodiment, the invention concerns a method for producing an anti-PRO187, anti PR0533, anti-PR0214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 10 antibody, wherein the method comprises culturing a host cell transformed with a nucleic acid molecule which encodes the antibody under conditions sufficient to allow expression of the antibody, and recovering the antibody from the cell culture. The invention further concerns antagonists of a PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 polypeptide that inhibit one or more of the biological and/or immunological 15 functions or activities of a PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 polypeptide. In a further embodiment, the invention concerns an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule encoding a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide or the complement thereof. The isolated nucleic acid molecule is preferably DNA, and 20 hybridization preferably occurs under stringent hybridization and wash conditions. Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein, which, in turn, can find use in the modulation of the transcription and/or translation of the respective amplified genes, or as antisense primers in amplification reactions. Furthermore, such sequences can be used as part of a ribozyme and/or a triple helix sequence which, in turn, may be used in regulation of the amplified genes. 25 In another embodiment, the invention provides a method for determining the presence of a PRO187, PRO533, PRO214, PR0240, PRO2 11, PRO230, PR0261, PR0246 or PRO317 polypeptide in a sample suspected of containing a PRO87, PR0533, PR0214, PR0240, PRO211, PR0230, PRO261, PR0246 or PR0317 polypeptide, wherein the method comprises exposing the sample to an anti-PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO2 11, anti-PR0230, anti-PR026 1, anti-PR0246 or anti-PRO317 antibody and determining 30 binding of the antibody to a PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 polypeptide in the sample. In another embodiment, the invention provides a method for determining the presenceofa PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide in a cell, wherein the method comprises exposing the cell to an anti-PRO 187, anti-PR0533, anti-PRO214, anti PRO240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody and determining 35 binding of the antibody to the cell. In yet another embodiment, the present invention concerns a method of diagnosing tumor in a mammal, comprising detecting the level of expression of a gene encoding a PRO187, PRO533, PRO214, PR0240, PRO211, -3- WO 00/15666 PCT/US99/20594 PR0230, PR0261, PR0246 or PRO3 17 polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test tissue cells were obtained. 5 In another embodiment, the present invention concerns a method of diagnosing tumor in a mammal, comprising(a)contactingan anti-PRO1 87,anti-PRO533, anti-PRO214, anti-PR0240, anti-PRO2 11, anti- PRO230, anti-PR026 1, anti-PR0246 or anti-PRO317 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the anti-PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody and a PRO187, PR0533, 10 PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal. The detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type. A larger quantity of complexes formed in the test sample indicates the presence of tumor in the mammal from which the test tissue cells were obtained. The antibody 15 preferably carries a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. The test sample is usually obtained from an individual suspected to have neoplastic cell growth or proliferation (e.g. cancerous cells). In another embodiment, the present invention concernsa cancer diagnostickit comprising an anti-PRO 187, 20 anti-PR0533, anti-PR0214, anti-PR0240, anti-PRO211, anti-PR0230, anti-PR0261, anti-PR0246 or anti PRO317 antibody and a carrier (e.g., a buffer) in suitable packaging. The kit preferably contains instructions for using the antibody to detect the presence of a PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide in a sample suspected of containing the same. In yet another embodiment, the invention concerns a method for inhibiting the growth of tumor cells 25 comprising exposing tumor cells which express a PRO187, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 polypeptide to an effective amount of an agent which inhibits a biological and/or immunological activity and/or the expression of a PRO 187, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 polypeptide, wherein growth of the tumor cells is thereby inhibited. The agent preferably is an anti-PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO211, anti-PR0230, anti 30 PR0261, anti-PRO246or anti-PRO317 antibody, a small organic and inorganic molecule, peptide, phosphopeptide, antisense or ribozyme molecule, or a triple helix molecule. In a specific aspect, the agent, e.g., the anti-PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti PRO317 antibody, induces cell death. In a further aspect, the tumor cells are further exposed to radiation treatment and/or a cytotoxic or chemotherapeutic agent. 35 In a further embodiment, the invention concerns an article of manufacture, comprising: a container; a label on the container; and -4- WO 00/15666 PCT/US99/20594 a composition comprising an active agent contained within the container; wherein the composition is effective for inhibiting the growth of tumor cells and the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PROl87, PRO533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide as compared to a normal cell ofthe same tissue type. 5 In particular aspects, the active agent in the composition is an agent which inhibits an activity and/or the expression of a PRO187, PR0533, PR0214, PR0240, PRO211, PR0230. PR0261, PR0246 or PR0317 polypeptide. In preferred aspects, the active agent is an anti-PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody or an antisense oligonucleotide. The invention also provides a method for identifying a compound that inhibits an activity of a PRO] 87, 10 PR0533, PRO214, PRO240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 polypeptide,comprisingcontacting a candidate compound with a PROL87, PR0533, PR0214, PRO240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide under conditions and for a time sufficient to allow these two components to interact and determining whether a biological and/or immunological activity of the PRO187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 polypeptide is inhibited. In a specific aspect, either the candidate 15 compound or the PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PR0317 polypeptide is immobilized on a solid support. In another aspect, the non-immobilized component carries a detectable label. In a preferred aspect, this method comprises the steps of (a) contacting cells and a candidate compound to be screened in the presence of PRO187, PRO533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide under conditions suitable for the induction of a cellular response normally induced 20 by aPROl87, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 orPRO317 polypeptide and (b) determining the induction of said cellular response to determine if the test compound is an effective antagonist. In another embodiment, the invention provides a method for identifying a compound that inhibits the expression of a PRO87, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PR0317 polypeptide in cells that express the polypeptide, wherein the method comprises contacting the cells with a 25 candidate compound and determining whether the expression of the PRO187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 polypeptide is inhibited. In a preferred aspect, this method comprises the steps of (a) contacting cells and a candidate compound to be screened under conditions suitable for allowing expression of the PRO187, PR0533, PRO214, PR0240, PRO211, PR0230, PR0261, PR0246 or PR0317 polypeptide and (b) determining the inhibition of expression of said polypeptide. 30 Brief Description of the Figures Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) of a cDNA containing a nucleotide sequence encoding native sequence PRO 187, wherein the nucleotide sequence (SEQ ID NO: 1) is a clone designated herein as DNA27864-1155. Also presented in bold font and underlined are the positions of the respective start and stop 35 codons. Figure 2 shows the amino acid sequence (SEQ ID NO:2) of a native sequence PRO187 polypeptide as derived from the coding sequence of SEQ ID NO: 1. -5- WO 00/15666 PCTIUS99/20594 Figure 3 shows the nucleotide sequence (SEQ ID NO:6) of a cDNA containing a nucleotide sequence encoding native sequence PRO533, wherein the nucleotide sequence (SEQ ID NO:6) is a clone designated herein as DNA49435-1219. Also presented in bold font and underlined are the positions of the respective start and stop codons. 5 Figure 4 shows the amino acid sequence (SEQ ID NO:7) of a native sequence PRO533 polypeptide as derived from the coding sequence of SEQ ID NO:6. Figure 5 shows the nucleotide sequence (SEQ ID NO: 11) of a cDNA containing a nucleotide sequence encoding native sequence PRO214, wherein the nucleotide sequence (SEQ ID NO: 11) is a clone designated herein as DNA32286-1191. Also presented in bold font and underlined are the positions of the respective start and stop 10 codons. Figure 6 shows the amino acid sequence (SEQ ID NO: 12) of a native sequence PR0214 polypeptide as derived from the coding sequence of SEQ ID NO: 11. Figure 7 shows the nucleotide sequence (SEQ ID NO:16) of a cDNA containing a nucleotide sequence encoding native sequence PRO240, wherein the nucleotide sequence (SEQ ID NO: 16) is a clone designated herein 15 as DNA34387-l 138. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 8 shows the amino acid sequence (SEQ ID NO: 17) of a native sequence PRO240 polypeptide as derived from the coding sequence of SEQ ID NO:16. Figure 9 shows the nucleotide sequence (SEQ ID NO:21) of a cDNA containing a nucleotide sequence 20 encoding native sequence PRO2 11, wherein the nucleotide sequence (SEQ ID NO:2 1) is a clone designated herein as DNA32292-1131. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 10 shows the amino acid sequence (SEQ ID NO:22) of a native sequence PRO21 I polypeptide as derived from the coding sequence of SEQ ID NO:2 1. 25 Figure I I shows the nucleotide sequence (SEQ ID NO:26) of a cDNA containing a nucleotide sequence encoding native sequence PR0230, wherein the nucleotide sequence (SEQ ID NO:26) is a clone designated herein as DNA33223-1136. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 12 shows the amino acid sequence (SEQ ID NO:27) of a native sequence PR0230 polypeptide as 30 derived from the coding sequence of SEQ ID NO:26. Figure 13 shows the nucleotide sequence (SEQ ID NO:3 1) of a cDNA containing a nucleotide sequence encoding native sequence PRO26 1, wherein the nucleotide sequence (SEQ ID NO:3 1) is a clone designated herein as DNA33473-1176. Also presented in bold font and underlined are the positions of the respective start and stop codons. 35 Figure 14 shows the amino acid sequence (SEQ ID NO:32) of a native sequence PR0261 polypeptide as derived from the coding sequence of SEQ ID NO:3 1. Figure 15 shows the nucleotide sequence (SEQ ID NO:36) of a cDNA containing a nucleotide sequence -6- WO 00/15666 PCT/US99/20594 encoding native sequence PR0246, wherein the nucleotide sequence (SEQ ID NO:36) is a clone designated herein as DNA35639-1172. Also presented in bold font and underlined are the positions of the respective start and stop codons. Figure 16 shows the amino acid sequence (SEQ ID NO:37) of a native sequence PR0246 polypeptide as 5 derived from the coding sequence of SEQ ID NO:36. Figure 17 shows the nucleotide sequence (SEQ ID NO:4 1) of a cDNA containing a nucleotide sequence encoding native sequence PRO317, wherein the nucleotide sequence (SEQ ID NO:4 1) is a clone designated herein as DNA33461-1199. Also presented in bold font and underlined are the positions of the respective start and stop codons. 10 Figure 18 shows the amino acid sequence (SEQ ID NO:42) of a native sequence PRO3 17 polypeptide as derived from the coding sequence of SEQ ID NO:4 1. Figures 19A through 19D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Figures 19A-B) and % nucleic acid sequence identity (Figures 19C D) using the ALIGN-2 sequence comparison computer program, wherein "PRO" represents the amino acid 15 sequence of a hypothetical PRO87, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared, "PRO-DNA" represents a hypothetical PRO 187-, PR0533-, PRO214-, PR0240-, PRO2 11-, PR0230-, PR026 1-, PR0246- or PRO317-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule 20 against which the "PRO-DNA" nucleic acid molecule of interest is being compared, "X, "Y" and "Z" each represent different hypothetical amino acid residues and "N", "L" and "V" each represent different hypothetical nucleotides. Figures 20A through 20Q provide the complete source code for the ALIGN-2 sequence comparison computer program. This source code may be routinely compiled for use on a UNIX operating system to provide 25 the ALIGN-2 sequence comparison computer program. Figure 21 is a map of Chromosome 8 showing the mapping region of DNA27864-1155. Figure 22 is a map of Chromosome 2 showing the mapping region of DNA34387-1138. Figure 23 is a map of Chromosome 1 showing the mapping region of DNA33223-l 136. 30 Detailed Description of the Invention I. Definitions The phrases "gene amplification" and "gene duplication" are used interchangeably and refer to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. The duplicated region (a stretch of amplified DNA) is often referred to as "amplicon." Usually, the amount of the messenger RNA 35 (mRNA) produced, i.e., the level of gene expression, also increases in the proportion of the number of copies made of the particular gene expressed. "Tumor", as used herein, refers to all neoplastic cell growth and proliferation, whethermalignant or benign, -7- WO 00/15666 PCT/US99/20594 and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, 5 prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer. "Treatment" is an intervention performed with the intention of preventing the development or altering the 10 pathology of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need oftreatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy. 15 The "pathology" of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, 20 domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cattle, pigs, sheep, etc. Preferably, the mammal is human. "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable 25 carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming 30 counterions such as sodium; and/or nonionic surfactants such as TWEEN T M , polyethylene glycol (PEG), and PLURONICSTM. Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of 35 cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., Ism, 112s, Yo and Re "6), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. -8- WO 00/15666 PCTIUS99/20594 A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rh6ne-PoulencRorer, Antony, Rnace), toxotere, methotrexate, 5 cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Pat. No. 4,675,187), 5-FU., 6-thioguanine, 6-mercaptopurine, actinomycin D, VP-16, chlorambucil, melphalan, and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone. 10 A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo. Thus, the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G I arrest and M-phase arrest. Classical M-phase blockers include the 15 vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G 1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5 fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al., (WB 20 Saunders: Philadelphia, 1995), especially p. 13. "Doxorubicin" is an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis)-10-(3 amino-2,3,6-trideoxy-a-L-lyxo-hexapyranosyl)oxy]-7,8,9, 10-tetrahydro-6,8,11 -trihydroxy-8-(hydroxyacetyl)-I methoxy-5,12-naphthacenedione. The term "cytokine" is a generic term for proteins released by one cell population which act on another 25 cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; 30 prolactin; placental lactogen; tumor necrosis factor-a and -P; mullerian-inhibiting substance; mouse gonadotropin associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-P; platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and TGF-P; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon -a, -P, and -y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte 35 macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL- la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-I 1, IL-12; a tumor necrosis factor such as TNF-a or TNF-B; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from -9- WO 00/15666 PCT/US99/20594 natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable 5 of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions 14:375-382, 615th Meeting, Belfast (1986), and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery", Directed Drug Delivery. Borchardt et al., (ed.), pp. 147-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, phosphate containingprodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containingprodrugs, 10 D-amino acid-modified prodrugs, glysocylated prodrugs, B-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5 fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrugs form for use in this invention include, but are not limited to, those chemotherapeutic agents described above. 15 An "effective amount" of a polypeptide disclosed herein or an antagonist thereof, in reference to inhibition of neoplastic cell growth, tumor growth or cancer cell growth, is an amount capable of inhibiting, to some extent, the growth of target cells. The term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells. An "effective amount" of a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide antagonist for purposes of inhibiting 20 neoplastic cell growth, tumor growth or cancer cell growth, may be determined empirically and in a routine manner. A "therapeutically effective amount", in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects: (1) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs; (5) 25 inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; and/or (7) relief, to some extent, of one or more symptoms associated with the disorder. A "therapeutically effective amount" of a PRO]87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide antagonist for purposes of treatment of tumor may be determined empirically and in a routine manner. 30 A "growth inhibitory amount" of a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 antagonist is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A "growth inhibitory amount" of a PRO187, PR0533, PR0214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner. 35 A "cytotoxic amount" of a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 antagonist is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A "cytotoxic amount" of a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, -10- WO 00/15666 PCTIUS99/20594 PR0261, PR0246 or PRO317 antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner. The terms "PROl 87", "PRO533", "PRO2 14", "PR0240", "PRO21 ", "PR0230", "PRO261", "PR0246" or "PRO317", "PRO187 polypeptide", "PR0533 polypeptide", "PR0214 polypeptide". "PR0240 polypeptide", 5 "PRO211 polypeptide", "PR0230 polypeptide", "PR0261 polypeptide", "PR0246 polypeptide" or "PRO317 polypeptide" when used herein encompass native sequence PRO187, PRO533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptides and PROl87, PR0533, PR0214, PR0240, PRO21 1, PRO230, PRO261, PR0246 or PRO317 polypeptide variants (which are further defined herein). The PRO187, PR0533, PRO214, PRO240, PRO21 1, PR0230, PRO261, PR0246 or PRO317 polypeptide may be isolated from 10 a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods. A "native sequence" PROl87, PR0533, PR0214, PRO240, PRO21 1, PRO230, PR0261, PRO246 or PRO317 polypeptide comprises a polypeptide having the same amino acid sequence as a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide derived from nature. Such 15 native sequence PRO87, PR0533, PR0214, PR0240, PRO211, PRO230, PR0261, PR0246 or PR0317 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means. The term "native sequence" PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide specifically encompasses naturally-occurring truncated or secreted forms (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic 20 variants of the PRO187, PRO533, PR0214, PR0240, PRO211, PRO230, PR0261, PR0246 and PR0317 polypeptide. In certain embodiments of the invention, the native sequence PRO 187, PR0533, PRO214, PRO240, PRO21 1, PRO230, PR026 1, PR0246 or PRO317 polypeptide is a mature or full-length native sequence PROl 87, PR0533, PRO214, PR0240, PRO2 11, PRO230, PR026 1, PR0246 or PRO317 polypeptide comprising the amino acid sequence of Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO:12), Figure 8 (SEQ 25 ID NO: 17), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:27), Figure 14 (SEQ ID NO:32), Figure 16 (SEQ ID NO:37), or Figure 18 (SEQ ID NO:42), respectively. Fragments of the respective native polypeptides herein include, but are not limited, to polypeptide variants from which the native N-terminal signal sequence has been fully or partially deleted or replaced by another sequence, and extracellular domains of the respective native sequences, regardless whether such truncated (secreted) forms occur in nature. Fragments are preferably sufficient in length 30 for the production of an antibody specifically binding the corresponding native "PRO" polypeptide. "PRO 187 variant polypeptide" means an active polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues I or about 23 to 205 of the PRO 187 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) X to 205 of the PRO 187 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 18 to 27 of Figure 2 (SEQ ID NO:2) or (c) another specifically 35 derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO:2). "PR0533 variant polypeptide" means an active polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 23 to 216 of the PR0533 -11- WO 00/15666 PCT/US99/20594 polypeptide shown in Figure 4 (SEQ ID NO:7), (b) X to 216 of the PR0533 polypeptide shown in Figure 4 (SEQ ID NO:7), wherein X is any amino acid residue from 18 to 27 of Figure 4 (SEQ ID NO:7) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7). "PR0214 variant polypeptide" means an active polypeptide as defined below having at least about 80% 5 amino acid sequence identity with the amino acid sequence of (a) residues I or about 30 to 420 of the PR0214 polypeptide shown in Figure 6 (SEQ ID NO: 12), (b) X to 420 of the PRO214 polypeptide shown in Figure 6 (SEQ ID NO: 12), wherein X is any amino acid residue from 25 to 34 of Figure 6 (SEQ ID NO: 12), (c) I or about 30 to X of Figure 6 (SEQ ID NO: 12), wherein X is any amino acid from amino acid 367 to amino acid 376 of Figure 6 (SEQ ID NO: 12) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ 10 ID NO:12). "PR0240 variant polypeptide" means an active polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues I or about 31 to 229 of the PR0240 polypeptide shown in Figure 8 (SEQ ID NO: 17), (b) X to 229 of the PR0240 polypeptide shown in Figure 8 (SEQ ID NO:17), wherein X is any amino acid residue from 26 to 35 of Figure 8 (SEQ ID NO:17), (c) I or about 31 to 15 X of Figure 8 (SEQ ID NO: 17), wherein X is any amino acid from amino acid 193 to amino acid 202 of Figure 8 (SEQ ID NO: 17) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 8 (SEQ ID NO:17). "PRO2 11 variant polypeptide" means an active polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues I or about 25 to 353 of the PRO211 20 polypeptide shown in Figure 10 (SEQ ID NO:22), (b) X to 353 of the PRO21 I polypeptide shown in Figure 10 (SEQ ID NO:22), wherein X is any amino acid residue from 20 to 29 of Figure 10 (SEQ ID NO:22) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 10 (SEQ ID NO:22). "PR0230 variant polypeptide" means an active polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 22 to 164 of the PR0230 25 polypeptide shown in Figure 12 (SEQ ID NO:27), (b) X to 164 of the PR0230 polypeptide shown in Figure 12 (SEQ ID NO:27), wherein X is any amino acid residue from 17 to 26 of Figure 12 (SEQ ID NO:27) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 12 (SEQ ID NO:27). "PR0261 variant polypeptide" means an active polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 24 to 250 of the PR0261 30 polypeptide shown in Figure 14 (SEQ ID NO:32), (b) X to 250 of the PR0261 polypeptide shown in Figure 14 (SEQ ID NO:32), wherein X is any amino acid residue from 19 to 28 of Figure 14 (SEQ ID NO:32) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 14 (SEQ ID NO:32). "PR0246 variant polypeptide" means an active polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues I or about 30 to 390 of the PR0246 35 polypeptide shown in Figure 16 (SEQ ID NO:37), (b) X to 390 of the PR0246 polypeptide shown in Figure 16 (SEQ ID NO:37), wherein X is any amino acid residue from 25 to 34 of Figure 16 (SEQ ID NO:37), (c) 1 or about 30 to X of Figure 16 (SEQ ID NO:37), wherein X is any amino acid from amino acid 242 to amino acid 251 of -12- WO 00/15666 PCTIUS99/20594 Figure 16 (SEQ ID NO:37) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 16 (SEQ ID NO:37). "PRO317 variant polypeptide" means an active polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues I or about 19 to 366 of the PRO317 5 polypeptide shown in Figure 18 (SEQ ID NO:42), (b) X to 366 of the PR0317 polypeptide shown in Figure 18 (SEQ ID NO:42), wherein X is any amino acid residue from 14 to 23 of Figure 18 (SEQ ID NO:42) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 18 (SEQ ID NO:42). Such PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 and PRO317 variant polypeptides include, for instance, PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 10 or PR0317 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C terminus, as well as within one or more internal domains, of the sequence of Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO:12), Figure 8 (SEQ ID NO:17), Figure 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:27), Figure 14 (SEQ ID NO:32), Figure 16 (SEQ ID NO:37), or Figure 18 (SEQ ID NO:42), respectively. 15 Ordinarily, a PRO 187 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid 20 sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more 25 preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues I or about 23 to 205 of the PROl 87 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) X to 205 of the PROl 87 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 18 to 27 of Figure 2 (SEQ ID NO:2) or (c) another specifically derived fragment 30 of the amino acid sequence shown in Figure 2 (SEQ ID NO:2). Ordinarily, a PR0533 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more 35 preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more -13- WO 00/15666 PCT/US99/20594 preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid 5 sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues I or about 23 to 216 of the PR0533 polypeptide shown in Figure 4 (SEQ ID NO:7), (b) X to 216 of the PR0533 polypeptide shown in Figure 4 (SEQ ID NO:7), wherein X is any amino acid residue from 18 to 27 of Figure 4 (SEQ ID NO:7) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7). 10 Ordinarily, a PRO214 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid 15 sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more 20 preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 30 to 420 of the PR0214 polypeptide shown in Figure 6 (SEQ ID NO: 12), (b) X to 420 of the PRO214 polypeptide shown in Figure 6 (SEQ ID NO: 12), wherein X is any amino acid residue from 25 to 34 of Figure 6 (SEQ ID NO:12), (c) 1 or about 30 to X of Figure 6 (SEQ 25 ID NO:12), wherein X is any amino acid from amino acid 367 to amino acid 376 of Figure 6 (SEQ ID NO:12) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO:12). Ordinarily, a PR0240 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 30 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid 35 sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid -14- WO 00/15666 PCTIUS99/20594 sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues I or about 31 to 229 of the PR0240 polypeptide shown in Figure 8 (SEQ ID NO: 17), (b) X to 229 of the PR0240 polypeptide shown in Figure 8 (SEQ ID NO: 17), wherein X is any amino acid residue from 26 to 35 of Figure 8 (SEQ ID NO:17), (c) 1 or about 31 to X of Figure 8 (SEQ 5 ID NO:17), wherein X is any amino acid from amino acid 193 to amino acid 202 of Figure 8 (SEQ ID NO:17) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 8 (SEQ ID NO:17). Ordinarily, a PRO2 11 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 10 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid 15 sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 25 to 353 of the PRO21 I polypeptide shown 20 in Figure 10 (SEQ ID NO:22), (b) X to 353 of the PRO21I polypeptide shown in Figure 10 (SEQ ID NO:22), wherein X is any amino acid residue from 20 to 29 of Figure 10 (SEQ ID NO:22) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 10 (SEQ ID NO:22). Ordinarily, a PR0230 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid 25 sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more 30 preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least 35 about 99% amino acid sequence identity with (a) residues I or about 22 to 164 of the PR0230 polypeptide shown in Figure 12 (SEQ ID NO:27), (b) X to 164 of the PR0230 polypeptide shown in Figure 12 (SEQ ID NO:27), wherein X is any amino acid residue from 17 to 26 of Figure 12 (SEQ ID NO:27) or (c) another specifically derived -15- WO 00/15666 PCT/US99/20594 fragment of the amino acid sequence shown in Figure 12 (SEQ ID NO:27). Ordinarily, a PR0261 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 5 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid 10 sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues I or about 24 to 250 of the PR0261 polypeptide shown 15 in Figure 14 (SEQ ID NO:32), (b) X to 250 of the PR0261 polypeptide shown in Figure 14 (SEQ ID NO:32), wherein X is any amino acid residue from 19 to 28 of Figure 14 (SEQ ID NO:32) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 14 (SEQ ID NO:32). Ordinarily, a PR0246 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid 20 sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more 25 preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least 30 about 99% amino acid sequence identity with (a) residues 1 or about 30 to 390 of the PR0246 polypeptide shown in Figure 16 (SEQ ID NO:37), (b) X to 390 of the PR0246 polypeptide shown in Figure 16 (SEQ ID NO:37), wherein X is any amino acid residue from 25 to 34 of Figure 16 (SEQ ID NO:37), (c) 1 or about 30 to X of Figure 16 (SEQ ID NO:37), wherein X is any amino acid from amino acid 242 to amino acid 251 of Figure 16 (SEQ ID NO:37) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 16 (SEQ ID 35 NO:37). Ordinarily, a PRO317 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid -16- WO 00/15666 PCT/US99/20594 sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 5 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid 10 sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 19 to 366 of the PRO317 polypeptide shown in Figure 18 (SEQ ID NO:42), (b) X to 366 of the PRO317 polypeptide shown in Figure 18 (SEQ ID NO:42), wherein X is any amino acid residue from 14 to 23 of Figure 18 (SEQ ID NO:42) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 18 (SEQ ID NO:42). 15 PRO87, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 and PR0317 variant polypeptides do not encompass the native PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide sequence. Ordinarily, PRO 187, PR0533, PRO21 4, PR0240, PRO21 1, PRO230, PR026 1, PR0246 or PRO317 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino 20 acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 300 amino acids in length, or more. 25 "Percent (%) amino acid sequence identity" with respect to the PRO187, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 and PR0317 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not 30 considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences 35 being compared. For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Figures 20A-Q. The ALIGN-2 sequence comparison computer -17- WO 00/15666 PCTIUS99/20594 program was authored by Genentech, Inc., and the source code shown in Figures 20A-Q has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Figures 20A-Q. The 5 ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid 10 sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program 15 ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations, Figures 19A-B demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to 20 the amino acid sequence designated "PRO". Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res.. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be 25 downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62. In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino 30 acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y 35 where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues -18- WO 00/15666 PCT/US99/20594 in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer 5 program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. For purposes herein, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of 10 interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (i.e., the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest. For example, in the statement "a polypeptide comprising an amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B", the amino acid sequence 15 A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest. "PROI87 variant polynucleotide" or "PROl87 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO] 87 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues I or about 23 to 205 of the 20 PROI 87 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) a nucleic acid sequence which encodes amino acids X to 205 of the PROl 87 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 18 to 27 of Figure 2 (SEQ ID NO:2), or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO:2). Ordinarily, a PRO187 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% 25 nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more 30 preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 35 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues I or about 23 to 205 of the PROl 87 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) a nucleic acid sequence which encodes amino acids X to 205 of the PROl87 -19- WO 00/15666 PCT/US99/20594 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 18 to 27 of Figure 2 (SEQ ID NO:2), or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO:2). PROI 87 polynucleotide variants do not encompass the native PRO 187 nucleotide sequence. 5 "PR0533 variant polynucleotide" or "PR0533 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0533 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) residues 1 or about 23 to 216 of the PR0533 polypeptide shown in Figure 4 (SEQ ID NO:7), (b) X to 216 of the PR0533 polypeptide shown in Figure 4 (SEQ ID NO:7), wherein X is any amino acid residue from 18 to 27 of Figure 4 (SEQ ID NO:7) or (c) another specifically derived fragment of the 10 amino acid sequence shown in Figure 4 (SEQ ID NO:7). Ordinarily, a PR0533 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more 15 preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 20 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) residues I or about 23 to 216 of the PRO533 polypeptide shown in Figure 4 (SEQ ID NO:7), (b) X to 216 of the PR0533 polypeptide shown in Figure 4 (SEQ ID NO:7), wherein X is any amino acid residue from 18 to 27 of Figure 4 25 (SEQ ID NO:7) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7). PR0533 polynucleotide variants do not encompass the native PR0533 nucleotide sequence. "PR0214 variant polynucleotide" or "PR0214 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0214 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) residues 1 or about 30 to 420 of the PR0214 polypeptide shown in Figure 30 6 (SEQ ID NO: 12), (b) X to 420 of the PRO214 polypeptide shown in Figure 6 (SEQ ID NO: 12), wherein X is any amino acid residue from 25 to 34 of Figure 6 (SEQ ID NO: 12), (c) I or about 30 to X of Figure 6 (SEQ ID NO: 12), wherein X is any amino acid from amino acid 367 to amino acid 376 of Figure 6 (SEQ ID NO:12) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO: 12). Ordinarily, a PRO214 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least 35 about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about -20- WO 00/15666 PCT/US99/20594 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more 5 preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) residues 1 or about 30 to 420 of the PRO214 polypeptide shown in Figure 6 (SEQ 10 ID NO: 12), (b) X to 420 of the PRO214 polypeptide shown in Figure 6 (SEQ ID NO: 12), wherein X is any amino acid residue from 25 to 34 of Figure 6 (SEQ ID NO:12), (c) 1 or about 30 to X of Figure 6 (SEQ ID NO:12), wherein X is any amino acid from amino acid 367 to amino acid 376 of Figure 6 (SEQ ID NO:12) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO:12). PR0214 polynucleotide variants do not encompass the native PR0214 nucleotide sequence. 15 "PR0240 variant polynucleotide" or "PR0240 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0240 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) residues I or about 31 to 229 of the PR0240 polypeptide shown in Figure 8 (SEQ ID NO: 17), (b) X to 229 of the PR0240 polypeptide shown in Figure 8 (SEQ ID NO: 17), wherein X is any amino acid residue from 26 to 35 of Figure 8 (SEQ ID NO: 17), (c) I or about 31 to X ofFigure 8 (SEQ ID NO: 17), 20 wherein X is any amino acid from amino acid 193 to amino acid 202 of Figure 8 (SEQ ID NO: 17) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 8 (SEQ ID NO: 17). Ordinarily, a PR0240 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid 25 sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more 30 preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) residues I or about 31 to 229 of the PR0240 polypeptide shown in Figure 8 (SEQ 35 ID NO: 17), (b) X to 229 of the PR0240 polypeptide shown in Figure 8 (SEQ ID NO: 17), wherein X is any amino acid residue from 26 to 35 of Figure 8 (SEQ ID NO:17), (c) 1 or about 31 to X of Figure 8 (SEQ ID NO:17), wherein X is any amino acid from amino acid 193 to amino acid 202 of Figure 8 (SEQ ID NO: 17) or (d) another -21- WO 00/15666 PCT/US99/20594 specifically derived fragment of the amino acid sequence shown in Figure 8 (SEQ ID NO:17). PR0240 polynucleotide variants do not encompass the native PR0240 nucleotide sequence. "PRO211 variant polynucleotide" or "PRO211 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO2 11 polypeptide as defined below and which has at least about 80% nucleic 5 acid sequence identity with either (a) residues I or about 25 to 353 of the PRO211 polypeptide shown in Figure 10 (SEQ ID NO:22), (b) X to 353 of the PRO211 polypeptide shown in Figure 10 (SEQ ID NO:22), wherein X is any amino acid residue from 20 to 29 of Figure 10 (SEQ ID NO:22) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 10 (SEQ ID NO:22). Ordinarily, a PRO21 1 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid 10 sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more 15 preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 20 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) residues I or about 25 to 353 of the PRO21 I polypeptide shown in Figure 10 (SEQ ID NO:22), (b) X to 353 of the PRO211 polypeptide shown in Figure 10 (SEQ ID NO:22), wherein X is any amino acid residue from 20 to 29 of Figure 10 (SEQ ID NO:22) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 10 (SEQ ID NO:22). PRO211 polynucleotide variants do not encompass the native PRO211 25 nucleotide sequence. "PR0230 variant polynucleotide" or "PR0230 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0230 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) residues I or about 22 to 164 of the PR0230 polypeptide shown in Figure 12 (SEQ ID NO:27), (b) X to 164 of the PR0230 polypeptide shown in Figure 12 (SEQ ID NO:27), wherein X is 30 any amino acid residue from 17 to 26 of Figure 12 (SEQ ID NO:27) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 12 (SEQ ID NO:27). Ordinarily, a PR0230 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more 35 preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more -22- WO 00/15666 PCT/US99/20594 preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid 5 sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) residues I or about 22 to 164 of the PR0230 polypeptide shown in Figure 12 (SEQ ID NO:27), (b) X to 164 of the PR0230 polypeptide shown in Figure 12 (SEQ ID NO:27), wherein X is any amino acid residue from 17 to 26 of Figure 12 (SEQ ID NO:27) or (c) another specifically derived fragment of the amino acid sequence 10 shown in Figure 12 (SEQ ID NO:27). PR0230 polynucleotide variants do not encompass the native PR0230 nucleotide sequence. "PR0261 variant polynucleotide" or "PR0261 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0261 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) residues I or about 24 to 250 of the PR0261 polypeptide shown in Figure 15 14 (SEQ ID NO:32), (b) X to 250 of the PR0261 polypeptide shown in Figure 14 (SEQ ID NO:32), wherein X is any amino acid residue from 19 to 28 of Figure 14 (SEQ ID NO:32) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 14 (SEQ ID NO:32). Ordinarily, a PR0261 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 20 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid 25 sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with 30 either (a) residues 1 or about 24 to 250 of the PR0261 polypeptide shown in Figure 14 (SEQ ID NO:32), (b) X to 250 of the PR0261 polypeptide shown in Figure 14 (SEQ ID NO:32), wherein X is any amino acid residue from 19 to 28 of Figure 14 (SEQ ID NO:32) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 14 (SEQ ID NO:32). PR0261 polynucleotide variants do not encompass the native PR0261 nucleotide sequence. 35 "PR0246 variant polynucleotide" or "PR0246 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0246 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) residues I or about 30 to 390 of the PR0246 polypeptide shown in Figure -23- WO 00/15666 PCT/US99/20594 16 (SEQ ID NO:37), (b) X to 390 of the PR0246 polypeptide shown in Figure 16 (SEQ ID NO:37), wherein X is any amino acid residue from 25 to 34 of Figure 16 (SEQ ID NO:37), (c) I or about 30 to X of Figure 16 (SEQ ID NO:37), wherein X is any amino acid from amino acid 242 to amino acid 251 of Figure 16 (SEQ ID NO:37) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 16 (SEQ ID NO:37). Ordinarily, 5 a PR0246 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more 10 preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 15 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) residues I or about 30 to 390 of the PR0246 polypeptide shown in Figure 16 (SEQ ID NO:37), (b) X to 390 of the PR0246 polypeptide shown in Figure 16 (SEQ ID NO:37), wherein X is any amino acid residue from 25 to 34 of Figure 16 (SEQ ID NO:37), (c) I or about 30 to X of Figure 16 (SEQ ID NO:37), 20 wherein X is any amino acid from amino acid 242 to amino acid 251 of Figure 16 (SEQ ID NO:37) or (d) another specifically derived fragment of the amino acid sequence shown in Figure 16 (SEQ ID NO:37). PR0246 polynucleotide variants do not encompass the native PR0246 nucleotide sequence. "PRO317 variant polynucleotide" or "PRO317 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO317 polypeptide as defined below and which has at least about 80% nucleic 25 acid sequence identity with either (a) residues 1 or about 19 to 366 of the PRO317 polypeptide shown in Figure 18 (SEQ ID NO:42), (b) X to 366 of the PRO317 polypeptide shown in Figure 18 (SEQ ID NO:42), wherein X is any amino acid residue from 14 to 23 of Figure 18 (SEQ ID NO:42) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 18 (SEQ ID NO:42). Ordinarily, a PRO317 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid 30 sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more 35 preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more -24- WO 00/15666 PCT/US99/20594 preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) residues I or about 19 to 366 of the PRO317 polypeptide shown in Figure 18 (SEQ ID NO:42), (b) X to 5 366 of the PRO317 polypeptide shown in Figure 18 (SEQ ID NO:42), wherein X is any amino acid residue from 14 to 23 of Figure 18 (SEQ ID NO:42) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 18 (SEQ ID NO:42). PRO317 polynucleotide variants do not encompass the native PRO317 nucleotide sequence. Ordinarily, PROl87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 and PR0317 10 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, 15 more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more. "Percent (%) nucleic acid sequence identity " with respect to the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR026 1, PRO246 and PRO317 polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO 187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide-encoding 20 nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to 25 achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Figures 20A-Q. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Figures 20A-Q has been filed with user documentation in the U.S. Copyright Office, 30 Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Figures 20A-Q. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. 35 For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid -25- WO 00/15666 PCTIUS99/20594 sequence D) is calculated as follows: 100 times the fraction W/Z 5 where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Figures 19C-D demonstrate how to calculate 10 the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO-DNA". Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et 15 al., Nucleic Acids Res.. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62. 20 In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 25 100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence 30 D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymologv 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are 35 set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. For purposes herein, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide -26- WO 00/15666 PCTIUS99/20594 encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide encoding nucleic acid and the comparison nucleic acid molecule of interest (i.e., the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide 5 encoding nucleic acid molecule of interest. For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest. In other embodiments, PRO187, PR0533, PR0214, PRO240, PRO211, PRO230, PR0261, PRO246 and 10 PRO317 variant polynucleotides are nucleic acid molecules that encode an active PRO]87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PRO261, PR0246 or PR0317 polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO187, PRO533, PRO214, PRO240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO: 12), Figure 8 (SEQ ID NO: 17), Figure 15 10 (SEQ ID NO:22), Figure 12 (SEQ ID NO:27), Figure 14 (SEQ ID NO:32), Figure 16 (SEQ ID NO:37), or Figure 18 (SEQ ID NO:42), respectively. PRO187, PR0533, PR0214, PRO240, PRO211, PRO230, PR0261, PR0246 or PRO317 variant polypeptides may be those that are encoded by a PRO187, PR0533, PR0214, PRO240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 variant polynucleotide. The term "positives", in the context of the amino acid sequence identity comparisons performed as 20 described above, includes amino acid residues in the sequences compared that are not only identical, but also those that have similar properties. Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 1 below) of the amino acid residue of interest. For purposes herein, the % value of positives of a given amino acid sequence A to, with, or against a 25 given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y 30 where X is the number of amino acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % positives of A to B will not equal the % positives of B to A. "Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide that has 35 been identified and separated and/or recovered from a component of its natural environment. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic -27- WO 00/15666 PCT/US99/20594 uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. 5 Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step. An "isolated" nucleic acid molecule encoding a PRO 187, PR0533, PRO214, PR0240, PRO2 11,PRO230, PR0261, PR0246 or PRO317 polypeptide or an "isolated" nucleic acid encoding an anti-PRO187, anti-PR0533, 10 anti-PR0214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody, is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PRO]87-, PR0533-, PRO214-, PR0240-, PRO21 1-, PR0230-, PR0261-, PR0246- or PR0317-encoding nucleic acid or the anti-PRO187-, anti-PR0533-, anti PRO214-, anti-PR0240-, anti-PRO2 11-, anti-PR0230-, anti-PR026 1-, anti-PR0246- or anti-PRO3 17-encoding 15 nucleic acid. Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated. An isolated PR0187-, PR0533-, PR0214-, PR0240-, PR021 1-, PR0230-, PR0261-, PR0246- or PRO317-encoding nucleic acid molecule or an anti-PRO 187-, anti-PR0533-, anti-PRO214-, anti PR0240-, anti-PRO21 1-, anti-PR0230-, anti-PR026 1-, anti-PR0246- or anti-PRO317-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore 20 are distinguished from the PROl87-, PR0533-, PR0214-, PR0240-, PRO21 1-, PR0230-, PR0261-, PR0246- or PRO317-encoding nucleic acid molecule or the anti-PRO 187-, anti-PR0533-, anti-PRO214-, anti-PR0240-, anti PRO2 11-, anti-PR0230-, anti-PR026 I-,anti-PRO246- or anti-PRO317-encoding nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule encoding a PRO187, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 polypeptide or an anti-PRO187, anti-PR0533, anti-PR0214, 25 anti-PR0240, anti-PRO2 11, anti-PR0230,anti-PRO26 I,anti-PRO246or anti-PRO3 17antibody includes PRO 187 ,PR0533-, PRO214-, PRO240-, PRO2 11-, PRO230-, PR0261-, PR0246- or PRO317-nucleic acid molecules and anti-PRO187-, anti-PR0533-, anti-PRO214-, anti-PR0240-, anti-PRO21 1-, anti-PR0230-, anti-PR0261-, anti PR0246- or anti-PRO317-encoding nucleic acid molecules contained in cells that ordinarily express PRO187, PR0533, PRO214, PRO240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptides or express anti 30 PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PRO317 antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, 35 include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid -28- WO 00/15666 PCTIUS99/20594 sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" 5 means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The term "antibody" is used in the broadest sense and specifically covers, for example, single anti 10 PRO187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PRO317 monoclonal antibodies (including agonist, antagonist,and neutralizingantibodies),anti-PRO187, anti PR0533, anti-PR0214, anti-PR0240, anti-PR021 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody compositions with polyepitopic specificity, single chain anti-PRO] 87, anti-PRO533, anti-PRO214, anti PR0240, anti-PRO2 11, anti-PR0230, anti-PR026 1, anti-PR0246 or anti-PRO317 antibodies, and fragments of 15 anti-PRO187, anti-PRO533, anti-PRO214,anti-PRO240, anti-PRO2 11, anti-PR0230, anti-PR026 1, anti-PR0246 or anti-PRO317 antibodies (see below). The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and 20 generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, 25 it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology Wiley Interscience Publishers, (1995). "Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.00 15 M 30 sodium citrate/0.1% sodium dodecyl sulfate at 50*C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 0 C; o- (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 35 pg/ml), 0.1% SDS, and 10% dextran sulfate at 42*C, with washes at 42 0 C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55 0 C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 0 C. -29- WO 00/15666 PCT/US99/20594 "Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37 0 C in a solution comprising: 20% 5 formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 35-50'C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like. The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a PRO 187, 10 PRO533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PRO246 or PRO317 polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 15 50 amino acid residues (preferably, between about 10 and 20 amino acid residues). "Active" or "activity" for the purposes herein refers to form(s) of PRO187, PR0533, PR0214, PR0240, PRO2 11, PR0230, PR026 1, PRO246 or PRO317 polypeptides which retain a biological and/or an immunological activity/property of a native or naturally-occurring PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PRO261, PRO246 or PRO317 polypeptide, wherein "biological" activity refers to a function (either inhibitory or 20 stimulatory) caused by a native or naturally-occurring PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR026 1, PRO246 or PRO3 17 polypeptide other than the ability to induce the production of an antibody against an antigenic epitope possessed by a a native or naturally-occurring PRO 187, PR0533, PRO21 4, PR0240, PRO211, PR0230, PR026 1, PR0246 or PRO317 polypeptide and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally- occurring PRO 187, 25 PRO533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide. "Biological activity" in the context of an antibody or another antagonist molecule that can be identified by the screening assays disclosed herein (e.g., an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to bind or complex with the polypeptides encoded by the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins or 30 otherwise interfere with the transcription or translation of a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PRO261, PRO246 or PRO317 polypeptide. A preferred biological activity is growth inhibition of a target tumor cell. Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor cell. The term "biological activity" in the context of a PROI87, PR0533, PRO214, PR0240, PRO21 1, 35 PR0230, PR026 1, PR0246 or PRO317 polypeptide means the ability of a PRO 187, PRO533, PRO214, PRO240, PRO2 11, PRO230, PRO26 1, PRO246 or PRO317 polypeptide to induce neoplastic cell growth or uncontrolled cell growth. -30- WO 00/15666 PCT/US99/20594 The phrase "immunological activity" means immunological cross-reactivity with at least one epitope of a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide. "Immunological cross-reactivity" as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO]87, PR0533, PRO214, PR0240, PRO21 1, 5 PR0230, PR0261, PR0246 or PRO3 17 polypeptide having this activity with polyclonal antisera raised against the known active PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PR0317 polypeptide. Such antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freunds. The immunological cross-reactivity preferably is "specific", 10 which means that the binding affinity of the immunologically cross-reactive molecule (e.g., antibody) identified, to the corresponding PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide is significantly higher (preferably at least about 2-times, more preferably at least about 4-times, even more preferably at least about 8-times, most preferably at least about 10-times higher) than the binding affinity of that molecule to any other known native polypeptide. 15 The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 polypeptide disclosed herein or the transcription or translation thereof. Suitableantagonistmoleculesspecifically include antagonist antibodies or antibody fragments, fragments, peptides, small organic molecules, anti-sense nucleic acids, etc. Included are methods for identifying antagonists of a 20 PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide. A "small molecule" is defined herein to have a molecular weight below about 500 Daltons. 25 "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas. The term "antibody" is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, 30 polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. "Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the 35 heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain -31- WO 00/15666 PCTIUS99/20594 disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain ofthe heavy chain. Particular amino acid residues are believed 5 to form an interface between the light- and heavy-chain variable domains. The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both 10 in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a P-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the P-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of 15 antibodies (see Kabat et al., NIH Publ. No.91-3242 Vol. 1, pages 647-669 (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a 20 "complementarity determining region" to "CDR" (i.e., residues 24-34 (L 1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H 1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD. [1 991]) and/or those residues from a "hypervariable loop" (i.e., residues 26-32 (L 1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain 25 variable domain ; Clothia and Lesk, J. Mol. Biol.. 196:901-917 [19871). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined. "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies(Zapata etal., Protein Eng., 8( 10): 1057-1062 [1995]); single-chain antibody molecules; 30 and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. 35 "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on -32- WO 00/15666 PCT/US99/20594 the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH 1) 5 of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH 1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab'), antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. 10 The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (A), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and 15 IgM, and several ofthese may be further divided into subclasses (isotypes), e.g., IgG 1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, 8, e, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of 20 substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparationswhich typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, 25 the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256:495 [1975], 30 or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 [1991] and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies 35 derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit WO 00/15666 PCT/US99/20594 the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 6855 [1984]). "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences 5 of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may 10 comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody 15 optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 [1988]; and Presta, Curr. O. Struct. Biol. 2:593-596 (1992). The humanized antibody includes a PRIMATIZEDT" antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest. 20 "Single-chain Fv" or "sFv" antibody fragments comprise the VH and V, domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and V, domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). 25 The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) Connected to a light-chain variable domain (V,) in the same polypeptide chain (V, - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and 30 Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by 35 weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie -34- WO 00/15666 PCT/US99/20594 blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. The word "label" when used herein refers to a detectable compound or composition which is conjugated 5 directly or indirectly to the antibody so as to generate a "labeled" antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. Radionuclides that can serve as detectable labels include, for example, 1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-21 1, Cu-67, Bi-212, and Pd-109. By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. 10 Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149. 15 A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a PRO187, PR0533, PR0214, PRO240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide or antibody thereto and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. 20 As used herein,the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a 25 contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG- 1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. II. Compositions and Methods of the Invention 30 A. Full-leneth PRO] 87. PR0533. PRO214, PR0240. PRO211. PRO230, PR026 1, PRO246and PRO317 polypentides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO 187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 and PRO317. In particular, cDNA encoding PRO 187, PR0533, PRO214, PR0240, PRO21 1, PR0230, 35 PR0261, PR0246 and PRO317 polypeptides has been identified and isolated, as disclosed in further detail in the Examples below. It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed. -35- WO 00/15666 PCTIUS99/20594 NOT FURNISHED UPON FILING 36 WO 00/15666 PCTIUS99/20594 acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about I to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the 5 sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 and PR0317 polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO187, PR0533, PRO214, PR0240, 10 PRO21 1, PRO230, PR026 1, PR0246 or PRO3 17 polypeptide. PRO187, PRO533, PRO214, PRO240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PRO187, PR0533, PR0214, PR0240, PRO211, PRO230, PR026 1, PR0246 or PRO317 fragments by enzymatic digestion, e.g., by treating the protein with an 15 enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, PRO187, PR0533, PRO214, PR0240, PRO21 I, PRO230, PR0261, PR0246 or PR0317 20 polypeptide fragments share at least one biological and/or immunological activity with the native PRO187, PR0533, PR0214, PR0240, PRO21 I, PRO230, PR0261, PR0246 or PR0317 polypeptide. In particular embodiments, conservative substitutions of interest are shown in Table I under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid 25 classes, are introduced and the products screened. Table I Original Exemplary Preferred Residue Substitutions Substitutions 30 Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu 35 Cys(C) ser ser GIn (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gin; lys; arg arg 40 Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile -37- WO 00/15666 PCT/US99/20594 Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala 5 Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; 10 ala; norleucine leu Substantial modifications in function or immunological identity of the polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or 15 hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; 20 (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into 25 the remaining (non-conserved) sites. The variations can be made using methods known in the art such as oligonucleotide-mediated (site directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res. 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells etal., Philos. Trans. R. Soc. London SerA. 317:415 30 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 variant DNA. Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid 35 among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins (W.H. Freeman & Co., N.Y.); Chothia, J. M iol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used. 40 -38- WO 00/15666 PCT/US99/20594 C. Modifications of PRO]87. PR0533. PR0214. PR0240. PRO21 1, PR0230. PRO261. PR0246 and PRO317 Covalent modifications of PRO187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR0261, PR0246 and PRO317 are included within the scope of this invention. One type of covalent modification includes reacting 5 targeted amino acid residues of a PRO 187, PR0533, PRO2 14, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317. Derivatization with bifunctional agents is useful, for instance, for crosslinking PRO187, PR0533, PRO2 14, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 to a water-insoluble support matrix or surface 10 for use in the method for purifying anti-PRO 187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO21 1, anti PR0230, anti-PR0261, anti-PRO246 or anti-PRO3 17 antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents 15 such as methyl-3-[(p-azidophenyl)dithio]propioimidate. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation ofthe a-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties W.H. Freeman & Co., San Francisco, pp. 79-86 20 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. Another type of covalent modification of the PR0212, PR0290, PR0341, PR0535, PR0619, PR0717, PR0809, PR0830, PRO848, PR0943, PRO005, PROl009, PRO1025, PROl030, PRO1097, PRO1107, PROl I 11, PROI 153, PROI 182, PRO] 184, PROl 187, PRO 1281, PR023,PRO39,PRO834,PRO1317,PROI710, PR02094, PRO2145 or PRO2198 polypeptide included within the scope of this invention comprises altering the 25 native glycosylation pattern of the polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO187, PR0533, PR0214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, 30 PR0246 or PRO317. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present. Addition of glycosylation sites to the PROl 87, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native 35 sequence PRO 187, PR0533, PRO214, PR0240, PRO2 11, PRO230, PR026 1, PR0246 or PRO3 17 (for O-linked glycosylation sites). The PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the -39- WO 00/15666 PCT/US99/20594 DNA encoding the PRO187, PRO533, PR0214, PRO240, PR021 1, PR0230, PR0261, PR0246 or PR0317 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids. Another means of increasing the number of carbohydrate moieties on the PRO187, PRO533, PR0214, PR0240, PRO2 11, PR0230, PRO26 1, PR0246 or PRO3 17 polypeptide is by chemical or enzymatic coupling of 5 glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 1 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem. pp. 259-306 (1981). Removal of carbohydrate moieties present on the PRO 187, PRO533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical 10 deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987). Another type of covalent modification of PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, 15 PR0261, PR0246 or PR0317 comprises linking the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The PRO187, PR0533, PR0214, PRO240, PRO211, PR0230, PR0261, PR0246 or PR0317 of the 20 present invention may also be modified in a way to form a chimeric molecule comprising PRO187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 with a tag polypeptide which provides an epitope to 25 which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl terminus of the PRO]87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317. The presence of such epitope-tagged forms of the PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 to be 30 readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E 10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes 35 Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky etal., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science 255:192-194 (1992)]; an a-tubulin epitope peptide [Skinner et al., J. Biol. -40- WO 00/15666 PCT/US99/20594 Chem. 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Nati. Acad. Sci. USA 87:6393-6397 (1990)]. In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 with an immunoglobulin or a particular 5 region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The 1g fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO] 87, PRO533, PRO214, PR0240, PRO2 11, PRO230, PRO26 1, PR0246 or PRO317 polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 10 and CH3, or the hinge, CH 1, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see also, US Patent No. 5,428,130 issued June 27, 1995. D. Preparation of PRO]87, PR0533, PR0214, PR0240, PRO21 1, PR0230. PR0261. PR0246 and PRO3 17 Polypeptides 15 The description below relates primarily to production of PROl 87, PRO533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 by culturing cells transformed or transfected with a vector containing PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PRO246 or PR0317. For instance, the 20 PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PRO246 or PR0317 sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 85:2149 2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) 25 using manufacturer's instructions. Various portions of the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO187, PR0533, PRO214, PR0240, PRO211, PR0230, PR026 1, PR0246 or PRO317. 30 a. Isolation of DNA Encoding a PRO87, PR0533, PR0214, PR0240, PRO211, PR0230, PR026 1, PR0246 or PRO317 Polypeptide DNA encoding PRO 187, PRO533, PRO214, PRO240, PRO2 11, PRO230, PR026 1, PRO246 or PRO317 may be obtained from a cDNA library prepared from tissue believed to possess the PRO187, PRO533, PRO214, PRO240, PRO211, PRO230, PRO261, PRO246 or PRO317 mRNA and to express it at a detectable level. 35 Accordingly, human PRO187, PRO533, PRO214, PRO240, PRO21 1, PRO230, PRO261, PRO246 or PRO317 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. PRO187, PRO533, PRO214, PRO240, PRO21 1, PRO230, PRO261, PRO246 or PRO317 encoding -41- WO 00/15666 PCTIUS99/20594 gene may also be obtained from a genomic library or by oligonucleotide synthesis. Libraries can be screened with probes (such as antibodies to the PROl87, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide, or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with 5 the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding PRO]87, PR0533, PRO214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)]. 10 The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 1 2 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, 15 are provided in Sambrook et al., supra. Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein. 20 Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA. 25 b. Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described herein for PROl 87, PR0533, PRO214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can 30 be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra. Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl 2 , CaPO 4 , liposome-mediated and electroporation. Depending on the host cell 35 used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as -42- WO 00/15666 PCT/US99/20594 described by Shaw et al., Gene 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456 457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Patent No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van 5 Solingen et al., J. Bact. 130:946 (1977) and Hsiao et al., Proc. Natil. Acad. Sci. (USA) 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see, Keown et al., Methods in Enzymology, 185:527-537(1990) and Mansour et al., Nature 336:348-352 (1988). 10 Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W31 10 (ATCC 27,325) and E. Coli strain K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include 15 Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia. Klebsiella, Proteus, Salmonella, e.g., Salmonella tvphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3 110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product 20 fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3 110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W31 10 strain IA2, which has the complete genotype tonA ; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3 110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompTkan'; E. coli W31 10 strain 37D6, which has 25 the complete genotype tonA ptr3 phoA E15 (argF-lac) J69 degP ompTrbs7 ilvG kan'; E. coli W31 10 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or 30 expression hosts for PRO187-, PR0533-, PR0214-, PR0240-, PRO21 1-, PR0230-, PR0261-, PR0246- or PRO317-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al., Bio/Technology, 9: 968-975 (199 1)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol. 737 [1983]), K. fragilis 35 (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Vanden Berg et al., Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichiapastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol. 28:265 -43- WO 00/15666 PCT/US99/20594 278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. 5 Common., 112:284-289 [1983]; Tilburn etal., Gene, 26:205-221 [1983]; Yelton etal., Proc. Natl. Acad. Sci. USA. 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of 10 Methylotrophs, 269 (1982). Suitable host cells for the expression of glycosylated PRO]87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO3 17 are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include 15 monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 3:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO), Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The selection of the 20 appropriate host cell is deemed to be within the skill in the art. c. Selection and Use of a Replicable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO 187, PR0533,PRO214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO3 17 may be inserted into a replicable vector for cloning (amplification of the 25 DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription 30 termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. The PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature 35 protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the PROl 87-,PRO533-,PRO214-, PR0240-, PRO2 11-, PR0230-, PR026 1-, PR0246- or PRO317-encodingDNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, -44- WO 00/15666 PCT/US99/20594 from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 5 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders. Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The 10 origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 p plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, 15 neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO 187-, PR0533-, PRO214-, PR0240-, PRO2 11-, PR0230-, PR026 I -, PR0246 or PRO317-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type 20 DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA. 77:4216 (1980). A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 2:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-l [Jones, Genetics 85:12 25 (1977)]. Expression and cloning vectors usually contain a promoter operably linked to the PRO187-, PR0533-, PRO2 14-, PR0240-, PRO21 I-, PR0230-, PR026 1-, PR0246- or PRO317-encodingnucleic acid sequenceto direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the p-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 30 (1978); Goeddel etal., Nature 281:544(1979)],alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res. 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer etal., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO187, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317. 35 Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3 phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem. 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry L7:4900 (1978)], such as enolase, glyceraldehyde -45- WO 00/15666 PCT/US99/20594 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription 5 controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3 phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PRO261, PR0246 or PR0317 transcription 10 from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems. 15 Transcription of a DNA encoding the PRO 187, PR0533, PRO214, PR0240, PRO2 1l, PR0230, PR026 1, PR0246 or PRO317 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples 20 include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side ofthe replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3 to the PRO 187, PR053 3, PRO214, PR0240, PRO211, PR0230, PR026 1, PR0246 or PRO317 coding sequence, but is preferably located at a site 5' from the promoter. Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated 25 cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PROl87, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317. 30 Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PROl87, PR0533, PR0214, PR0240, PR021 1, PR0230, PR0261, PR0246 or PR0317 in recombinant vertebrate cell culture are described in Gething et al., Nature 293:620-625 (198 1); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058. 35 d. Detecting Gene Amplification/Expression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Nat]. Acad. Sci. USA, -46- WO 00/15666 PCT/US99/20594 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so 5 that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected. Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the 10 antibodies may be prepared against a native sequence PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against an exogenous sequence fused to PRO 187, PR0533, PRO214, PRO240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 DNA and encoding a specific antibody epitope. 15 e. Purification of Polypeptide Forms of PROI 87, PR0533, PRO214, PR0240, PRO2 11, PRO230, PR0261, PR0246 or PRO317 may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PRO187, PRO533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 can be 20 disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. It may be desired to purify PRO187, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; 25 chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the PRO187, PRO533, PRO214, PR0240, PRO2 11, PRO230, PR026 1, PR0246 or PRO317. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in 30 Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO187, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 produced. E. Amplification ofGenes Encoding the PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, 35 PRO261, PR0246 or PR0317 Polypeptides in Tumor Tissues and Cell Lines The present invention is based on the identification and characterization of genes that are amplified in certain cancer cells. -47- WO 00/15666 PCT/US99/20594 The genome of prokarvotic and eukaryotic organisms is subjected to two seemingly conflicting requirements. One is the preservation and propagation of DNA as the genetic information in its original form, to guarantee stable inheritance through multiple generations. On the other hand, cells or organisms must be able to adapt to lasting environmental changes. The adaptive mechanisms can include qualitative or quantitative 5 modifications of the genetic material. Qualitative modifications include DNA mutations, in which coding sequences are altered resulting in a structurally and/or functionally different protein. Gene amplification is a quantitative modification, whereby the actual number of complete coding sequence, i.e., a gene, increases, leading to an increased number of available templates for transcription, an increased number of translatable transcripts, and, ultimately, to an increased abundance of the protein encoded by the amplified gene. 10 The phenomenon of gene amplification and its underlying mechanisms have been investigated in vitro in several prokaryotic and eukaryotic culture systems. The best-characterized example of gene amplification involves the culture of eukaryotic cells in medium containing variable concentrations of the cytotoxic drug methotrexate (MTX). MTX is a folic acid analogue and interferes with DNA synthesis by blocking the enzyme dihydrofolate reductase (DHFR). During the initial exposure to low concentrations of MTX most cells (>99.9%) will die. A 15 small number of cells survive, and are capable of growing in increasing concentrations of MTX by producing large amounts of DHFR-RNA and protein. The basis of this overproduction is the amplification of the single DHFR gene. The additional copies of the gene are found as extrachromosomal copies in the form of small, supernumerary chromosomes (double minutes) or as integrated chromosomal copies. Gene amplification is most commonly encountered in the development of resistance to cytotoxic drugs 20 (antibiotics for bacteria and chemotherapeutic agents for eukaryotic cells) and neoplastic transformation. Transformation of a eukaryotic cell as a spontaneous event or due to a viral or chemical/environmental insult is typically associated with changes in the genetic material of that cell. One of the most common genetic changes observed in human malignancies are mutations of the p53 protein. p53 controls the transition of cells from the stationary (G 1) to the replicative (S) phase and prevents this transition in the presence of DNA damage. In other 25 words, one of the main consequences of disabling p53 mutations is the accumulation and propagation of DNA damage, i.e., genetic changes. Common types of genetic changes in neoplastic cells are, in addition to point mutations, amplifications and gross, structural alterations, such as translocations. The amplification of DNA sequences may indicate specific functional requirement as illustrated in the DHFR experimental system. Therefore, the amplification of certain oncogenes in malignancies points toward a 30 causative role of these genes in the process of malignant transformation and maintenance of the transformed phenotype. This hypothesis has gained support in recent studies. For example, the bcl-2 protein was found to be amplified in certain types of non-Hodgkin's lymphoma. This protein inhibits apoptosis and leads to the progressive accumulation of neoplastic cells. Members of the gene family of growth factor receptors have been found to be amplified in various types of cancers suggesting that overexpression of these receptors may make neoplastic cells 35 less susceptible to limiting amounts of available growth factor. Examples include the amplification ofthe androgen receptor in recurrent prostate cancer during androgen deprivation therapy and the amplification ofthe growth factor receptor homologue ERB2 in breast cancer. Lastly, genes involved in intracellular signaling and control of cell -48- WO 00/15666 PCT/US99/20594 cycle progression can undergo amplification during malignant transformation. This is illustrated by the amplification of the bcl-/ and ras genes in various epithelial and lymphoid neoplasms. These earlier studies illustrate the feasibility of identifying amplified DNA sequences in neoplasms, because this approach can identify genes important for malignant transformation. The case of ERB2 also 5 demonstrates the feasibility from a therapeutic standpoint, since transforming proteins may represent novel and specific targets for tumor therapy. Several different techniques can be used to demonstrate amplified genomic sequences. Classical cytogenetic analysis of chromosome spreads prepared from cancer cells is adequate to identify gross structural alterations, such as translocations, deletions and inversions. Amplified genomic regions can only be visualized, 10 if they involve large regions with high copy numbers or are present as extrachromosomal material. While cytogenetics was the first techniqueto demonstrate the consistent association of specific chromosomal changes with particular neoplasms, it is inadequate for the identification and isolation of manageable DNA sequences. The more recently developed technique of comparative genomic hybridization (CGH) has illustrated the widespread phenomenon of genomic amplification in neoplasms. Tumor and normal DNA are hybridized simultaneously onto 15 metaphases of normal cells and the entire genome can be screened by image analysis for DNA sequences that are present in the tumor at an increased frequency. (WO 93/18,186; Gray et al., Radiation Res. 137:275-289 [1994]). As a screening method, this type of analysis has revealed a large number of recurring amplicons (a stretch of amplified DNA) in a variety of human neoplasms. Although CGH is more sensitive than classical cytogenetic analysis in identifying amplified stretches of DNA, it does not allow a rapid identification and isolation of coding 20 sequences within the amplicon by standard molecular genetic techniques. The most sensitive methods to detect gene amplification are polymerase chain reaction (PCR)-based assays. These assays utilize very small amount of tumor DNA as starting material, are exquisitely sensitive, provide DNA that is amenable to further analysis, such as sequencing and are suitable for high-volume throughput analysis. The above-mentioned assays are not mutually exclusive, but are frequently used in combination to identify 25 amplifications in neoplasms. While cytogenetic analysis and CGH represent screening methods to survey the entire genome for amplified regions, PCR-based assays are most suitable for the final identification of coding sequences, i.e., genes in amplified regions. According to the present invention, such genes have been identified by quantitative PCR (S. Gelmini et al., Clin. Chem. 43:752 [1997]), by comparing DNA from a variety of primary tumors, including breast, lung, 30 colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc. tumor, or tumor cell lines, with pooled DNA from healthy donors. Quantitative PCR was performed using a TaqMan instrument (ABI). Gene-specific primers and fluorogenic probes were designed based upon the coding sequences of the DNAs. Human lung carcinomacell lines include A549 (SRCC768), Calu- I (SRCC769), Calu-6(SRCC770),H 157 (SRCC771), H441 (SRCC772), H460 (SRCC773), H522 (SRCC832), H810 (SRCC833), SKMES-I (SRCC774) 35 and SW900 (SRCC775), all available from ATCC. Primary human lung tumor cells usually derive from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, non-small cell carcinomas, small cell carcinomas, and broncho alveolar carcinomas, and include, for example, SRCC724 (adenocarcinoma, abbreviated -49- WO 00/15666 PCTIUS99/20594 as "AdenoCa")(LT1), SRCC725 (squamous cell carcinoma, abbreviated as "SqCCa)(LTIa), SRCC726 (adenocarcinoma)(LT2), SRCC727 (adenocarcinoma)(LT3), SRCC728 (adenocarcinoma)(LT4), SRCC729 (squamous cell carcinoma)(LT6), SRCC730 (adeno/squamous cell carcinoma)(LT7), SRCC825 (adenocarcinoma)(LT8), SRCC731 (adenocarcinoma)(LT9), SRCC732 (squamous cell carcinoma)(LT10), 5 SRCC733 (squamous cell carcinoma)(LT 11), SRCC734 (adenocarcinoma)(LT12), SRCC735 (adeno/squamous cell carcinoma)(LT13), SRCC736 (squamous cell carcinoma)(LTl 5),SRCC737(squamouscellcarcinoma)(LT16), SRCC738 (squamous cell carcinoma)(LT17), SRCC739 (squamous cell carcinoma)(L T18), SRCC740 (squamous cell carcinoma)(LT19), SRCC741 (lung cell carcinoma, abbreviated as "LCCa")(LT21), SRCC8I1 (adenocarcinoma)(LT22), SRCC887 (squamous cell carcinoma) (LT26), SRCC888 (adeno-BAC carcinoma) 10 (LT27), SRCC889 (squamous cell carcinoma) (LT28), SRCC890 (squamous cell carcinoma) (LT29), SRCC891 (adenocarcinoma) (LT30), SRCC892 (squamous cell carcinoma) (LT3 1), SRCC894 (adenocarcinoma) (LT33). Also included are human lung tumors designated SRCC 1125 [HF-000631], SRCC 1129 [HF-000643], SRCCI 133 [HF-000840] and SRCC1135 [HF-000842]. Colon cancer cell lines include, for example, ATCC cell lines SW480 (adenocarcinoma, SRCC776), 15 SW620 (lymph node metastasis of colon adenocarcinoma, SRCC777), Colo320 (carcinoma, SRCC778), Colo205 (carcinoma, SRCC828), HCC2998 (carcinoma, SRCC830), HT29 (adenocarcinoma, SRCC779), HM7 (carcinoma, SRCC780),KM 12 (carcinoma,SRCC83 1),CaWiDr (adenocarcinoma, SRCC78 1), HCT 15 (carcinoma,SRCC829), HCTI 16 (carcinoma, SRCC782), SKCO1 (adenocarcinoma, SRCC783), SW403 (adenocarcinoma, SRCC784), LS174T (carcinoma, SRCC785), and HM7 (a high mucin producing variant of ATCC colon adenocarcinoma cell 20 line LS 174T, SRCC780, obtained from Dr. Robert Warren, UCSF). Primary colon tumors include colon adenocarcinomas designated CTI (SRCC751), CT2 (SRCC742), CT3 (SRCC743), CT4 (SRCC752), CT5 (SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT8 (SRCC744), CT9 (SRCC756), CTIO (SRCC745), CTI 1 (SRCC757), CT12(SRCC746), CT14 (SRCC747), CT15 (SRCC748),CT16(SRCC749),CT1 7(SRCC750), CT18 (SRCC758), CT25 (adenocarcinoma, SRCC912), CT28 (adenocarcinoma, SRCC915) CT35 (adenocarcinoma, 25 SRCC92 1). Also included are human colon tumor centers designated SRCC 1051 [HF-000499], SRCC 1052 [HF 0005391, SRCC 1053 [HF-000575], SRCC 1054 [HF-000698], SRCC 1060 [HF-000756], SRCC 1144 [HF-000789] and SRCC1 148[HF-00081 1] and human colon tumor margin designated SRCC 1059 [HF-000755]. Human breast carcinoma cell lines include, for example, HBLIOO (SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468(SRCC762), MB175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 30 (SRCC766), and SKBR3 (SRCC767), and human breast tumor center designated SRCC1057 [HF-000545]. Also included are human breast tumors designated SRCC1094, SRCC1095, SRCCl096, SRCC1097, SRCC1098, SRCC1099, SRCCI 100 and SRCCI 101. Human kidney tumor centers include SRCC989 [HF-0006 11] and SRCC 10 14 [HF-000613]. Lymph node tumor includes SRCC 1004 [HF-000854]. Rectal tumor margin includes SRCC82 [HF-00055 1]. Testis tumor 35 center includes SRCCOOI [HF-000733] and testis tumor margin SRCC999 [HF-000716]. -50- WO 00/15666 PCT/US99/20594 F. Tissue Distribution The results of the gene amplification assays herein can be verified by further studies, such as, by determining mRNA expression in various human tissues. As noted before, gene amplification and/or gene expression in various tissues may be measured by 5 conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA 77:5201-5205 [1980]), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. 10 Gene expression in various tissues, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 15 or PRO3 17 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to sequence PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 DNA and encoding a specific antibody epitope. General techniques for generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided hereinbelow. 20 G. Chromosome Mapping If the amplification of a given gene is functionally relevant, then that gene should be amplified more than neighboring genomic regions which are not important for tumor survival. To test this, the gene can be mapped to a particular chromosome, e.g., by radiation-hybrid analysis. The amplification level is then determined at the location identified, and at neighboring genomicregion. Selective or preferential amplification at the genomic region 25 to which the gene has been mapped is consistent with the possibility that the gene amplification observed promotes tumor growth or survival. Chromosome mapping includes both framework and epicenter mapping. For further details see e.g., Stewart et al., Genome Research 7:422-433 (1997). H. Antibody Binding Studies The results of the gene amplification study can be further verified by antibody binding studies, in which 30 the ability of anti-PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO2 11, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibodies to inhibit the expression of the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptides on tumor (cancer) cells is tested. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow. 35 Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987). -51- WO 00/15666 PCT/US99/20594 Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of target protein (encoded by a gene amplified in a tumor cell) in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies preferably are 5 insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound. Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus 10 forming an insoluble three-part complex. See, e.g., U.S. Patent No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. For immunohistochemistry, the tumor sample may be fresh or frozen or may be embedded in paraffin and 15 fixed with a preservative such as formalin, for example. I. Cell-Based Tumor Assays Cell-based assays and animal models for tumors (e.g., cancers) can be used to verify the findings of the gene amplification assay, and further understand the relationship between the genes identified herein and the 20 development and pathogenesis of neoplastic cell growth. The role of gene products identified herein in the development and pathology of tumor or cancer can be tested by using primary tumor cells or cells lines that have been identified to amplify the genes herein. Such cells include, for example, the breast, colon and lung cancer cells and cell lines listed above. In a different approach, cells of a cell type known to be involved in a particular tumor are transfected with 25 the cDNAs herein, and the ability of these cDNAs to induce excessive growth is analyzed. Suitable cells include, for example, stable tumor cells lines such as, the B 104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and ras-transfected NIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogenic growth. Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumorogenic cell growth by exerting cytostatic or 30 cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC). Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of cancer. In addition, primary cultures derived from tumors in transgenic animals (as described below) can be used in the cell-based assays herein, although stable cell lines are preferred. Techniques to derive continuous cell lines 35 from transgenic animals are well known in the art (see, e.g., Small et al., Mol. Cell. Biol., 5:642-648 [1985]). J. Animal Models -52- WO 00/15666 PCT/US99/20594 A variety of well known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other antagonists of the native polypeptides, including small molecule antagonists. The in vivo nature of such models makes them particularly predictive of responses in human patients. Animal models 5 of tumors and cancers (e.g., breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g., murine models. Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g., colon cancer cells implanted in colonic tissue. 10 (See, e.g., PCT publication No. WO 97/3355 1, published September 18, 1997). Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice. The observation that the nude mouse with hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose. The autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, ASW, 15 A/He, AKR, BALB/c, BIO.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL. In addition, a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts. For further details see, e.g., The Nude Mouse in Oncology Research E. Boven and B. Winograd, eds., CRC Press, Inc., 1991. The cells introduced into such animals can be derived from known tumor/cancer cell lines, such as, any 20 of the above-listed tumor cell lines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene); ras-transfected NIH-3T3 cells; Caco-2 (ATCC HTB-37); a moderately well differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-3 8), or from tumors and cancers. Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al., Br. J. Cancer 48:689-696 [1983]). 25 Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures. The subcutaneous (s.c.) space in mice is very suitable for tumor implantation. Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions. For solid block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s.c. space. Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously. Tumor cells can also be injected as 30 subdermal implants. In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s.c. tissue. Boven and Winograd (1991), supra. Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogen was initially isolated), or neu-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al., PNAS USA 83:9129-9133 (1986). 35 Similarly, animal models of colon cancer can be generated by passaging colon cancer cells in animals, e.g., nude mice, leading to the appearance of tumors in these animals. An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et al., Cancer Research 54:4726-4728 (1994) and -53- WO 00/15666 PCT/US99/20594 Too et al., Cancer Research 55:681-684 (1995). This model is based on the so-called "METAMOUSE" sold by AntiCancer, Inc. (San Diego, California). Tumors that arise in animals can be removed and cultured in vitro. Cells from the in vitro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening. Alternatively, the 5 tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest. Such passaging techniques can be performed with any known tumor or cancer cell lines. For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al., J. Exp. Med. 146:720 [1977]), which provide a highly controllable model 10 system for studying the anti-tumor activities of various agents (Palladino et al., J. Immunol., 138:4023-4032 [1987]). Briefly, tumor cells are propagated in vitro in cell culture. Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about 10x10 6 to 10x10 7 cells/ml. The animals are then infected subcutaneously with 10 to 100 pl ofthe cell suspension, allowing one to three weeks for a tumor to appear. In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most thoroughly studied 15 experimental tumors, can be used as an investigational tumor model. Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL). This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al., Br. J. Cancer 4 :suppl. 4:309 [1980]), and evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells 20 survive. For further information about this tumor model see, Zacharski, Haemostasis 16:300-320 [1986]). One way of evaluating the efficacy of a test compound in an animal model is implanted tumor is to measure the size of the tumor before and after treatment. Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions. The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using 25 a mathematical formula. However, the measurement of tumor size is very inaccurate. The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay. Another important variable in the description of tumor growth is the tumor volume doubling time. Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals Wu and Sheng eds., Basel, 1989, 301. 30 It is noted, however, that necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially. Therefore, these changes need to be carefully monitored, by a combination of a morphometric method and flow cytometric analysis. Recombinant (transgenic) animal models can be engineered by introducing the coding portion ofthe genes identified herein into the genome of animals ofinterest, using standard techniques for producing transgenic animals. 35 Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees and monkeys. Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U.S. -54- WO 00/15666 PCT/US99/20594 Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al., Proc. Nati. Acad. Sci. USA 82:6148-615 [1985]); gene targeting in embryonic stem cells (Thompson et al., .56, 5:313-321 [1989]); electroporation of embryos (Lo, Mol. Cell Biol., 3:1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Ce 5:717-73 [1989]). For review, see, for example, U.S. Patent No. 4,736,866. 5 For the purpose of the present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals"). The transgene can be integrated either as a single transgene, or in concatamers, e.g., head-to-head or head-to-tail tandems. Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al., Proc. Natl. Acad. Sci. USA, 89:6232 636 (1992). 10 The expression of the transgene in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry. The animals are further examined for signs of tumor or cancer development. 15 Alternatively, "knock out" animals can be constructed which have a defective or altered gene encoding a PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal. For example, cDNA encoding a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 20 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques. A portion ofthe genomic DNA encoding a particular PRO 187, PR0533, PRO214, PRO240, PRO211, PRO230, PR026 1, PR0246 or PRO317 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchi, Cell, 5L:503 25 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A 30 chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to absence of the 35 PRO187, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 polypeptide. The efficacy of antibodies specifically binding the polypeptides identified herein and other drug candidates, can be tested also in the treatment of spontaneous animal tumors. A suitable target for such studies is -55- WO 00/15666 PCT/US99/20594 the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor. These tumors are usually not amenable to surgery, primarily because of the 5 anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor. Prior to entry into the study, each cat undergoes complete clinical examination, biopsy, and is scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves. Each cat is treated repeatedly, over a longer period of time. Photographs of the tumors will be taken 10 daily during the treatment period, and at each subsequent recheck. After treatment, each cat undergoes another CT scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter. The data are evaluated for differences in survival, response and toxicity as compared to control groups. Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span. In addition, other spontaneous animal tumors, such as fibrosarcoma, adenocarcinoma, lymphoma, 15 chrondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested. Of these mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans. However, the use of this model is limited by the rare occurrence of this type of tumor in animals. K. Screening Assays for Drug Candidates 20 Screening assays for drug candidates are designed to identify compounds that bind or complex with the polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably 25 soluble peptides,(poly)peptide-immunoglobulinfusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art. 30 All assays are common in that they call for contacting the drug candidate with a polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact. In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. 35 Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized -56- WO 00/15666 PCT/US99/20594 component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing 5 occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex. If the candidate compound interacts with but does not bind to a particular PRO]87, PR0533, PR0214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. 10 Such assays include traditional approaches, such as, cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88: 9578-9582 (1991)] as disclosed by Chevray and Nathans, Proc. Nati. Acad. Sci. USA 89:5789-5793 (1991)]. Many transcriptional activators, such as yeast GAL4, consist of two 15 physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl -iacZ reporter gene under control of a 20 GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for p-galactosidase. A complete kit

(MATCHMAKER

T M ) for identifying protein-protein interactions between two specific proteins using the two hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these 25 interactions. Compounds that interfere with the interaction of a PRO187-, PR0533-, PRO214-, PR0240-, PRO21 1-, PR0230-, PR0261-, PR0246- or PRO317-encoding gene identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the amplified gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and 30 binding of the two products. To test the ability of a test compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test 35 compound interferes with the interaction of the test compound and its reaction partner. To assay for antagonists, the PRO 187, PR0533, PRO214,PRO240,PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide may be added to a cell along with the compound to be screened for a particular activity and -57- WO 00/15666 PCTIUS99/20594 the ability of the compound to inhibit the activity of interest in the presence of the PRO]87, PR0533, PR0214, PR0240, PR021 1, PR0230, PR0261, PR0246 or PR0317 polypeptide indicates that the compound is an antagonist to the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PRO246 or PR0317 polypeptide. Alternatively, antagonists may be detected by combining the PROI 87, PR0533, PR0214, PR0240, 5 PRO21 1, PR0230, PR026 1, PR0246 or PRO317 polypeptide and a potential antagonist with membrane-bound PRO1 87, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The PRO187, PRO533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide can be labeled, such as by radioactivity, such that the number of PROl 87, PR0533, PRO214, PRO240, PRO21 1, PR0230, PR0261, PR0246 10 or PRO317 polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun.. 1(2): Chapter 5 (1991). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PRO261, PR0246 or PR0317 15 polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide. Transfected cells that are grown on glass slides are exposed to labeled PRO 187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide. The PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide can be labeled by a 20 variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor. As an alternative approach for receptor identification, labeled PRO 187, PR0533, PRO214, PR0240, 25 PRO21 1, PRO230, PRO261, PRO246 or PRO317 polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro-sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the 30 putative receptor. In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO187, PRO533, PRO214, PRO240, PRO21 1, PR0230, PR0261, PRO246 or PRO317 polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured. 35 More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with PRO187, PRO533, PRO214, PRO240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and -58- WO 00/15666 PCT/US99/20594 antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the PRO187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 polypeptide that recognizes the receptor but imparts no effect, 5 thereby competitively inhibiting the action of the PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO3 17 polypeptide. Another potential PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 polypeptide antagonist is an antisenseRNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted 10 mRNA and preventing protein translation. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA 15 oligonucleotide is designed to be complementary to a 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); Dervan et al., Science 251:1360 (1991)), thereby preventingtranscriptionand the production ofthe PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO187, PR0533, PRO214, PRO240, 20 PRO21 1, PRO230, PR0261, PR0246 or PR0317 polypeptide (antisense - Okano, Neurochem., 56560 (1991); Oligodeoxvnucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the 25 translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred. Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, 30 about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more. Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO187, PR0533, PR0214, PR0240, PRO21 1, PRO230, 35 PR0261, PR0246 or PR0317 polypeptide, thereby blocking the normal biological activity of the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and -59- WO 00/15666 PCT/US99/20594 synthetic non-peptidyl organic or inorganic compounds. Ribozymes are enzymatic RNA moleculescapable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further 5 details see, e.g., Rossi, Current Biology, 4:469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997). Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or 10 pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551, supra. These small molecules can be identified by any one or more ofthe screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art. L. Compositions and Methods for the Treatment of Tumors 15 The compositions useful in the treatment of tumors associatedwith the amplification of the genes identified herein include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc. that inhibit the expression and/or activity of the target gene product. For example, antisense RNA and RNA molecule act to directly block the translation of mRNA by 20 hybridizing to targeted mRNA and preventing protein translation. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred. Ribozymes are enzymatic RNA molecules capable ofcatalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. 25 Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biology. 4:469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997). Nucleic acid molecules in triple helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes 30 triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/3355 1, supra. These molecules can be identified by any or any combination of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art. 35 M. Antibodies Some ofthe most promising drug candidates according to the present invention are antibodies and antibody fragments which may inhibit the production or the gene product of the amplified genes identified herein and/or -60- WO 00/15666 PCT/US99/20594 reduce the activity of the gene products. 1. Polvclonal Antibodies Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can 5 be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the PRO 187, PR0533, PRO214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such 10 immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetictrehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. 15 2. Monoclonal Antibodies The anti-PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO211, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PRO317 antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an 20 immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizingagentwill typically include the PRO187, PR0533, PRO21 4, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 polypeptide, including fragments, or a fusion protein of such protein or a fragment thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or 25 spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture 30 medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells. Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of 35 antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection (ATCC), -61- WO 00/15666 PCT/US99/20594 Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984): Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63]. The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of 5 monoclonal antibodies directed against PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, 10 Anal. Biochem., 107:220 (1980). After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. 15 The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and 20 sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal 25 antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Patent No. 4,816,567; Morrison et al., supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable 30 domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue 35 or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. -62- WO 00/15666 PCT/US99/20594 3. Human and Humanized Antibodies The anti-PRO 187, anti-PR0533, anti-PRO214, anti-PRO240, anti-PRO21 1, anti-PR0230, anti-PR026 1, anti-PR0246 or anti-PRO317 antibodies may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains 5 or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human 10 immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized 15 antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non 20 human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 25 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Human antibodies can also be produced using various techniques known in the art, including phage display 30 libraries [Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol., 2:581 (1991)]. The techniques of Cole et al., and Boerner et al., are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been 35 partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; -63- WO 00/15666 PCTIUS99/20594 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology, 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature. 368:812-13 (1994); Fishwild et al., Nature Biotechnology, 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995). 5 4. Antibody Dependent Enzyme Mediated Prodruc Therapy (ADEPT) The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U. S. Patent No. 4,975,278. 10 The enzyme component ofthe immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such as way so as to convert it into its more active, cytotoxic form. Enzymes that are useful in the method ofthis invention include, but are not limited to, glycosidase, glucose oxidase, human lysosyme, human glucuronidase, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine 15 deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2 and carboxypeptidase A) and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as p-galactosidase and neuraminidase useful for converting glycosylated 20 prodrugs into free drugs; p-lactamase useful for converting drugs derivatized with p-lactams into free drugs; and penicillin amidases, such as penicillin Vamidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes" can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can 25 be prepared as described herein for delivery of the abzyme to a tumor cell population. The enzymes of this invention can be covalently bound to the anti-PRO187, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO2 11, anti-PR0230, anti-PR026 1, anti-PR0246 or anti-PRO317 antibodies by techniques well known in the art such as the use of the heterobifunctional cross-linking agents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of the antibody of the invention linked to at least a 30 functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312:604-608 (1984)). 5. Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding 35 specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit. -64- WO 00/15666 PCT/US99/20594 Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537-539 [1983]). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a 5 potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker etal., EMBOJ. 10:3655-3659(1991). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy 10 chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210(1986). 15 According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size 20 to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been 25 described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan etal., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab' 30 thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Fab' fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized 35 bispecific antibody F(ab') 2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity -65- WO 00/15666 PCTIUS99/20594 of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins 5 were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain 10 variable domain (V,) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary V, and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol., 152:5368 (1994). 15 Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol., 147:60 (1991). Exemplary bispecific antibodies may bind to two different epitopes on a given polypeptide herein. Alternatively, an anti-polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such 20 as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular polypeptide. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular polypeptide. These antibodies possess a polypeptide-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the polypeptide and further binds tissue factor (TF). 25 6. Heteroconjugate Antibodies Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may 30 be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4 mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980. 35 7. Effector function engineering It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example. For example, cysteine residue(s) may -66- WO 00/15666 PCT/US99/20594 be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992). Homodimeric antibodies with enhanced 5 anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See, Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989). 10 8. Immunoconiugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin), or a radioactive isotope (i.e., a radioconjugate). Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. 15 Enzymatically active protein toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, saporin, mitogellin, restrictocin, phenomycin, enomycin and the 20 tricothecenes. Small molecule toxins include, for example, calicheamicins, maytansinoids, palytoxin and CC1065. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include mBi, 1311, 'in, 9 0 Y and ' 86 Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctionalderivatives 25 of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3 30 methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, W094/11026. In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" 35 (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide). -67- WO 00/15666 PCT/US99/20594 9. Immunoliposomes The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980): and U.S. Patent Nos. 4,485,045 and 5 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., 10 J. Biol. Chem., 257:286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See, Gabizon et al., J. National Cancer Inst. 81(19):1484 (1989). N. Pharmaceutical Compositions 15 Antibodies specifically binding the product of an amplified gene identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions. If the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, 20 or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable region sequences of an antibody, peptide molecules can be designed which retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e.g., Marasco et al., Proc. Nat. Acad. Sci. USA 90:7889-7893 [1993]). 25 Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic 30 acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylorpropyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight(less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or 35 lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN T M , -68- WO 00/15666 PCT/US99/20594

PLURONICS

T

I or polyethylene glycol (PEG). Non-antibody compounds identified by the screening assays of the present invention can be formulated in an analogous manner, using standard techniques well known in the art. The formulation herein may also contain more than one active compound as necessary for the particular 5 indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and 10 poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980). The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. 15 Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, 20 degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37*C, resulting in a loss 25 of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. 30 0. Methods of Treatment It is contemplated that the antibodies and other anti-tumor compounds ofthe present invention may be used to treat various conditions, including those characterized by overexpression and/or activation of the amplified genes identified herein. Exemplary conditions or disorders to be treated with such antibodies and other compounds, 35 including, but not limited to, small organic and inorganic molecules, peptides, antisense molecules, etc., include benign or malignant tumors (e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas; and various head and neck tumors); -69- WO 00/15666 PCT/US99/20594 leukemias and lymphoid malignancies; other disorders such as neuronal, glial, astrocytal. hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders. The anti-tumor agents of the present invention, e.g., antibodies, are administered to a mammal, preferably 5 a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred. Other therapeutic regimens may be combined with the administration of the anti-cancer agents, e.g., antibodies of the instant invention. For example, the patient to be treated with such anti-cancer agents may also 10 receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams& Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may precede, or follow administration of the anti-tumor agent, 15 e.g., antibody, or may be given simultaneously therewith. The antibody may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules. It may be desirable to also administer antibodies against other tumor associated antigens, such as antibodies which bind to the ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, 20 or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to the patient. Sometimes, it may be beneficial to also administer one or more cytokines to the patient. In a preferred embodiment, the antibodies herein are co-administered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by an antibody of the present invention. However, simultaneous administration or administration of the antibody of the present invention first is also 25 contemplated. Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the antibody herein. For the prevention or treatment of disease, the appropriate dosage of an anti-tumor agent, e.g., an antibody herein will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical 30 history and response to the agent, and the discretion of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments. For example, depending on the type and severity of the disease, about I pg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 pg/kg 35 to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by -70- WO 00/15666 PCTIUS99/20594 conventional techniques and assays. P. Articles of Manufacture In another embodiment of the invention, an article of manufacture containing materials useful for the 5 diagnosis or treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection 10 needle). The active agent in the composition is usually an anti-tumor agent capable of interfering with the activity of a gene product identified herein, e.g., an antibody. The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and 15 user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Q. Diagnosis and Prognosis of Tumors While cell surface proteins, such as growth receptors overexpressed in certain tumors are excellent targets 20 for drug candidates or tumor (e.g., cancer) treatment, the same proteins along with secreted proteins encoded by the genes amplified in tumor cells find additional use in the diagnosis and prognosis of tumors. For example, antibodies directed against the protein products of genes amplified in tumor cells can be used as tumor diagnostics or prognostics. For example, antibodies, including antibody fragments, can be used to qualitatively or quantitatively detect 25 the expression of proteins encoded by the amplified genes ("marker gene products"). The antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. These techniques are particularly suitable, if the amplified gene encodes a cell surface protein, e.g., a growth factor. Such binding assays are performed essentially as described in section 5 above. 30 In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in 35 situ detection. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. -71- WO 00/15666 PCTIUS99/20594 All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. EXAMPLES 5 Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209. All original deposits referred to in the present application were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for 10 the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc., and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, 15 whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC § 122 and the Commissioner's rules pursuant thereto (including 37 CFR § 1.14 with particular reference to 886 OG 638). Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks: Sambrook et al., Molecular Cloning: A 20 Laboratory Manual, Cold Spring Harbor Press N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989; Innis et al., PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., N.Y., 1990; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Press Cold Spring Harbor, 1988; Gait, Oligonucleotide Synthesis IRL Press, Oxford, 1984; R.I. Freshney, Animal Cell Culture. 1987; Coligan et al., Current Protocols in Immunology 1991. 25 EXAMPLE 1 Isolation of cDNA clones Encoding a Human PRO] 87 An expressed sequence tag (EST) DNA database ( LIFESEQ@, Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST [#843193] was identified which showed homology to fibroblast growth factor (FGF-8) 30 also known as androgen-induced growth factor. RNA for construction of cDNA libraries was then isolated from human fetal lung tissue. The cDNA libraries used to isolate the cDNA clones encoding human PRO 187 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sall hemikinased adaptors, cleaved with NotI, sized appropriately 35 by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique XhoI and NotI. -72- WO 00/15666 PCTIUS99/20594 Oligonucleotide probes based upon the above described EST sequence were then synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO 187. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences 5 are typically 40-55 bp in length. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs. The oligonucleotide probes employed were as follows: 10 forward PCR primer: 5'-CAGTACGTGAGGGACCAGGGCGCCATGA-3'(SEQ ID NO:3) reverse PCR primer: 5'-CCGGTGACCTGCACGTGCTTGCCA-3' (SEQ ID NO:4) hybridization probe: 15 5'-GCGGATCTGCCGCCTGCTCANCTGGTCGGTCATGGCGCCCT-3' (SEQ ID NO:5) A full length clone was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 26-28 and a stop signal at nucleotide positions 641-643 (Figure 1, SEQ ID NO: 1). The predicted polypeptide precursor is 205 amino acids long and is shown in Figure 2 (SEQ ID NO:2) Analysis of the full-length PRO187 sequence shown in Figure 2 (SEQ ID NO:2) evidences the presence of an 20 important polypeptide domain as shown in Figure 2, wherein the location given for that important polypeptide domain is approximate as described above. Analysis of the full-length PRO 187 sequence evidenced a signal peptide from about amino acid I to about amino acid 22. Clone DNA27864-1155 has been deposited with ATCC on October 16, 1997 and is assigned ATCC deposit no. 209375. An analysis ofthe Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment 25 analysis of the full-length sequence shown in Figure 2 (SEQ ID NO:2), evidenced 74% sequence identity between the PRO 187 amino acid sequence and human fibroblast growth factor-8 (androgen-induced growth factor). EXAMPLE 2 Isolation of cDNA Clones Encoding a Human PR0533 30 The EST sequence accession number AF007268, a murine fibroblast growth factor (FGF-15) was used to search various public EST databases (e.g., GenBank, Dayhoff, etc.). The search was performed using the computer program BLAST or BLAST2 [Altschul et aL., Methods in Enzymology 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. The search resulted in a hit with GenBank EST AA220994, which has been identified as stratagene NT2 neuronal precursor 937230. 35 RNA for construction of cDNA libraries was then isolated from human fetal retina. The cDNA libraries used to isolate the cDNA clones encoding human PR0533 were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo -73- WO 00/15666 PCT/US99/20594 dT containing a Notl site, linked with blunt to Sall hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science 253:1278-1280 (1991)) in the unique Xhol and Notl. 5 Oligonucleotide probes based upon the above described EST sequence were then synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0533. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In order to screen several libraries for a full-length clone, DNA from the libraries 10 was screened by PCR amplification, as per Ausubel et aL, Current Protocols in Molecular Biology supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs. The oligonucleotide probes employed were as follows: FGFI5.f (forward PCR primer): 15 5'-ATCCGCCCAGATGGCTACAATGTGTA-3'(SEQ ID NO:8) FGF15.r (reverse PCR primer): 5'-CCAGTCCGGTGACAAGCCCAAA-3' (SEQ ID NO:9) FGF15.p (hybridization probe): 5'-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3' (SEQ ID NO: 10) 20 A full length clone was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 464-466 and a stop signal at nucleotide positions 1112-1114 (Figure 3, SEQ ID NO:6). The predicted polypeptide precursor is 216 amino acids long and is shown in Figure 4 (SEQ ID NO:7) Analysis of the full-length PR0533 sequence shown in Figure 4 (SEQ ID NO:7) evidences the presence of an important polypeptide domain as shown in Figure 4, wherein the location given for that important polypeptide 25 domain is approximate as described above. Analysis ofthe full-length PR0533 sequence evidences a signal peptide from about amino acid I to about amino acid 22. Clone DNA49435-1219 has been deposited with ATCC on November 21, 1997 and is assigned ATCC deposit no. 209480. An analysis ofthe Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 4 (SEQ ID NO:7), evidenced 53% sequence identity between 30 the PR0533 amino acid sequence and fibroblast growth factor. EXAMPLE 3 Isolation of cDNA Clones Encoding a Human PR0214 The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 35 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ@, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 -74- WO 00/15666 PCT/US99/20594 [Altschul et al., Methods in Enzymology 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). 5 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA28744. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above. 10 Based on the DNA28744 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO214. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater 15 than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs. PCR primers (forward and reverse) were synthesized: 20 forward PCR primer: 5'-ATTCTGCGTGAACACTGAGGGC-3' (SEQ ID NO: 13) reverse PCR primer: 5'-ATCTGCTTGTAGCCCTCGGCAC-3' (SEQ ID NO: 14) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28744 25 sequence which had the following nucleotide sequence: hybridization probe: 5'-CCTGGCTATCAGCAGGTGGGCTCCAAGTGTCTCGATGTGGATGAGTGTGA-3' (SEQ ID NO:15) RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. The cDNA 30 libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a NotI site, linked with blunt to Sall hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD: pRK5B is a precursor of pRK5D that does not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI 35 and NotI sites. DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PRO214 polypeptide (designatedhereinas DNA32286-1191 [Figure 5, SEQ ID NO: 11]) and the derived -75- WO 00/15666 PCT/US99/20594 protein sequence for that PRO214 polypeptide. The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 103-105 and a stop signal at nucleotide positions 1363-1365 (Figure 5, SEQ ID NO: 11). The predicted polypeptide precursor is 420 amino acids long and is shown in Figure 6 (SEQ ID 5 NO: 12). Analysis of the full-length PRO214 sequence shown in Figure 6 (SEQ ID NO: 12) evidences the presence of a variety of important polypeptide domains as shown in Figure 6, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PRO214 sequence evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 29 and a transmembrane domain from about amino acid 372 to about amino acid 392. Clone DNA32286-1 191 has been 10 deposited with ATCC on October 16, 1997 and is assigned ATCC deposit no. 209385. An analysis of the Dayhoff database (version35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 6 (SEQ ID NO: 12), evidenced sequence identity between the PR0214 amino acid sequence and HT protein and/or Fibulin (49% and 38%, respectively). 15 EXAMPLE 4 Isolation of cDNA Clones Encoding a Human PR0240 Polypeptide The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ@, Incyte 20 Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). 25 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA30873. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above. 30 Based on the DNA30873 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0240. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater 35 than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe -76- WO 00/15666 PCT/US99/20594 oligonucleotide and one of the primer pairs. PCR primers (forward and reverse) were synthesized: forward PCR primer: 5'-TCAGCTCCAGACTCTGATACTGCC-3' (SEQ ID NO:18) 5 reverse PCR primer: 5'-TGCCTTTCTAGGAGGCAGAGCTCC-3' (SEQ ID NO:19) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30873 sequence which had the following nucleotide sequence: hybridization probe: 10 5'-GGACCCAGAAATGTGTCCTGAGAATGGATCTTGTGTACCTGATGGTCCAG-3' (SEQ ID NO:20) RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a NotI 15 site, linked with blunt to Sall hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique Xhol and NotI sites. DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a 20 full-length PR0240 polypeptide (designated herein as DNA343 87-113 8[Figure 7, SEQ ID NO: 16]) and the derived protein sequence for that PR0240 polypeptide. The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 12-14 and a stop signal at nucleotide positions 699-701 (Figure 7, SEQ ID NO: 16). The predicted polypeptide precursor is 229 amino acids long and is shown in Figure 8 (SEQ ID NO: 17). 25 Analysis of the full-length PR0240 sequence shown in Figure 8 (SEQ ID NO: 17) evidences the presence of a variety of important polypeptide domains as shown in Figure 8, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PRO240 sequence evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 30 and a transmembrane domain from about amino acid 198 to about amino acid 212. Clone DNA34387-1138 has been 30 deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no. 209260. An analysisofthe Dayhoffdatabase (version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 8 (SEQ ID NO: 17), evidenced sequence identity between the PRO240 amino acid sequence and the serrate precursor protein from Drosophilia melanogaster and the C-serrate-I protein from Gallus gallus (30% and 35%, respectively). 35 -77- WO 00/15666 PCTIUS99/20594 EXAMPLE 5 Isolation of cDNA Clones Encoding a Human PRO211 The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST 5 databases included public EST databases (e.g., Dayhoff, GenBank) and a proprietary EST database (LIFESEQ@, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in Enzymology 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA 10 sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA28730. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed 15 above. Based on the DNA28730 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO2 11. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 20 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs. 25 PCR primers (forward and reverse) were synthesized: forward PCR primer: 5'-AGAGTGTATCTCTGGCTACGC-3' (SEQ ID NO:23) reverse PCR primer: 5'-TAAGTCCGGCACATTACAGGTC-3' (SEQ ID NO:24) 30 Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28730 sequence which had the following nucleotide sequence: hybridization probe: 5'-AGGGAGCACGGACAGTGTGCAGATGTGGACGAGTGCTCACTAGCA-3' (SEQ ID NO:25) RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. The cDNA 35 libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a NotI site, linked with blunt to SalI hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, -78- WO 00/15666 PCTIUS99/20594 and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique Xhol and NotI sites. DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a 5 full-length PRO2 11 polypeptide (designated herein as DNA32292- 1131 [Figure 9, SEQ ID NO:2 1]) and the derived protein sequence for that PRO2 11 polypeptide. The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 65-67and a stop signal at nucleotide positions 1124-1126 (Figure 9, SEQ ID NO:21). The predicted polypeptide precursor is 353 amino acids long with a molecular weight of approximately 10 38,190 daltons [Figure 10; (SEQ IDNO:22)]. Analysis of the full-length PRO211 sequence shown in Figure 10 (SEQ ID NO:22) evidences the presence of an important polypeptide domain as shown in Figure 10, wherein the location given for that important polypeptide domain is approximate as described above. Analysis of the full-length PRO2 11 sequence evidences the presence of a signal peptide from about amino acid 1 to about amino acid 24. Clone DNA32292-1131 has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no. 15 209258. An analysis ofthe Dayhoff database (version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis ofthe full-length sequence shown in Figure 10 (SEQ ID NO:22), evidenced sequence identity between the PRO21I1 amino acid sequence and EGF. 20 EXAMPLE 6 Isolation of cDNA Clones Encoding a Human PR0230 The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ@, Incyte 25 Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in EnzVmology 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). 30 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA30857. An EST proprietary to Genentech was employed in the consensus assembly and is herein designated DNA20088. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed 35 above. Based on the DNA30857 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length -79- WO 00/15666 PCT/US99/20594 coding sequence for PR0230. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was 5 screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs. PCR primers (forward and reverse) were synthesized: forward PCR primer: 10 5'-TTCGAGGCCTCTGAGAAGTGGCCC-3' (SEQ ID NO:28) reverse PCR primer: 5'-GGCGGTATCTCTCTGGCCTCCC-3' (SEQ ID NO:29) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30857 sequence which had the following nucleotide sequence: 15 hybridization probe: 5'-TTCTCCACAGCAGCTGTGGCATCCGATCGTGTCTCAATCCATTCTCTGGG-3' (SEQ ID NO:30) RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available 20 reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Notl site, linked with blunt to SalI hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes etal., Science, 253:1278-1280 (1991)) in the unique Xhol and Notl sites. 25 DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR0230 polypeptide (designated herein as DNA33223-1136 [Figure 11, SEQ ID NO:26]) and the derived protein sequence for that PR0230 polypeptide. The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 100-102 and a stop signal at nucleotide positions 592-594 (Figure 11, SEQ 30 ID NO:26). The predicted polypeptide precursor is 164 amino acids long [Figure 12; (SEQ ID NO:27)]. Analysis of the full-length PR0230 sequence shown in Figure 12 (SEQ ID NO:27) evidences the presence of an important polypeptide domain as shown in Figure 12, wherein the location given for that important polypeptide domain is approximate as described above. Analysis of the full-length PR0230 sequence evidences the presence of a signal peptide from about amino acid 1 to about amino acid 21. Clone DNA33223-1136 has been deposited with ATCC 35 on September 16, 1997 and is assigned ATCC deposit no. 209264. An analysis ofthe Dayhoff database (version 35.45 SwissProt35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 12 (SEQ ID NO:27), evidenced sequence identity between the -80- WO 00/15666 PCT/US99/20594 PR0230 amino acid sequence and a rabbit tubulointerstitial nephritis antigen precursor. EXAMPLE 7 Isolation of cDNA Clones Encodin2 a Human PR0261 5 The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ@, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as a comparison of the ECD protein sequences to 10 a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA30843. In some cases, the consensus sequence derives 15 from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above. Based on the DNA30843 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length 20 coding sequence for PR026 1. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, supra, with the PCR 25 primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs. PCR primers (forward and reverse) were synthesized: forward PCR primer: 5'-AAAGGTGCGTACCCAGCTGTGCC-3' (SEQ ID NO:33) 30 reverse PCR primer: 5'-TCCAGTCGGCAGAAGCGGTTCTGG-3' (SEQ ID NO:34) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30843 sequence which had the following nucleotide sequence: hybridization probe: 35 5'-CCTGGTGCTGGATGGCTGTGGCTGCTGCCGGGTATGTGCACGGCGGCTGGG-3' (SEQ ID NO:35) -81- WO 00/15666 PCT/US99/20594 RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a NotI site, linked with blunt to Sall hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, 5 and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science, 253:1278-1280 (199 1)) in the unique Xhol and NotI sites. DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR0261 polypeptide (designated herein as DNA33473-1 176 [Figure 13, SEQ ID NO:3 1]) and the 10 derived protein sequence for that PR0261 polypeptide. The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 10-12 and a stop signal at nucleotide positions 760-762 (Figure 13, SEQ ID NO:3 1). The predicted polypeptide precursor is 250 amino acids long [Figure 14; (SEQ ID NO:32)]. Analysis of the full-length PR0261 sequence shown in Figure 14 (SEQ ID NO:32) evidences the presence of an important 15 polypeptide domain as shown in Figure 14, wherein the location given for that important polypeptide domain is approximate as described above. Analysis of the full-length PR0261 sequence evidences the presence of a signal peptide from about amino acid I to about amino acid 23. Clone DNA33473-1176 has been deposited with ATCC on October 17, 1997 and is assigned ATCC deposit no. 209391. An analysis ofthe Dayhoffdatabase(version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment 20 analysis of the full-length sequence shown in Figure 14 (SEQ IDNO:32), evidenced sequence identity between the PR0261 amino acid sequence and CTCF, thereby indicating that PR0261 is a novel growth factor. EXAMPLE 8 Isolation of cDNA Clones Encoding a Human PR0246 25 The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ@, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in Enzymology 266:460-480 (1996)] as a comparison of the ECD protein sequences to 30 a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA30955. In some cases, the consensus sequence derives 35 from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above. -82- WO 00/15666 PCTIUS99/20594 Based on the DNA30955 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0246. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 5 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs. 10 PCR primers (forward and reverse) were synthesized: forward PCR primer: 5'-AGGGTCTCCAGGAGAAAGACTC-3' (SEQ ID NO:38) reverse PCR primer: 5'-ATTGTGGGCCTTGCAGACATAGAC-3' (SEQ ID NO:39) 15 Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30955 sequence which had the following nucleotide sequence: hybridization probe: 5'-GGCCACAGCATCAAAACCTTAGAACTCAATGTACTGGTTCCTCCAGCTCC-3' (SEQ ID NO:40) 20 RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a NotI site, linked with blunt to Sall hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor 25 of pRK5D that does not contain the SfiI site; see, Holmes et al., Science 253:1278-1280 (1991)) in the unique Xhol and NotI sites. DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR0246 polypeptide (designated herein as DNA35639-1172 [Figure 15, SEQ ID NO:36]) and the derived protein sequence for that PR0246 polypeptide. 30 The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 126-128 and a stop signal at nucleotide positions 1296-1298 (Figure 15, SEQ ID NO:36). The predicted polypeptide precursor is 390 amino acids long [Figure 16; (SEQ ID NO:37)]. Analysis of the full-length PR0246 sequence shown in Figure 16 (SEQ ID NO:37) evidences the presence of a variety of important polypeptide domains as shown in Figure 16, wherein the locations given for those important polypeptide 35 domains are approximate as described above. Analysis ofthe full-length PR0246 sequence evidences the presence of the following: a signal peptide from about amino acid 1 to about amino acid 29 and a transmembrane domain from about amino acid 247 to about amino acid 266. Clone DNA35639-1176 has been deposited with ATCC on -83- WO 00/15666 PCT/US99/20594 October 17, 1997 and is assigned ATCC deposit no. 209396. An analysisofthe Dayhoff database (version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 16 (SEQ ID NO:37), evidenced sequence identity between the PR0246 amino acid sequence and the human cell surface protein HCAR, thereby indicating that PR0246 may be 5 a novel cell surface virus receptor. EXAMPLE 9 Isolation of cDNA Clones Encoding a Human PR0317 The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 10 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public EST databases (e.g., GenBank)and a proprietary EST database (LIFESEQ@, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in Enzymology 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some 15 cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington). A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA28722. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap 20 to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above. Based on the DNA28722 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO317. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and 25 are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe 30 oligonucleotide and one of the primer pairs. PCR primers (forward and reverse) were synthesized: forward PCR primer: 5'-AGGACTGCCATAACTTGCCTG-3' (SEQ ID NO:43) reverse PCR primer: 35 5'-ATAGGAGTTGAAGCAGCGCTGC-3' (SEQ ID NO:44) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28722 sequence which had the following nucleotide sequence: -84- WO 00/15666 PCTIUS99/20594 hybridization probe: 5'-TGTGTGGACATAGACGAGTGCCGCTACCGCTACTGCCAGCACCGC-3' (SEQ ID NO:45) RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue. The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available 5 reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sall hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science, 253:1278-1280 (199 1)) in the unique Xhol and NotI sites. 10 DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR0317 polypeptide (designated herein as DNA33461-l199 [Figure 17, SEQ ID NO:41]) and the derived protein sequence for that PRO317 polypeptide. The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 68-70 and a stop signal at nucleotide positions 1166-1168 (Figure 17, SEQ 15 ID NO:4 1). The predicted polypeptide precursor is 366 amino acids long [Figure 18; (SEQ ID NO:42)]. Analysis of the full-length PRO317 sequence shown in Figure 18 (SEQ ID NO:42) evidences the presence of important polypeptide domains as shown in Figure 18, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PR0317 sequence (Figure 18, SEQ ID NO:42) evidences the following: a signal peptide from about amino acid I to about amino acid 18, and an N-linked 20 glycosylation site at amino acid 160. Clone DNA33461-1199 has been deposited with ATCC on October 15, 1997 and is assigned ATCC deposit no. 209367. An analysis ofthe Dayhoff database (version 35.45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 18 (SEQ ID NO:42), evidenced 92% sequence identity between the PRO317 amino acid sequence and human EBAF- 1. A significant homology also exists between the 25 PRO317 amino acid sequence and human EBAF-2 and mouse LEFTY protein. PRO317 shows amino acid sequence alignment with other members of the TGF-P superfamily. The C-terminal end of the protein contains many conserved sequences and the pattern expected of the TGF-P superfamily. EXAMPLE 10 30 Gene Amplification This example shows that the PRO187-, PR0533-, PR0214-, PR0240-, PRO21 1-, PR0230-, PR0261-, PR0246- or PRO317-encoding genes are amplified in the genome of certain human lung, colon and/or breast cancers and/or cell lines. Amplification is associated with overexpression of the gene product, indicating that the polypeptides are useful targets for therapeutic intervention in certain cancers such as colon, lung, breast and other 35 cancers. Therapeutic agents may take the form of antagonists of PROl87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 polypeptide, for example, murine-human chimeric, humanized or human antibodies against a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 -85- WO 00/15666 PCTIUS99/20594 polypeptide. The starting material for the screen was genomic DNA isolated from a variety cancers. The DNA is quantitated precisely, e.g., fluorometrically. As a negative control, DNA was isolated from the cells of ten normal healthy individuals which was pooled and used as assay controls for the gene copy in healthy individuals (not 5 shown). The 5' nuclease assay (for example, TaqMan

T

M) and real-time quantitative PCR (for example, ABI Prizm 7700 Sequence Detection System

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M

1 (Perkin Elmer, Applied Biosystems Division, Foster City, CA)), were used to find genes potentially amplified in certain cancers. The results were used to determine whether the DNA encoding PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PR0317 is over-represented in any of the primary lung or colon cancers or cancer cell lines or breast cancer cell lines that were 10 screened. The primary lung cancers were obtained from individuals with tumors of the type and stage as indicated in Table 2. An explanation of the abbreviations used for the designation of the primary tumors listed in Table 2 and the primary tumors and cell lines referred to throughout this example has been given hereinbefore. The results of the TaqMan T M are reported in delta (A) Ct units. One unit corresponds to I PCR cycle or approximately a 2-fold amplification relative to normal, two units corresponds to 4-fold, 3 units to 8-fold 15 amplification and so on. Quantitation was obtained using primers and a TaqMan

T

' fluorescent probe derived from the PRO 187-, PR0533-,PRO214-, PR0240-, PRO2 11-, PR0230-, PR026 I -, PR0246- or PRO3 17-encodinggene. Regions of PRO]87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 which are most likely to contain unique nucleic acid sequences and which are least likely to have spliced out introns are preferred for the primer and probe derivation, e.g., 3-untranslated regions. The sequences for the primers and 20 probes (forward, reverse and probe) used for the PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 gene amplification analysis were as follows: PRO187 (DNA27864- 1155) 27864.tm.p: 5'-GCAGATTTTGAGGACAGCCACCTCCA-3' (SEQ ID NO:46) 25 27864.tm.f: 5'-GGCCTTGCAGACAACCGT-3' (SEQ ID NO:47) 27864.tm.r: 5'-CAGACTGAGGGAGATCCGAGA-3' (SEQ ID NO:48) 27864.tm.p2: 30 5'-CAGCTGCCCTTCCCCAACCA-3' (SEQ ID NO:49) 27864.tm.f2: 5'-CATCAAGCGCCTCTACCA-3' (SEQ ID NO:50) 27864.tm.r2: 5'-CACAAACTCGAACTGCTTCTG-3' (SEQ ID NO:5 1) 35 -86- WO 00/1 5666 PCTIUS99/20594 PR0533 (DNA49435-12 19): 49435.tm.f:. 5'-GGGACGTGCTTCTACAAGAACAG-3' (SEQ ID NO:52) 49435.tm.r: 5 5'-CAGGCTTACAATGTTATGATCAGACA-3 (SEQ ID NO:53) 49435.tm.p: 5'-TATTCAGAGTTTTCCATTGGCAGTGCCAGTT-3' (SEQ ID NO:54) PR0214 (DNA32286-1 191): 10 32286.3utr-5: 5'-GGGCCATCACAGCTCCCT-3' (SEQ ID NO:55) 32286.3utr-3b: 5'-GGGATGTGGTGAACACAGAACA-3' (SEQ ID NO:56) 32286.3utr-probe: 15 5'-TGCCAGCTGCATGCTGCCAGTT-3' (SEQ ID NO:57) PR0240 (DNA34387-1 138): 343 87.tm.p 1: 5'-CAGCGCCGAAAAGCCAAGACTTCAT-3' (SEQ ID NO:58) 20 34387.tm.fl: 5'-GATTCTGGGAGCCACCACTCTAT-3' (SEQ ID NO:59) 343 87.tm.rI: 5'-AGCTCCCTGACTGGGCTAAGATA-3' (SEQ ID NO:60) 34387.3utr-5: 25 5'-GTCAGGGAGCTCTGCTTCCTAG-3' (SEQ ID NO:6 1) 343 87.3utr-3: 5'-AATGGCGGCCTCAACCTT-3' (SEQ ID NO:62) 343 87.3utr-probe.rc: 5'-CGAATCCACTGGCGAAAGATGCCTT-3' (SEQ ID NO:63) 30 PRO211I (DNA32292-1 131): 32292.3utr-5: 5'-CAGAAGGATGTCCCGTGGAA-3' (SEQ ID NO:64) 32292.3utr-3: 35 5'-GCCGCTGTCCACTGCAG-3' (SEQ ID NO:65) 32292.3utr-probe.rc: 5'-GACGGCATCCTCAGGGCCACA-3' (SEQ ID NO:66) -87- WO 00/15666 PCT/US99/20594 PR0230 (DNA33223-1136): 33223.tm.p3: 5'-ATGTCCTCCATGCCCACGCG-3' (SEQ ID NO:67) 33223.tm.f3: 5 5'-GAGTGCGACATCGAGAGCTT-3' (SEQ ID NO:68) 33223.tm.r3: 5'-CCGCAGCCTCAGTGATGA-3' (SEQ ID NO:69) 33223.3utr-5: 5'-GAAGAGCACAGCTGCAGATCC-3' (SEQ ID NO:70) 10 33223.3utr-3: 5'-GAGGTGTCCTGGCTTTGGTAGT-3' (SEQ ID NO:71) 33223.3 utr-probe: 5'-CCTCTGGCGCCCCCACTCAA-3' (SEQ ID NO:72) 15 PR0261 (DNA33473-1176): 33473.3utr-5: 5'-TCTAGCCCACTCCCTGCCT-3' (SEQ ID NO:73) 33473.3utr-3: 5'-GAAGTCGGAGAGAAAGCTCGC-3' (SEQ ID NO:74) 20 33473.3utr-probe: 5'-CACACACAGCCTATATCAAACATGCACACG-3' (SEQ ID NO:75) PR0246 (DNA35639-1172): 35639.3utr-5: 25 5'-GGCAGAGACTTCCAGTCACTGA-3' (SEQ ID NO:76) 35639.3utr-3: 5'-GCCAAGGGTGGTGTTAGATAGG-3' (SEQ ID NO:77) 35639.3utr-probe: 5'-CAGGCCCCCTTGATCTGTACCCCA-3' (SEQ ID NO:78) 30 PR0317 (DNA33461-1 199): 33461.tm.f: 5'-CCAGGAGAGCTGGCGATG-3' (SEQ ID NO:79) 33461.tm.r: 35 5'-GCAAATTCAGGGCTCACTAGAGA-3' (SEQ ID NO:80) 3346 I.tm.p: 5'-CACAGAGCATTTGTCCATCAGCAGTTCAG-3' (SEQ ID NO:81) -88- WO 00/15666 PCT/US99/20594 The 5'nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5' exonuclease activity of Taq DNA polymerase enzyme to monitor amplification in real time. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase 5 enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated 10 for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data. The 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism 7700TM Sequence Detection. The system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, 15 laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data. 5' Nuclease assay data are initially expressed as Ct, or the threshold cycle. This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence. The ACt values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid 20 sample when comparing cancer DNA results to normal human DNA results. Table 2 describes the stage, T stage and N stage of various primary tumors which were used to screen the PRO187, PR0533, PR0214, PRO240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 compounds of the invention. -89- WO 00/15666 PCT/US99/20594 Table 2 Primary Lung and Colon Tumor Profiles Primary Tumor Stage Other Stage Dukes Stage T Stae N Stage 5 Human lung tumor AdenoCa (SRCC724) [LTI] IIA TI NI Human lung tumor SqCCa (SRCC725) [LTla] IIB T3 NO Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 NO Human lung tumor AdenoCa (SRCC727) [LT3] IIIA TI N2 Human lung tumor AdenoCa (SRCC728) [LT4] IB T2 NO 10 Human lung tumor SqCCa (SRCC729) [LT6] IB T2 NO Human lung tumor Aden/SqCCa (SRCC730) [LT7] IA TI NO Human lung tumor AdenoCa (SRCC73 I) [LT9] IB T2 NO Human lung tumor SqCCa (SRCC732) [LT 10] IIB T2 NI Human lung tumor SqCCa (SRCC733) [LT 1I] IIA TI NI 15 Human lung tumor AdenoCa (SRCC734) [LTI2] IV T2 NO Human lung tumor AdenoSqCCa (SRCC735)[LTI3] LB T2 NO Human lung tumor SqCCa (SRCC736) [LT15] IB T2 NO Human lung tumor SqCCa (SRCC737) [LT16] IB T2 NO Human lung tumor SqCCa (SRCC738) [LT17] IIB T2 NI 20 Human lung tumor SqCCa (SRCC739) [LT18] IB T2 NO Human lung tumor SqCCa (SRCC740) [LT 19] IB T2 NO Human lung tumor LCCa (SRCC741) [LT21] IIB T3 NI Human lung AdenoCa (SRCC81 I) [LT22] IA TI NO Human colon AdenoCa (SRCC742) [CT2] MI D pT4 NO 25 Human colon AdenoCa (SRCC743) [CT3] B pT3 NO Human colon AdenoCa (SRCC 744) [CT8] B T3 NO Human colon AdenoCa (SRCC745) [CT IO] A pT2 NO Human colon AdenoCa (SRCC746) [CT12] MO, RI B T3 NO Human colon AdenoCa (SRCC747) [CT14] pMO, RO B pT3 pNO 30 Human colon AdenoCa (SRCC748) [CT15] Ml, R2 D T4 N2 Human colon AdenoCa (SRCC749) [CT16] pMO B pT3 pNO Human colon AdenoCa (SRCC750) [CT17] CI pT3 pN1 Human colon AdenoCa (SRCC751) [CTl] MO, RI B pT3 NO Human colon AdenoCa (SRCC752) [CT4] B pT3 MO 35 Human colon AdenoCa (SRCC753) [CT5] G2 Cl pT3 pNO Human colon AdenoCa (SRCC754) [CT6] pMO, RO B pT3 pNO Human colon AdenoCa (SRCC755) [CT7] G I A pT2 pNO Human colon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human colon AdenoCa (SRCC757) [CTI 1] B T3 NO 40 Human colon AdenoCa (SRCC758) [CTI 8] MO, RO B pT3 pNO DNA Preparation: DNA was prepared from cultured cell lines, primary tumors, normal human blood. The isolation was 45 performed using purification kit, buffer set and protease and all from Quiagen, according to the manufacturer's instructions and the description below. Cell culture lysis: Cells were washed and trypsinized at a concentration of 7.5 x 108 per tip and pelleted by centrifuging at 1000 rpm for 5 minutes at 4'C, followed by washing again with 1/2 volume of PBS recentrifugation. The pellets 50 were washed a third time, the suspended cells collected and washed 2x with PBS. The cells were then suspended into 10 ml PBS. Buffer Cl was equilibrated at 4 0 C. Qiagen protease #1 9155 was diluted into 6.25 ml cold ddH 2 O -90- WO 00/15666 PCTIUS99/20594 to a final concentration of 20 mg/ml and equilibrated at4 0 C. 10 ml of G2 Buffer was prepared by diluting Qiagen RNAse A stock (100 mg/ml) to a final concentration of 200 pg/ml. Buffer Cl (10 ml, 4 0 C) and ddH20 (40 ml, 4 0 C) were then added to the 10 ml of cell suspension, mixed by inverting and incubated on ice for 10 minutes. The cell nuclei were pelleted by centrifuging in a Beckman 5 swinging bucket rotor at 2500 rpm at 4'C for 15 minutes. The supernatant was discarded and the nuclei were suspended with a vortex into 2 ml Buffer C l (at 4"C) and 6 ml ddHO, followed by a second 4'C centrifugation at 2500 rpm for 15 minutes. The nuclei were then resuspended into the residual buffer using 200 pl per tip. G2 buffer (10 ml) was added to the suspended nuclei while gentle vortexing was applied. Upon completion of buffer addition, vigorous vortexing was applied for 30 seconds. Quiagen protease (200 pl, prepared as indicated above) was added 10 and incubated at 50'C for 60 minutes. The incubation and centrifugation was repeated until the lysates were clear (e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min., 4"C). Solid human tumor sample preparation and lysis: Tumor samples were weighed and placed into 50 ml conical tubes and held on ice. Processing was limited to no more than 250 mg tissue per preparation (1 tip/preparation). The protease solution was freshly prepared by 15 diluting into 6.25 ml cold ddH 2 0 to a final concentration of 20 mg/mI and stored at 4*C. G2 buffer (20 ml) was prepared by diluting DNAse A to a final concentration of 200 mg/mI (from 100 mg/mI stock). The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds using the large tip of the polytron in a laminar-flow TC hood in order to avoid inhalation of aerosols, and held at room temperature. Between samples, the polytron was cleaned by spinning at 2 x 30 seconds each in 2L ddH.0, followed by G2 buffer (50 ml). If tissue was still present on the 20 generator tip, the apparatus was disassembled and cleaned. Quiagen protease (prepared as indicated above, 1.0 ml) was added, followed by vortexing and incubation at 50"C for 3 hours. The incubation and centrifugation was repeated until the lysates were clear (e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min., 4C). Human blood preparation and lysis: 25 Blood was drawn from healthy volunteers using standard infectious agent protocols and citrated into 10 ml samples per tip. Quiagen protease was freshly prepared by dilution into 6.25 ml cold ddH 2 O to a final concentration of 20 mg/mI and stored at 4 0 C. G2 buffer was prepared by diluting RNAse A to a final concentration of 200 pg/ml from 100 mg/mI stock. The blood (10 ml) was placed into a 50 ml conical tube and 10 ml Cl buffer and 30 ml ddHO (both previously equilibrated to 4 0 C) were added, and the components mixed by inverting and 30 held on ice for 10 minutes. The nuclei were pelleted with a Beckman swinging bucket rotor at 2500 rpm, 4 0 C for 15 minutes and the supernatant discarded. With a vortex, the nuclei were suspended into 2 ml CI buffer (4*C) and 6 ml ddH 2 O (4 0 C). Vortexing was repeated until the pellet was white. The nuclei were then suspended into the residual buffer using a 200 pl tip. G2 buffer (10 ml) were added to the suspended nuclei while gently vortexing, followed by vigorous vortexing for 30 seconds. Quiagen protease was added (200 pl) and incubated at 50 0 C for 35 60 minutes. The incubation and centrifugation was repeated until the lysates were clear (e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min., 4 0 C). Purification of cleared lysates: -91- WO 00/15666 PCT/US99/20594 (1) Isolation of cenomic DNA: Genomic DNA was equilibrated (1 sample per maxi tip preparation) with 10 ml QBT buffer. QF elution buffer was equilibrated at 50'C. The samples were vortexed for 30 seconds, then loaded onto equilibrated tips and drained by gravity. The tips were washed with 2 x 15 ml QC buffer. The DNA was eluted into 30 ml silanized, 5 autoclaved 30 ml Corex tubes with 15 ml QF buffer (50"C). Isopropanol (10.5 ml) was added to each sample, the tubes covered with parafin and mixed by repeated inversion until the DNA precipitated. Samples were pelleted by centrifugation in the SS-34 rotor at 15,000 rpm for 10 minutes at 4'C. The pellet location was marked, the supernatant discarded, and 10 ml 70% ethanol (4"C) was added. Samples were pelleted again by centrifugation on the SS-34 rotor at 10,000 rpm for 10 minutes at 4 0 C. The pellet location was marked and the supernatant discarded. 10 The tubes were then placed on their side in a drying rack and dried 10 minutes at 37"C, taking care not to overdry the samples. After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5) and placed at 504C for 1-2 hours. Samples were held overnight at 4"C as dissolution continued. The DNA solution was then transferred to 1.5 ml tubes with a 26 gauge needle on a tuberculin syringe. The transfer was repeated 5x in order to shear the DNA. Samples were 15 then placed at 50*C for 1-2 hours. (2) Quantitation of genomic DNA and preparation for gene amplification assay: The DNA levels in each tube were quantified by standard A2 60 , A, 0 spectrophotometry on a 1:20 dilution (5 pl DNA + 95 pl ddH 2 O) using the 0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer. A 2 6

O/A

280 ratios were in the range of 1.8-1.9. Each DNA samples was then diluted further to approximately 200 ng/ml in TE 20 (pH 8.5). If the original material was highly concentrated (about 700 ng/il), the material was placed at 50 0 C for several hours until resuspended. Fluorometric DNA quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer's guidelines as modified below. This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometer to warm-up for about 15 minutes. The Hoechst dye working solution (#H33258, 10 pl, prepared within 25 12 hours of use) was diluted into 100 ml I x TNE buffer. A 2 ml cuvette was filled with the fluorometer solution, placed into the machine, and the machine was zeroed. pGEM 3Zf(+) (2 pl, lot #360851026) was added to 2 ml of fluorometer solution and calibrated at 200 units. An additional 2 pl of pGEM 3Zf(+) DNA was then tested and the reading confirmed at 400 +/- 10 units. Each sample was then read at least in triplicate. When 3 samples were found to be within 10% of each other, their average was taken and this value was used as the quantification value. 30 The fluorometricly determined concentration was then used to dilute each sample to 10 ng/pl in ddH 2 O. This was done simultaneously on all template samples for a single TaqMan plate assay, and with enough material to run 500-1000 assays. The samples were tested in triplicate with Taqman"' primers and probe both B-actin and GAPDH on a single plate with normal human DNA and no-template controls. The diluted samples were used provided that the CT value of normal human DNA subtracted from test DNA was +/- 1 Ct. The diluted, lot 35 qualified genomic DNA was stored in 1.0 ml aliquots at -80 0 C. Aliquots which were subsequently to be used in the gene amplification assay were stored at 4"C. Each 1 ml aliquot is enough for 8-9 plates or 64 tests. -92- WO 00/15666 PCTIUS99/20594 Gene amplification assay: The PRO 187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR026 1, PRO246or PRO3 17 compounds of the invention were screened in the following primary tumors and the resulting ACt values are reported in Table 5 10 15 20 25 30 35 -93- WO 00/1 5666 PCT/US99/20594 -q 00l00 0~a -4 09O 00 0 '0 Cl000 CDo In.00 0 r- 100 -- Cl 00 ci66 66 r00 0 E CC 0- -0 -Tc m 0 Cc cc C 0 ~ 00rqCD \Cc m-o 00-'0 0 C C- 0 0 00 Wo ::- r-oIn 0- r -~r 00 r4 9C C 66R0 =a Cl4 r40 0'C 0nC k)t 000C C4" l 00 WO 00/1 5666 PCT/US99/20594 m0~ LO 0n -0 -DC 0-0 -0C 0q 4 m0 LO 0l m 00 m . m 0 E m r- 10 t- 000 0 E c3 . -o CCD 'n 00 Cq'rr -C -o00 66, 00 6 0)0 CO C> 0 0 0 0OC r4 010mr q - 2 C - -0 m Zr a.00 C) 6 6 m0m WO 00/1 5666 PCT/US99/20594 00 10 0'n .l 7. -? ~0 0NN -0 CD~ 000n22 N N 0 01) E~0 .o -oo~- Lo o CO C 0 E - -0 -01) ir 00 C)c00 C3 0 r0 -t' N00 oo c =~~~c 0 N O N CO~c c 0 0 0 -'~~~C -o6 Co 0 00 0 N 0mD N ' E.n~ uOen -O o00 0 C0e WO 00/15666 PCTIUS99/20594 m r- C7, m ' 'o 00 ' C 0 '0 q (1! 0'0 - '0 r 00 'r 11 m~ -0n04 . I -'It-0 CON0 qC C> 0o -0'0 00 C ~ ~ 0 'o r -c -ooo - 1 or- )0 , CDo 000e " - 000 -0 00 -00eN 00 9' m0U o.-- E~ 0 00 WO 00/1 5666 PCTIUS99/20594 00 N 0,'o00 m( 00 -0 mmmD : 0 00 m -IT 0 CD a, meen (N o r- -,r 0 IO O n E oE U C ~ 0 en 0 < rq C4-90oCO0mMr C7, 0 ~r- C) C7, ~ 0r-en 0'0 m-r -C CD(N- CD00 0 Cnnne 0 E :3 -- 000 a,'DN on en ' C 00) WO 00/1 5666 PCT/US99/20594

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00 'C 0 0 0 CD 000 El)l Uu 0 ce. Q cnc .0 kn 0N WO 00/1 5666 PCTIUS99/20594 CCl =R 0 00 Cl 0 000e - 0e a30 len' 0 C4 0, 00 m 0 -7D* Ei uCl ~ 0 tn% Dk 0k enn WO 00/1 5666 PCTIUS99/20594 00 ca '0 0 W r C 00 00 E CD CD V)0 Cr Ci 0n C) 0 -~~ 16 Ar WO 00/15666 PCTIUS99/20594 PRO187: PRO187 (DNA27864-1155) was reexamined along with selected tumors from the above initial screen with framework mapping. Table 4 describes the framework markers that were employed in association with PRO187 (DNA27864-1155). The framework markers are located approximately every 20 megabases along 5 Chromosome 8 (Figure 21), and are used to control aneuploidy. The ACt values for the described framework markers along Chromosome 8 relative to PRO 187 (DNA27864) are indicated for selected tumors in Table 6. PRO 187 (DNA27864-1 155) was also reexamined along with selected tumors from the above initial screen with epicenter mapping. Table 5 describes the epicenter markers that were employed in association with PRO187 (DNA27864-1155). These markers are located in close proximity to DNA27864 and are used to assess the 10 amplification status of the region of Chromosome 8 in which DNA27864 is located. The distance between markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1% chance of a breakage between two markers. One cR is very roughly equivalent to 20 kilobases. The marker SHGC-9963 is the marker found to be the closest to the location on Chromosome 8 where DNA27864-1 155 closely maps. Table 7 indicates the ACt values for results of epicenter mapping relative to DNA27864, indicating the 15 relative amplification in the region more immediate to the actual location of DNA27864 along Chromosome 8 (Figure 21). Table 4 Framework Markers Used for DNA27864 Map Position on Chromosome 8 Stanford Genome Center Marker Name 20 H9 EST00040 H59 WI-961 H121 SHGC- 11323 H200 SHGC-7433 H256 AFMal83zfl 25 Table 5 Epicenter Markers Along Chromosome 8 Used for DNA27864 Map Position on Chromosome 8 Stanford Human Genome Distance to Next Marker Center Marker Name (cR) H60 SHGC-32556 9 30 H61 SHGC-15994 5 H62 SHGC-9963 5 H63 SHGC-33989 10 H64 UT5371 33 H65 SHGC-9507 -115- WO 00/15666 PCTIUS99/20594 Table 6 Amplification of framework markers relative to DNA27864 (ACt) Tumor H9 DNA27864 H59 H121 H200 H256 CT] 0.00 0.50 0.07 -0.07 -0.13 0.58 5 CT4 -0.42 0.20 -0.18 -0.49 -0.08 0.27 CT5 -0.07 2.03 -0.54 -0.34 0.14 0.43 CT6 -0.08 0.13 0.04 -0.02 -0.09 -0.13 CT7 0.03 0.86 0.02 -0.06 -0.10 0.07 CT9 -0.39 -1.39 -0.14 -0.24 -0.29 0.08 10 CTl 1 -0.08 1.90 0.04 -0.05 -0.12 0.12 CTI8 0.12 0.11 0.02 -0.06 0.09 0.09 CT2 -0.25 2.92 0.23 0.16 0.14 0.34 CT3 -1.47 -0.05 0.09 0.07 -0.22 -0.01 CT8 -0.80 0.84 0.04 0.08 -0.11 0.29 15 CT1O -0.41 1.51 -0.48 0.03 0.05 0.25 CT12 -1.69 -0.57 -0.60 -0.07 0.43 0.83 CT14 -1.17 1.62 0.24 0.14 0.21 1.14 CT15 -1.05 0.30 -0.65 0.32 0.02 -15.63 CT16 -1.24 -0.09 0.13 0.16 0.07 0.11 20 CT17 -0.12 0.26 -0.03 0.15 0.19 0.16 LTl1.1 -0.11 -0.30 -0.17 0.10 -0.11 0.06 LT12.1 0.26 2.26 0.59 -0.37 0.40 0.48 LT13.1 0.24 2.58 0.45 0.14 0.08 0.28 LT15.1 0.21 3.54 0.39 -0.16 -0.09 0.30 25 LT16.2 0.03 2.07 -0.14 -0.66 -0.34 0.99 LT17.2 0.64 -0.10 0.39 0.03 -0.01 0.38 LT18.2 0.82 -0.73 0.27 0.09 -0.10 0.19 LT22.1 0.07 -1.57 -0.27 -0.22 0.43 0.45 LTI.1 0.05 -1.94 -0.62 -0.12 0.25 0.25 30 LT1a.1 -0.15 -0.71 -0.65 0.00 0.29 0.56 LT2.2 0.13 -0.56 0.22 0.21 0.25 0.43 LT3.1 -0.19 0.26 -0.10 0.12 0.20 0.13 -116- WO 00/15666 PCT/US99/20594 Tumor H9 DNA27864 H59 H121 H200 H256 LT4.2 0.17 -1.35 -0.19 0.36 0.81 0.58 LT6.1 -0.38 -0.81 -0.82 0.25 0.15 0.36 LT7.1 -0.94 -1.12 -0.89 -0.50 0.35 0.73 LT9.1 0.11 -0.27 -0.10 -0.01 0.26 -0.01 5 LT10.1 0.10 0.25 0.00 0.10 -0.10 -0.04 Table 7 Amplification of Epicenter Markers Relative to DNA27864 (ACt) Tumors H60 H61 H62 DNA H63 H64 H65 27864 10 LT1.1 -0.32 -0.56 0.00 -2.38 -0.77 -3.07 -0.22 LTIa.1 -0.09 -0.32 0.00 -0.66 -0.29 -1.14 -0.09 LT2.2 0.16 -0.31 0.00 -0.55 -0.52 1.14 -0.09 LT3.1 -0.47 -0.63 0.00 -0.45 -0.75 0.29 -0.13 LT4.2 -0.32 -0.29 0.00 -1.61 -0.09 -3.46 -0.05 15 LT6.1 -0.52 -0.69 0.00 -0.99 -0.20 -0.65 -0.30 LT7.1 -1.16 -1.51 0.00 -1.54 -1.19 -2.79 -1.18 LT9.1 0.16 -0.15 0.00 -1.13 -0.61 -0.88 -0.05 LT0.1 -0.27 -0.46 0.00 -0.41 -0.66 -1.25 -0.28 LTI1.1 1.87 0.20 0.00 -0.53 0.55 0.92 0.51 20 LT12.1 0.48 0.02 0.00 1.96 -0.21 -0.54 0.11 LT13.1 0.23 -0.19 0.00 2.32 0.11 1.74 0.10 LT15.1 0.23 -0.27 0.00 3.43 -0.41 1.61 -0.03 LT16.2 -0.16 -0.72 0.00 1.88 -0.59 1.05 -0.32 LT17.2 2.44 0.20 0.00 0.14 0.71 0.63 0.01 25 LT18.2 0.17 -0.29 0.00 -0.60 -0.27 0.01 -0.05 LT22.1 0.38 -0.40 0.00 -1.42 -0.35 -9.53 -0.20 CT2 0.19 -0.10 0.00 3.25 0.13 3.15 0.05 CT3 0.15 -0.18 0.00 -0.02 0.15 2.34 0.09 CT8 0.33 0.18 0.00 1.05 -8.10 0.34 0.05 30 CT1O 0.34 0.02 0.00 1.81 0.32 3.52 0.05 -117- WO 00/15666 PCT/US99/20594 Tumors H60 H61 H62 DNA H63 H64 H65 27864 CT12 -0.14 -0.27 0.00 -0.44 -0.46 0.71 0.29 CT14 0.90 0.24 0.00 1.86 0.45 0.34 0.02 CT16 0.28 0.09 0.00 0.27 0.33 1.88 -0.07 CT17 0.13 -0.22 0.00 0.32 0.40 0.29 -0.15 5 CTl -0.05 -0.08 0.00 0.76 -0.25 -0.66 0.16 CT4 -0.47 -0.24 0.00 0.28 -0.47 -0.15 -0.36 CT5 -0.35 -0.15 0.00 2.19 -0.47 0.00 0.06 CT6 -0.17 0.11 0.00 0.41 -0.06 -1.50 0.31 CT7 -0.29 -0.22 0.00 0.98 -0.25 0.81 0.07 10 CT9 -0.33 -0.36 0.00 -1.89 0.06 -1.74 0.09 CT1 1 -0.23 -0.30 0.00 1.89 0.33 0.36 0.38 CT18 -0.15 -0.12 0.00 -0.05 0.16 -1.36 0.31 PR0240: 15 PR0240 (DNA34387-1138) was reexamined along with selected tumors from the above initial screen with framework mapping. Table 8 describes the framework markers that were employed in association with PR0240 (DNA34387-1138). The framework markers are located approximately every 20 megabases along Chromosome 2 (Figure 22), and are used to control aneuploidy. The ACt values for the described framework markers along Chromosome 2 relative to PR0240 (DNA34387) are indicated for selected tumors in Table 10. 20 PR0240 (DNA34387-1138) was also reexamined along with selected tumors from the above initial screen with epicenter mapping. Table 9 describes the epicenter markers that were employed in association with PR0240 (DNA34387-1138). These markers are located in close proximity to DNA34387 and are used to assess the amplification status of the region of Chromosome 2 (Figure 22) in which DNA34387 is located. The distance between individual markers is measured in centirays, which is a radiation breakage unit approximately equal to a 25 1% chance of a breakage between two markers. One cR is very roughly equivalent to 20 kilobases. The marker SHGC-1 4626 is the marker along Chromosome 2 which most closely maps to DNA34387; however, the TaqMan T M primers and probes for SHGC-14626 failed in our assay, due to technical difficulties related to PCR. DNA34387 was also found to be contained within a BAC (Bacterial Artifical Chromosome). The full BAC was about 100Kb. A BAC containing DNA34387 was identified by screening a BAC library with the TaqMan T M primers and probe 30 for DNA34387. The ends of a DNA34387-positive clone were sequenced, and two sets of TaqMan T M primers and probes were made for the BAC end sequence (Tables 9 & 11). This confirms the validity of our original epicenter mapping results. Table I1 indicates the ACt values for results of epicenter mapping relative to DNA34387, indicating the relative amplification in the immediate chromosomal region along Chromosome 2 (Figure 22). -118- WO 00/15666 PCT/US99/20594 Table 8 Framework Markers Used for DNA34387 Map Position on Chromosome 2 Stanford Human Genome Center Marker Name B3 SHGC-30098 5 B45 SHGC-33615 B90 WI-7936 B 125 WI-469 B169 SHGC-31624 B213 SHGC-10074 10 B251 SHGC-30979 B290 SHGC-9794 B331 AFM2 I Oxb2 B386 SHGC-13676 B441 AFMI99yf2 15 B494 SHGC-33448 Table 9 Epicenter Markers Along Chromosome 2 Used for DNA34387 Map Position on Chromosome 2 Stanford Human Genome Distance to next marker (cR) Center Marker Name 20 B60 AFMal36wh9 65 (gap) B63 AFM254vc9 6 B64 SHGC-14574 28 B65 SHGC-14626 7 B66 SHGC-1 1736 7 25 B67 SHGC-35430 17 B68 AFM234YA9 32 B71 CHLC.GATA8FO7.440 189D13FOR1 Marker from forward end of BAC sequence 189D13REVI Marker from forward end of BAC sequence -119- WO 00/15666 PCT/US99/20594 0 C 0 0 0 V)~ Cfl Vj) tn~ B/n B V) Br) kn Bn Bn Bn V) V C) 9 C? C? 9 1 Ci _e0 C) n 00 0C 0 0 C7, r- C> M 'I ri kn M 9 o0 C 0 0 0 0 0 0 666 666 666 660 cci E. C? B- -1 D D r nr- 00-------0------ -- -- M - < [00 ONo 00 -k n ~ r 0 0 r) q 00 0V ) 00 0 , C; 5 6 i C E 000 o-'T r -0 0 66006 ~C 0) O 0 0 N Br N 00 e - 0 0 ON N; N CD ~~a :2 enk i c O 9 - e N N ~ i \ WO 00/1 5666 PCTIUS99/20594 00 00O &0 C- -0 0 0 -, 0- 00 00ONt ~~0 000 0 00 C - - 0, 00Z -- : "O ON C- C 00 m -r 0 0 () ' - 00 ON C:O Cl < C, N: N 0 0;N 00 0l 0l 0l C0 C u CD ON 0, 'I < ;jr ON Cl V0 00 -D NO O :)l -: c-. 0D 00 Cl0 Cll 0~C 6C___ C 00 '- C O iN l N 6CI6D0 0 I-- _121 WO 00/15666 PCT1US99/20594 E C C < 00 cl <n C - 00 0 00 m 00 l - 0 NCl - O m cc mc Cl C )N0N c U 0 0 Cl ml C) 00 Ni C oN ON Cl Q5 0c -t - 0 - - \- & t00 Cl cc ) C-1 C 5 6Z '3r- rc9 00 0 ~ ' ~ ~ '3 c . 3 C5Cl CD 6 -0; 6 6 C5 06 6 6 '3k' c ln r CD D C 0 C ON C D '3 - - l C Cl -D CD0& ~ l oo 0 0 0 0) 0m 03 Cl -- - r -C 0 E 1 IV) WO 00/1 5666 PCTIUS99/20594 E 00 LM 00 ;- 0 r0-T N m 60C i 0 co 0 to c6 WO 00/15666 PCT/US99/20594 PR0230: PR0230 (DNA33223-1136) was reexamined along with selected tumors from the above initial screen with framework mapping. Table 12 describes the framework markers that were employed in association with PR0230 (DNA33223-1136). The framework markers are located approximately every 20 megabases along 5 Chromosome I (Figure 23), and are used to control aneuploidy. The ACt values for the described framework markers along Chromosome I relative to PR0230 (DNA33223) are indicated for selected tumors in Table 14. PR0230 (DNA33223-1136) was also reexamined with epicenter mapping. A Bacterial Artificial Chromosome (BAC) containing DNA33223 was identified by screening a BAC library. The ends of a DNA33223 positive clone were sequenced, and two sets of TaqMan

T

I' primers and probes were made for the BAC end 10 sequence. TaqMan T M primers and probes to the BAC ends were thus used as markers to assess the amplification status of the region of Chromosome I in which DNA33223 is located. BAC clones are typically 100 to 150 Kb. DNA40625 (a novel gene) is also located on the BAC containing DNA33223. Table 13 describes the epicenter markers that were employed in association with PR0230 (DNA33223). These markers are located in close proximity to DNA33223 and are used to assess the amplification status of the region of Chromosome I in which 15 DNA33223 is located. The distance between markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1% chance of a breakage between two markers. One cR is very roughly equivalent to 20 kilobases. The marker SHGC-35321 is the marker found to be the closest to the location on Chromosome I where DNA33223-1136 maps (Figure 23). Table 15 indicates the ACt values for results of epicenter mapping to DNA33223, indicating the relative 20 amplification in the region more immediate to the actual location of DNA33223 along Chromosome I (Figure 23). Table 12 Framework Markers Used for DNA33223 25 Map Position Stanford Human Genome Center Marker Name Al SHGC-33169 A40 SHGC-3901 A84 AFM234tb6 A 129 ACTIB03 30 A180 SHGC-34001 A220 AFM338wb5 A263 EST00691 A312 SHGC-7599 A355 SHGC-32839 35 A398 SHGC-37552 -124- WO 00/15666 PCT/US99/20594 Map Position Stanford Human Genome Center Marker Name A443 SHGC-11921 A485 SHGC-9327 A520 SHGC-30345 A553 SHGC-3055 5 Table 13 Epicenter Markers Along Chromosome I Used for DNA33223 Map Position on Stanford Human Genome Center Distance to Next Marker (cR) Chromosome 1 Marker Name 10 A83 SHGC-37693 97 (gap) A84 AFM234tb6 9 A85 AFM240za9 46 A86 AFMI99zd2 10 A87 SHGC-35321 11 15 A88 SHGC-3252 12 A89 SHGC-11204 173 A 102 SHGC- 11219 189D13FOR1 Marker from forward end of BAC sequence 189D13FOR2 Marker from forward end of BAC sequence 20 DNA33223 DNA40625 189D13REVI Marker from forward end of BAC sequence 189D13REV2 Marker from forward end of BAC sequence 25 -125- WO 00/15666 PCT/US99/20594 NN -o r- m z 6 00 00 o 5 C ) - Cl 't) Cl C 0l0 C < 6 6 6 6 0 0 6 CD oo o 0 0 - 6 N - No _ 0 Cl Cl l -l C? C? 00 0 osA o6 6 o o 6 00 ca o o) o o o 00 N 00 C CN Clm E m Cm 00 6 6 6 O6 Cll o oN o Cool o E CA CD C -CD C o <6o 6 6 6 o o CO) c: : 0s o oo N - o N M Mo 013 CO C O 0 0 0 0 0D 0 0 E C O0 C ) CC O Cl~~~~C C Cl C 00 ICD 0) 0 0 0 6 6 0 CO1 WO 00/15666 PCTIUS99/20594 00 - - C- o Cl0 00 00 kN Cl - - 00- o o o o, o , o o S o o , 6 o0 0 0 0 a) d 0l -D C C)l 0 O 0 en 00 '0 arC'0 a N ON N d/ d C 9 9 0 0 0 9 o 6 6 6 6 6 o o o o ON 0 O O O O o l ol - o0 0 O 0 0 - Cl 6 Cl C0 Cl Cl0 o o0 o - 0 O O ol o - - 6 6 'a 1VV 00 a/ C 00 r- Nl- O - C. 0) ON C)00 a E cc C l 6) o (0 0) 6D 0 C) 6C0 o. CD <D C/N CD ON C Cl CD ON ON 0D C6 6) ClaD0 0l 0% 00 0 0m - aCN a, O 65 6 9 9 6 0 00 ClCl - -~~ o 0 65 6 00 -q Cl) 0, ON ON N ' -: cCN Cl CN CDN Cl 00 m 67 6o 0 C\ 6 0 00 q ON \0 ON %q C ~ 0 - Cl a/N C0 N) 00 ON 1 1)7 WO 00/1 5666 PCT/US99/20594 - (-1 00 00 m 0 C- Cl C ON mII 00 cqtn r N 0II <1 r'! CD CD - 0 I0. c'0 I { N - -rl O 0- C (= . . O 000 P 0 0' 0 6C 0, C ~ C C O Cl - 0 Ir C, 6 6 6 - 5 0 D -C) 00 Cl 0D. 0 0 S< qo 606 a 6 060 C;6 000 K0 00 . ON - CD m0 0 cq -T q -I 00 Cl ON0D C) N' l* C> 6~6 ~ 0 E 0 m 64N R 0 '0 ;7a 0 00 0 ~ Cl - ~ ra ' - 0 N _ ' ~ c Ir * 0~ 0 0 C ~ 0 -Cl 2 WO 00/1 5666 PCT/US99/20594 > 00 ON 0 C 00 >l m O ON LU C DC 00 C 00 N nO O 0 -D - 0 0S2C Z l E ON N, Cl* ON CD C 6- .- C ~~6 Cl .. ~ 9 ON ~ 00 ON r- -1 00 00 00 00 C 00 0: 0: - 0 90! 6q 66 66 60 c 6 0 6? C : : 00 "C ICO =O (q 0: 0 & N 00 0: C' 0I 0 - 0 0D r - ON Cl 0 C N 00 00 000 N -) 00 c,~ C:) - - \O Clr N ON 00 c) - jD 0 N C O 00 00 trF 0; (q !2C 00 Cl 0- 0 Cl Cl Cl c) Cl 1) 00) C) OND Cl ON C WO 00/15666 PCTIUS99/20594 00 ~ 0 0 0 00r N 0 00 N- 00 :2 n - 0 - CD en CD N 0r00 4 n - -f -l ar Cr: o r n O ON 0 0 00 00 C)a 00 m. m0 r-- NN rq N r- 04 a\ WO gr 00 arD g 0 00 N r 00 ON " a ) D 0. ON 0) kn a a a a 00 0 m 00 00 6 & A ~ ~ O *00 O N \ o 0 q 0 1 W0 0N 0 0_ 0000 l: r " :t N r 6C? 66 a 0l aD 6CD C=6 6 NO~~r 4r' ON -q ,I0- 0 O ~ N N 0 00 0ITe - C 00 - Ci* 6 : 0? 69 0 0 6 0D 6D 05 0D CD CD a a a aa a a - ~ ~ ~ ~ r r- N0 n e a~e r WO 00/15666 PCT/US99/20594 -CD 0 0 - D 00 00 0D CD C0 0D -D 0 0 C C C 00i 0 0 0 0 0 0 0 00 r N - Cr. 0 n (ON' O oo0 N l C 0 - -: - m 0 0 0 0D 0 0 0 00 0' 0I 0 0 0 0 NOCl - N - ;7 ;7ON 00 Cl ON Cl 9 u 600000? 6 WO 00/15666 PCTIUS99/20594 DISCUSSION AND CONCLUSION: PRO]87 (DNA27864-1 155): The ACt values for DNA27864-1155 in a variety of tumors are reported in Table 3. A ACt of >1 was 5 typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 3 indicates that significant amplification of nucleic acid DNA27864-l155 encoding PRO187 occurred (1) in primary lung tumors: LT12, LT13, LT15, LT16, LT19 and LT30; and (2) in primary colon tumors: CT2, CT8, CT1O, CTl4, CT1, CT5, CT6, CT7, CT9 and CT1 1. Amplification has been confirmed by framework mapping (Table 6) for DNA27864: in primary lung tumors: LT12, LT13, LT15 and LTI6; and in primary colon tumors: 10 CT2, CT5, CT 10, CTI 1 and CT 14. The framework markers analysis reports the relative amplification of particular regions of Chromosome 8 in the indicated tumors, while the epicenter markers analysis gives a more precise reading of the relative amplification in the region immediately in the vicinity of the gene of interest. The amplification of the closest known framework markers (Table 6) does not occur to a greater extent than that of DNA27864. 15 Amplification has been confirmed by epicenter mapping (Table 7) for DNA27864-1155 and resulted in significant amplification: in primary colon tumors: CT2, CT5, CT8, CTIO, CTl land CTI4; and in primary lung tumors: LT12, LT13, LT15, and LT16. In contrast, the amplification of the closest known epicenter markers (with the exception of H60 and H64) does not occur to a greater extent than that of DNA27864 (Table 7). This strongly suggests that DNA27864 is the gene responsible for the amplification of the particular region on Chromosome 8. 20 Because amplification of DNA27864 occurs in various lung and colon tumors, it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA27864 (PRO 187) would be expected to have utility in cancer therapy. PR0533 (DNA49435-121 9): 25 The ACt values for DNA49435-1219 in a variety of lung tumors are reported in Table 3. A ACt of> I was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 3 indicates that significant amplification of nucleic acid DNA49435-1219 encoding PR0533 occurred in primary lung tumors: LTla, LT7, LTI 1, LT16, LT17, and LT19. Because amplification of DNA49435-1219 occurs in various lung tumors, it is likely associated with tumor formation and/or growth. As a result, antagonists (e.g., 30 antibodies) directed against the protein encoded by DNA49435 (PR0533) would be expected to be useful in cancer therapy. PR0214 (DNA32286-1 191): The ACt values for DNA32286-1191 in a variety of tumors are reported in Table 3. A ACt of >1 was 35 typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 3 indicates that significant amplification of nucleic acid DNA32286-11I9lencoding PR0214 occurred: (1) in primary lung tumors: LT3, LTI 1, LT12, LT13, LT15, LT17 and LT19; and (2) in primary colon tumors: CT2, -132- WO 00/15666 PCT/US99/20594 CT3, CT8, CTlO, CT12, CT14, CT15, CT16, CTl7, CT1, CT4, CT5, CT6, CT9 and CTI 1. Because amplification of DNA32286-119loccurs in various tumors, it is likely associated with tumor formation and/or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA32286 (PR0214) would be expected to be useful in cancer therapy. 5 PR0240 (DNA34387-1 138): The ACt values for DNA34387-1138 in a variety of tumors are reported in Table 3. A ACt of>l was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 3 indicates that significant amplification of nucleic acid DNA34387-1138 encoding PR0240 occurred: (1) in 10 primary lung tumors: LT I a, LT3, LT6, LT 10, LTl 1, LT12, LT13, LT 15, LT16, LT 17, LT19, LT21, LT26, LT28, LT30 and in HF000840; (2) in primary colon tumors: CT2, CT3, CT8, CT1O, CT12, CTI4, CT15, CT16, CT17, CT1, CT4, CT5, CT6, CT7, CTI 1 and colon tumor centers HF000539, HF000575 and HF000698; (3) in breast tumors SRCC1097, SRCC1098, SRCCl 100, SRCCI 101and breast tumor center HF000575; (4) in kidney tumor center HF00061 1; (5) in lymph node HF000854; and (6) in testis tumor center HF000733 and testis tumor margin 15 HF000716. Amplification has been confirmed by framework mapping (Table 10) for DNA34387-1138: in primary lung tumors: LTla, LT3, LT1 1, LT12, LT13, LT15, LT16, LT17 and LT19. In contrast, the amplification of the closest known epicenter markers (with the exception of markers B3 and B90) does not occur to a greater extent than that of DNA34387 (Table 10). The framework markers analysis reports the relative amplification of particular 20 regions of Chromosome 2 in the indicated tumors, while the epicenter markers analysis gives a more precise reading of the relative amplification in the region immediately in the vicinity of the gene of interest. Amplification has also been confirmed by epicenter mapping (Table 11) for DNA343 87-1138 and resulted in significant amplification: in primary lung tumors: LTla, LT2, LT3, LT4, LT6, LT7, LT9, LT1O, LT1 1, LT12, LT13, LTI5, LT16, LT17, LT19 and LT21. It appears that DNA34387 is very close to the BAC marker 25 208K2lForl as the marker shows amplification in a similar pattern of tumors as DNA34387, and the degree of amplification is similar (Table 11). The amplification shown by framework and epicenter mapping strongly suggests that DNA34387 is the gene responsible for the amplification of the particular region on Chromosome 2. Because amplification of DNA34387 occurs in various tumors, it is highly probable to play a significant role in tumor formation or growth. 30 As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA34387 (PR0240) would be expected to have utility in cancer therapy. PR0211 (DNA32292-1 131): The ACt values for DNA32292-1131 in a variety of tumors are reported in Table 3. A ACt of >1 was 35 typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 3 indicates that significant amplification of nucleic acid DNA32292-1131 encoding PRO211 occurred: (1) in primary lung tumors: LT Ia, LT3, LT4, LT9, LT10, LTI 1, LT12, LT13, LT15, LT16, LT17, LT19 and LT21; (2) -133- WO 00/15666 PCT/US99/20594 in primary colon tumors: CT2. CTl, CT4, CT5, CT6 and CT1 1; and (3) in lung tumor cell line SW900. Because amplification of DNA32292-1 131 occurs in various tumors, it is likely associated with tumor formation and/or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA32292 (PRO21 1) would be expected to be useful in cancer therapy. 5 PR0230 (DNA33223-1136): The ACt values for DNA33223-1136 in a variety of tumors are reported in Table 3. A ACt of>I was typically used as the threshold..value for amplification scoring, as this represents a doubling of gene copy. Table 3 indicates that significant amplification of nucleic acid DNA33223-1136 encoding PR0230 occurred: (1) in 10 primary lung tumors: LTI 1, LT12, LT13, LT15, LT16, LT17, LT19, LT21, LT3O and HF000840; (2) in primary colon tumors: CT3, CT12, CT16, CTl7, CTI, CT4, CT5, CT7, CTI 1 and colon tumor center HF000539; (3) in lung tumor cell line Calu- 1; (4) in colon tumor cell line SW620; (5) in kidney tumor center HF00061 1; and (6) in testis tumor center and tumor margin HF000733 and HF000716, respectively. Amplification has been confirmed by framework mapping (Table 14) for DNA33223: in primary lung 15 tumors: LT1 1, LT12, LTI3, LT15, LTI7 and LTI9. Epicenter mapping (Table 15) for DNA33223 resulted in significant amplification: in primary lung tumors: LT1 a, LT3, LT6, LT9, LTI 0, LT 11, LTl2, LTI 3, LTI 5, LTl 6, LTI 7and LT19; and in primary colon tumors: CT2, CT5, CT10 and CT 11. The framework markers analysis reports the relative amplification of particular regions of Chromosome I in the indicated tumors, while the epicenter markers analysis gives a more precise reading of the relative amplification in the region immediately in the vicinity 20 of the gene of interest. The amplification of the closest known framework markers (Table 14) does not occur to a greater extent than that of DNA33223. This strongly suggests that DNA33223 is the gene responsible for the amplification of the particular region on Chromosome 1. Because amplification of DNA33223 occurs in various tumors and cell lines (especially lung), it is highly probable to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA33223 (PR0230) would be 25 expected to have utility in cancer therapy. PR0261 (DNA33473-1 176): The ACt values for DNA33473-1 176 in a variety of tumors are reported in Table 3. A ACt of >l was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 30 3 indicates that significant amplification of nucleic acid DNA33473-1176 encoding PRO26loccurred: (1) in primary lung tumors: LTI a, LT1O, LTI 1, LT12, LT13, LT15, LT16, LTI7, LTI8, LTI9 and LT21; (2) in primary colon tumors: CT2, CT3, CT14, and CT5; (3) in colon tumor cell lines: SW480, SW620, Colo320, HT29, WiDr, HCTI 16, SKCO 1, SW403 and LS I 74T; and (4) in breast tumor cell lines HBL 100, MB435s, BT20, and SKBR3. Because amplification of DNA33473-1 176 occurs in various tumors, it is likely associated with tumor formation 35 and/or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA33473 (PR0261) would be expected to be useful in cancer therapy. -134- WO 00/15666 PCTIUS99/20594 PR0246 (DNA35639-1 172): The ACt values for DNA35639-1172 in a variety of tumors are reported in Table 3. A ACt of>1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 3 indicates that significant amplification of nucleic acid DNA35639-1172 encoding PR0246 occurred: (1) in 5 primary lung tumors: LT3, LTIO, LTI1, LT12, LT13, LT15, LT17, LT19 and LT21; and (2) in primary colon tumors: CT4, CT5, CT9 and CT11. Because amplification of DNA35639-1172 occurs in various tumors, it is likely associated with tumor formation and/or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA35639 (PR0246) would be expected to be useful in cancer therapy. 10 PR0317 (DNA33461-1199): The ACt values for DNA33461-1199 in a variety of tumors are reported in Table 3. A ACt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 3 indicates that significant amplification of nucleic acid DNA33461-1199 encoding PRO317 occurred: (1) in primary lung tumors: LTla, LT3, LT4, LT6, LT7, LT9, LTIO, LTl 1, LT12, LT13, LTl5, LTI6, LT17 and LTI9; 15 and (2) in primary colon tumors: CT2, CT1O, CT14, CT15, CT4 and CT9. Because amplification of DNA33461 1199 occurs in various tumors, it is likely associated with tumor formation and/or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA33461 (PRO317) would be expected to be useful in cancer therapy. 20 EXAMPLE I I In situ Hybridization In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific 25 mRNA synthesis and aid in chromosome mapping. In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision [,:169-176(1994), using PCR-generated "P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37'C , and further processed for in situ hybridization as described by Lu and Gillett, supra. A ["-P] UTP-labeled antisense 30 riboprobe was generated from a PCR product and hybridized at 55'C overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks. "P-Riboprobe synthesis 6.0 pl (125 mCi) of "P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vac dried. To each tube containing dried "P-UTP, the following ingredients were added: 35 2.0 pl 5x transcription buffer 1.0 pA DTT (100 mM) 2.0 pl NTP mix (2.5 mM : 10 A; each of 10 mM GTP, CTP & ATP + 10 pl H 2 O) -135- WO 00/15666 PCT/US99/20594 1.0 yl UTP (50 yM) 1.0 yl Rnasin 1.0 pl DNA template (lug) 1.0,pl HO 5 1.0 pl RNA polymerase (for PCR products T3 = AS, T7 = S, usually) The tubes were incubated at 37'C for one hour. 1.0 pl RQI DNase were added, followed by incubation at 37 0 C for 15 minutes. 90 Ml TE (10 mM Tris pH 7.6/1mM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Microcon-50 ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). 10 After the final recovery spin, 100 pl TE were added. I MI of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II. The probe was run on a TBE/urea gel. 1-3 pl of the probe or 5 pl of RNA Mrk III were added to 3 yl of loading buffer. After heating on a 37 0 C heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes. The gel was wrapped in 15 saran wrap and exposed to XAR film with an intensifying screen in -70*C freezer one hour to overnight. 1 3 P-Hybridization Pretreatment of frozen sections: The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for 5 minutes. The trays were placed in a 551C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed 20 in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ HO). After deproteination in 0.5 pg/ml proteinase K for 10 minutes at 37*C (12.5 Ml of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in 0.5 x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each. Pretreatment of paraffin-embedded sections: The slides were deparaffinized,placed in SQ H2O, and rinsed 25 twice in 2 x SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 pg/ml proteinase K (500 pl of 10 mg/ml in 250 ml RNase-free RNase buffer; 371C, 15 minutes ) - human embryo, or 8 x proteinase K (100 pl in 250 ml Rnase buffer, 37'C, 30 minutes) - formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above. Prehybridization: The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% 30 formamide) - saturated filter paper. The tissue was covered with 50 pl of hybridization buffer (3.75g Dextran Sulfate +6 ml SQ H 2 0), vortexed and heated in the microwave for 2 minutes with the cap loosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20 x SSC and 9 ml SQ HO were added, the tissue was vortexed well, and incubated at 42'C for 1-4 hours. Hybridization: 1.0 x 106 cpm probe and 1.0 Ml tRNA (50 mg/ml stock) per slide were heated at 95'C 35 for 3 minutes. The slides were cooled on ice, and 48 pl hybridization buffer were added per slide. After vortexing, 50 pl 3 P mix were added to 50 pl prehybridization on slide. The slides were incubated overnight at 55 0 C. Washes: Washing was done 2x10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC -136- WO 00/15666 PCT/US99/20594 + 16 ml 0.25M EDTA, Vf=4L), followed by RNaseA treatment at 37 0 C for 30 minutes (500 pl of 10 mg/ml in 250 ml Rnase buffer = 20 pg/ml), The slides were washed 2x 10 minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55 0 C, 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA, Vf=4L). 5 DNA49435-1219 (FGF homolog, FGF receptor 3 ligand) Oligo A-252G 46 mer: 5'-GGATTCTAATACGACTCACTATAGGGCGGATCCTGGCCGGCCTCGG-3' (SEQ ID NO:82) Oligo A-25 I H 48 mer: 10 5'-CTATGAAATTAACCCTCACTAAAGGGAGCCCGGGCATGGTCTCAGTTA-3 (SEQ ID NO:83) Moderate expression was observed over cortical neurons in the fetal brain. Expression was observed over the inner aspect of the fetal retina, and possibly in the developing lens. Expression was seen over fetal skin, cartilage, small intestine, placental villi and umbilical cord. In adult tissues, there was an extremely high level of expression over the gall bladder epithelium. Moderate expression was seen over the adult kidney, gastric and 15 colonic epithelia. These data are consistent with the potential role of this molecule in cartilage and bone growth. DNA32286-1191 (EGF-like homolog): Oligo B-138U 47mer: 5'-GGATTCTAATACGACTCACTATAGGGCCCCTCCTGCCTTCCCTGTCC-3' (SEQ ID NO:84) 20 Oligo A-I 34R 48mer: 5'-CTATGAAATTAACCCTCACTAAAGGGAGTGGTGGCCGCGATTATCTGC-3' (SEQ ID NO:85) In fetal tissues, low level expression was observed throughout the mesenchyme. Moderate expression was seen in placental stromal cells in membraneous tissues, and in thyroid. Low level expression was seen in cortical neurons. 25 DNA34387-1138 (Jagged/EGF homolog): Oligo B-23 I W 48mer: 5'-GGATTCTAATACGACTCACTATAGGGCCCGAGATATGCACCCAATGTC-3 (SEQ ID NO:86) Oligo B-231-X 47mer: 30 5'-CTATGAAATTAACCCTCACTAAAGGGATCCCAGAATCCCGAAGAACA-3' (SEQ ID NO:87) Expression pattern in human adult and fetal tissues Elevated signal was observed at the following sites: Fetal tissues: thyroid epithelium, small intestinal epithelium, gonad, pancreatic epithelium, hepatocytes in liver and renal tubules; expression was also seen in vascular tissue in developing bones. 35 Adult tissues: moderate signal in placental cytotrophoblast, renal tubular epithelium, bladder epithelium, parathyroid and epithelial tumors. -137- WO 00/15666 PCT/US99/20594 Expression in lung adenocarcinoma and squamous carcinoma Expression was observed in all eight squamous carcinomas and in six out of eight adenocarcinomas. Expression was seen in in-situ and infiltrating components. Expression levels were low to moderate in the adenocarcinomas. In general, expression was higher in the squamous carcinomas and in two the expression was 5 strong. No expression was seen in the tumor stroma, alveoli or normal respiratory epithelium. There was possible low level expression in lymph nodes. DNA33223-1 136 (tubulointerstitial nephritis antigen homolog): DNA33223p1: 10 5'-GGATTCTAATACGACTCACTATAGGGCGGCGATGTCCACTGGGGCTAC-3' (SEQ ID NO:88) DNA33223p2: 5'-CTATGAAATTAACCCTCACTAAAGGGACGAGGAAGATGGGCGGATGGT-3' (SEQ ID NO:89) Expression in human and fetal tissues Tissue sections showed an intense signal associated with arterial and venous vessels in the fetus. In 15 arteries, the signal appeared to be confined to smooth muscle/pericytic cells. The signal was also seen in capillary vessels and in glomeruli. Expression was also observed in epithelium cells in the fetal lens. Strong expression was also seen in cells within placental trophoblastic villi. These cells lie between the trophoblast and the fibroblast-like cells that express HGF, and have an uncertain histogenesis. In the adult, there was no evidence of expression in the wall of the aorta and most vessels appeared to be 20 negative. However, expression was seen over vascular channels in the normal prostate and in the epithelium lining the gallbladder. Expression was seen in the vessels of the soft-tissue sarcoma and the renal cell carcinoma. In summary, this molecule showed relatively specific vascular expression in the fetus as well as in some adult organs. Expression was also observed in the fetal lens and the adult gallbladder. Expression using breast and lung tumor tissues 25 Vascular expression similar to the above results was seen in fetal blocks. Expression was in vascular smooth muscle, rather than epithelium. Expression was also seen in smooth muscle of the developing esophagus, hence this molecule is not vascular specific. Expression was examined in 4 lung and 4 breast carcinomas. Substantial expression was seen in vascular smooth muscle of at least 3 out of 4 lung cancers and 2 out of 4 breast cancers. In addition, in one breast carcinoma (IF97-06551 3E), expression was observed in peritumoral stromal 30 cells of uncertain histogenesis (possibly myofibroblasts). Expression in lung adenocarcinoma and squamous carcinoma One of sixteen (1/16) tumors showed a strong hybridization signal, and 2 out of 16 showed positive signals of weak to moderate intensity. All three tumors were classified as poorly differentiated squamous cell carcinomas. The remaining 6 squamous carcinomas and all 7 adenocarcinomas were negative. As seen in previous studies 35 described above, expression was present in endothelial and smooth muscle cells of small and medium-sized vascular channels and one case showed weak, focal expression in benign glandular cells. -138- WO 00/15666 PCT/US99/20594 DNA33473-1176 (CTGF homolog): Oligo D- 1 70R 45 mer: 5'-GGATTCTAATACGACTCACTATAGGGCGCGAGGACGGCGGCTTCA-3' (SEQ ID NO:90) Oligo D-170V 48 mer: 5 5'-CTATGAAATTAACCCTCACTAAAGGGAAGAGTCGCGGCCGCCCTTTTT (SEQ ID NO:91) Strong expression was observed in dermal fibroblasts in normal adult skin. Strong expression was also seen in two cirrhotic livers, at sites of active hepatic fibrosis. Moderate expression was found over fasiculata cells of the adrenal cortex. This localization supports a role for this molecule in extracellular matrix formation or turnover. 10 DNA35639-1 172 (HCAR homolog): DNA35639p1: 5'-GGATTCTAATACGACTCACTATAGGGCTTGCTGCGGTTTTTGTTCCTG-3' (SEQ ID NO:92) DNA35639p2: 15 5'-CTATGAAATTAACCCTCACTAAAGGGAGCTGCCGATCCCACTGGTATT-3' (SEQ ID NO:93) This molecule was strongly expressed in fetal vascular endothelium, including tissues ofthe CNS. Lower level of expression was observed in adult vasculature, including CNS. It was not obviously expressed at higher levels in tumor vascular endothelium. -Signal was also seen over bone matrix and adult spleen, however, this signal was obviously cell associated. 20 EXAMPLE 12 Use of PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 as a hybridization probe The following method describes use of a nucleotide sequence encoding a PRO187, PR0533, PRO214, 25 PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide as a hybridization probe. DNA comprising the coding sequence of a full-length or mature "PRO" polypeptide as disclosed herein and/or fragments thereof may be employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PROl87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 in human tissue cDNA libraries or human tissue genomic libraries. 30 Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled PR0187-, PR0533-, PR0214-, PR0240-, PRO211-, PR0230-, PR0261-, PR0246- or PRO317-derived probe to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42 0 C for 20 hours. Washing of the filters is performed in an aqueous solution 35 ofO.lxSSCandO.I%SDSat42*C. DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO187, PRO533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 can then be identified using -139- WO 00/15666 PCT/US99/20594 standard techniques known in the art. EXAMPLE 13 Expression of "PRO" Polypeptides in E. coli. 5 This example illustrates preparation of an unglycosylated form of PRO187, PR0533, PRO214, PRO240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 by recombinant expression in E. coli. The DNA sequence encoding the PRO polypeptide of interest is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector 10 is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a poly-His leader (including the first six STII codons, poly-His sequence, and enterokinase cleavage site), the PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 15 or PRO317 coding region, lambda transcriptional terminator, and an argU gene. The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing. 20 Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on. After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized 25 PRO187, PRO533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein. PRO 187, PRO533, PR0240 and PRO317 were successfully expressed in E. coli in a poly-His tagged form using the following procedure. The DNA encoding PRO 187, PRO533, PRO240 or PRO317 was initially amplified using selected PCR primers. The primers contained restriction enzyme sites which correspond to the restriction 30 enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences were then ligated into an expression vector, which was used to transform an E. coli host based on strain 52 (W3 110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(laclq). Transformants were first grown in LB containing 50 mg/ml carbenicillin at 30*C with shaking until an O.D.

0 0 of 35 3-5 was reached. Cultures were then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH 4

)

2

SO

4 , 0.71 g sodium citrate-2H 2 0, 1.07 g KCI, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO 4 ) and grown for approximately 20-30 -140- WO 00/15666 PCT/US99/20594 hours at 30*C with shaking. Samples were removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets were frozen until purification and refolding. E. coli paste from 0.5 to I L fermentations (6-10g pellets) was resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final 5 concentrations of 0.1M and 0.02 M, respectively, and the solution was stirred overnight at 4'C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution was centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant was diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract was loaded onto a 5 ml Qiagen Ni 2 1-NTA metal chelate column equilibrated in the metal chelate column 10 buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4'C. Protein concentration was estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence. The proteins were refolded by diluting sample slowly into freshly prepared refolding buffer consisting of: 15 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and I mM EDTA. Refolding volumes were chosen so that the final protein concentration was between 50 to 100 micrograms/mi. The refolding solution was stirred gently at 4'C for 12-36 hours. The refolding reaction was quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to 2-10% final concentration. The refolded 20 protein was chromatographed on a Poros RI/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A,, absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. 25 Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples. Fractions containing the desired folded PRO187, PR0533, PR0240 and PR0317 proteins, respectively, were pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins were formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration 30 using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered. EXAMPLE 14 Expression of PRO]87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 in mammalian cells 35 This example illustrates preparation of a potentially glycosylated form of PRO187, PR0533, PR0214, PR0240, PR021 1, PR0230, PR0261, PR0246 or PR0317 by recombinant expression in mammalian cells. The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector. -141- WO 00/15666 PCT/US99/20594 Optionally, the PRO]87, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO]87, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRK5-PRO187, pRK5-PRO533, pRK5-PRO214, pRK5 5 PRO240, pRK5-PRO21 1, pRK5-PRO230, pRK5-PRO261, pRK5-PRO246 or pRK5-PRO317. In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 pg pRK5-PRO 187, pRK5-PRO533, pRK5-PRO214, pRK5-PRO240,pRK5-PRO211 ,pRK5-PRO230,pRK5-PRO26 1,pRK5-PRO246or pRK5-PRO317 DNA is mixed 10 with about 1 pg DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 Ml of 1 mM Tris-HCI, 0.1 mM EDTA, 0.227 M CaCl 2 . To this mixture is added, dropwise, 500 /A of 50 mM HEPES (pH 7.35), 280 mM NaC1, 1.5 mM NaPO 4 , and a precipitate is allowed to form for 10 minutes at 25 0 C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37 0 C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed 15 with serum free medium, fresh medium is added and the cells are incubated for about 5 days. Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 pCi/ml "S-cysteine and 200 MCi/ml "S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of 20 PRO187, PR0533, PR0214, PRO240, PRO211, PR0230, PR0261, PR0246 or PR0317 polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays. In an alternative technique, PRO 187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 DNA may be introduced into 293 cells transiently using the dextran sulfate method described by 25 Somparyrac etal., Proc. Nati. Acad. Sci. 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 Mg pRK5-PRO187, pRK5-PRO533, pRK5-PR0214, pRK5-PRO240, pRK5-PRO21I, pRK5-PRO230, pRK5-PRO26 1, pRK5-PRO246 or pRK5-PRO317 DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re 30 introduced into the spinner flask containing tissue culture medium, 5 pg/ml bovine insulin and 0.1 pg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PRO]87, PR0533, PR0214, PRO240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography. 35 In another embodiment PRO]87, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 can be expressed in CHO cells. The pRK5-PRO187, pRK5-PRO533, pRK5-PRO214, pRK5-PRO240, pRK5-PRO2 11, pRK5-PRO230, pRK5-PRO261, pRK5-PRO246 or pRK5-PRO317 vector can be transfected into -142- WO 00/15666 PCT/US99/20594 CHO cells using known reagents such as CaPO 4 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as "S methionine. After determiningthe presence of PRO] 87, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide, the culture medium may be replaced with serum free medium. Preferably, the 5 cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed PROI 87, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 can then be concentrated and purified by any selected method. Epitope-tagged PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 may also be expressed in host CHO cells. The PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, 10 PRO246 or PR0317 may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-His tag into a Baculovirus expression vector. The poly-His tagged PRO187, PRO533, PR0214, PR0240, PRO21 1, PR0230, PRO261, PR0246 or PR0317 insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be 15 performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged PRO 187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR026 1, PR0246 or PRO317 can then be concentrated and purified by any selected method, such as by Ni 2 -chelate affinity chromatography. Expression in CHO and/or COS cells may also be accomplised by a transient expression procedure. PRO214, PR0240, PRO21 1, PR0230 and PR0261 were expressed in CHO cells by both a transient and 20 a stable expression procedure. In addition, PR0246 was transiently expressed in CHO cells. Stable expression in CHO cells was performed using the following procedure. The proteins were expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g., extracellular domains) of the respective proteins were fused to an IgGI constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form. 25 Following PCR amplification, the respective DNAs were subcloned in a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used for expression in CHO cells is as described in Lucas et al., Nucl. Acids Res., 24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer 30 to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection. Twelve micrograms ofthe desired plasmid DNA were introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect* (Quiagen), Dosper* or Fugene* (Boehringer Mannheim). The cells were grown as described in Lucas et al., supra. Approximately 3 x 10' cells are frozen in 35 an ampule for further growth and production as described below. The ampules containing the plasmid DNA were thawed by placement into water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 mis of media and centrifuged at 1000 -143- WO 00/15666 PCT/US99/20594 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 ml of selective media (0.2 pm filtered PS20 with 5% 0.2 ym diafiltered fetal bovine serum). The cells were then aliquoted into a 100 ml spinner containing 90 ml of selective media. After 1-2 days, the cells were transferred into a 250 ml spinner filled with 150 ml selective growth medium and incubated at 37'C. After another 2-3 days, 250 ml, 500 ml and 2000 ml 5 spinners were seeded with 3 x 10' cells/ml. The cell media was exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in US Patent No. 5,122,469, issued June 16, 1992 was actually used. 3L production spinner is seeded at 1.2 x 106 cells/ml. On day 0, the cell number and pH were determined. On day 1, the spinner was sampled and sparging with filtered air was commenced. On day 2, the spinner was sampled, the temperature shifted to 33"C, 10 and 30 ml of 500 g/L glucose and 0.6 ml of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Coming 365 Medical Grade Emulsion). Throughout the production, pH was adjusted as necessary to keep at around 7.2. After 10 days, or until viability dropped below 70%, the cell culture was harvested by centrifugation and filtered through a 0.22 4m filter. The filtrate was either stored at 4 0 C or immediately loaded onto columns for purification. For the poly-His tagged constructs, the proteins were purified using a Ni 2 '-NTA column (Qiagen). Before 15 purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni 2 -NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4 0 C. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCI and 4% 20 mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80 0 C. Immunoadhesin (Fc containing) constructs were purified from the conditioned media as follows. The conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml 25 fractions into tubes containing 275 pl of I M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation. EXAMPLE 15 30 Expression of PRO87, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 in Yeast The following method describes recombinant expression of PROl87, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 in yeast. First, yeast expression vectors are constructed for intracellular production or secretion of PRO 187, 35 PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 from the ADH2/GAPDH promoter. DNA encoding PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct -144- WO 00/15666 PCT/US99/20594 intracellular expression of PRO187, PR0533, PR0214. PR0240, PRO21 1, PR0230, PR0261, PRO246 or PR0317. For secretion, DNA encoding PRO187, PR0533, PR0214, PR0240, PRO2I 1, PR0230, PR0261, PR0246 or PRO317 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PROl 87, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 5 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PRO187, PR0533, PRO214, PR0240, PRO2 11, PRO230, PR026 1, PR0246 or PRO317. Yeast cells, such as yeast strain ABI 10, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be 10 analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain. Recombinant PRO187, PR0533, PR0214, PR0240, PR021 1, PR0230, PR0261, PR0246 or PRO317 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate 15 containing PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 may further be purified using selected column chromatography resins. EXAMPLE 16 Expression of PRO] 87, PR0533, PR0214, PR0240. PRO211, PR0230, PR0261, PR0246 or PR0317 in 20 Baculovirus-infected Insect Cells The following method describes recombinant expression in Baculovirus-infected insect cells. The sequence coding for PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, 25 including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 or the desired portion of the coding sequence of PRO 187, PRO533, PRO214, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers 30 complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector. Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into Spodopterafrugiperda ("Sf9")cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28*C, the released viruses are harvested and used for further 35 amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual Oxford: Oxford University Press (1994). Expressed poly-His tagged PROl 87, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 -145- WO 00/15666 PCT/US99/20594 or PRO317 can then be purified, for example, by Ni 2 *-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 36.:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCl,; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCI), and sonicated twice for 20 seconds on ice. The sonicates are 5 cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 pm filter. A Ni 2 +-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of loading buffer. The filtered cell extract is loaded onto the column at 0.5 ml per minute. The column is washed to baseline A,,, with loading buffer, at which point fraction collection is started. Next, the column is 10 washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A,,, baseline again, the column is developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer. One ml fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni 2 *-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His,,-tagged PRO 187, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317, 15 respectively, are pooled and dialyzed against loading buffer. Alternatively, purification of the IgG tagged (or Fc tagged) PRO187, PR0533, PRO214, PRO240, PRO2 11, PR0230, PR0261, PR0246 or PRO317 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography. PRO187, PR0533, PR0214, PRO240, PRO211, PR0230, PR0246 and PR0317 were expressed in 20 baculovirus infected Sf9 insect cells. While expression was actually performed in a 0.5-2 L scale, it can be readily scaled up for larger (e.g., 8 L) preparations. The proteins were expressed as an IgG construct (immunoadhesin), in which the protein extracellular region was fused to an IgG I constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms. Following PCR amplification, the respective coding sequences were subcloned into a baculovirus 25 expression vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins), and the vector and Baculogold@ baculovirus DNA (Pharmingen) were co-transfected into 105 Spodopterafrugiperda ("Sf9") cells (ATCC CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences. The cells were grown in Hink's TNM-FH medium supplemented with 10% 30 FBS (Hyclone). Cells were incubated for 5 days at 28'C. The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells were incubated for 3 days at 28*C. The supernatant was harvested and the expression of the constructs in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 ml of Ni 2 +-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A 35 Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining. The first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown -146- WO 00/15666 PCTIUS99/20594 in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells were incubated for 3 days at 28*C. The supernatant was harvested and filtered. Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed. The conditioned medium from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove 5 the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein construct were purified using a Ni 2 +-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media were pumped onto a 6 ml Ni 2 +-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4'C. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration 10 buffer containing 0.25 M imidazole. The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80*C. Immunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows. The conditioned media were pumped onto a 5 ml Protein A column (Pharmacia) which had been 15 equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting I ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the proteins was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid 20 sequencing by Edman degradation. Alternatively, a modified baculovirus procedure may be used incorporating high 5 cells. In this procedure, the DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector. Such epitope tags include poly His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including 25 plasmids derived from commercially available plasmids such as plE 1-1 (Novagen). The plE I-I and plE 1-2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus iel promoter in stably transformed insect cells. The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for ie1-mediated gene expression in uninfected insect cells as well as the hr5 enhancer element. pIE I -1 and plE1-2 include the translation initiation site and can be used to produce fusion 30 proteins. Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product was then digested with those selected restriction enzymes and subcloned into the expression vector. For example, derivatives ofplE 1-1 can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3'-of) 35 the desired sequence. Preferably, the vector construct is sequenced for confirmation. High 5 cells are grown to a confluency of 50% under the conditions of, 27*C, no C0 2 , NO pen/strep. For each 150 mm plate, 30 pg of pIE based vector containing the sequence was mixed with I ml Ex-Cell medium -147- WO 00/15666 PCT/US99/20594 (Media: Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401-78P (note: this media is light sensitive)), and in a separate tube, 100 pl of CellFectin (CelIFECTIN (GibcoBRL #10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium. The two solutions were combined and allowed to incubate at room temperature for 15 minutes. 8 ml of Ex-Cell media was added to the 2 ml of DNA/CellFECTIN mix and this is layered on High 5 5 cells that have been washed once with Ex-Cell media. The plate is then incubated in darkness for 1 hour at room temperature. The DNA/CelIFECTIN mix is then aspirated, and the cells are washed once with Ex-Cell to remove excess CelIFECTIN, 30 ml of fresh Ex-Cell media was added and the cells are incubated for 3 days at 28 0 C. The supernatant was harvested and the expression of the sequence in the baculovirus expression vector was determined by batch binding of I ml of supernatant to 25 ml of Ni 2 '-NTA beads (QIAGEN) for histidine tagged proteins or 10 Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining. The conditioned media from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein comprising the sequence is purified using aNi 2 '-NTA column (Qiagen). Before purification, imidazole is added to the conditioned 15 media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni 2 '-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 48 0 C. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) 20 column and stored at -80 0 C. Immunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows. The conditioned media was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized 25 by collecting I ml fractions into tubes containing 275 ml of I M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the sequence was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary. PRO187, PR0533, PRO214, PR0240, PRO21 1 and PR0246 were successfully expressed by the above 30 modified baculovirus procedure incorporating high 5 cells. EXAMPLE 17 Demonstration of binding of PR0533 to FGF Recptor 3 PR0533 was expressed in baculovirus in a C-terminal His8 epitope tagged form as described in Example 35 16, as was a control C-terminal His8 epitope protein. The extracellular domains of FGF receptors 1-4 and TIEl receptor were expressed as Fc fusion proteins. Proteins were allowed to interact in binding buffer (DMEM media + 10mM Hepes pH 7.4 + 0.1% albumin + 200 ng/ml heparin) at room temperature for one hour. Protein A -148- WO 00/15666 PCTIUS99/20594 Sepharose (Pharmacia) was added (0.01 ml) and binding continued for 30 minutes. Protein A Sepharose beads were collected and washed twice in binding buffer. Samples were then resolved by SDS PAGE under reducing conditions. Western blot analysis was conducted with anti-His antibody (Qiagen) as recommended by the manufacturer. The results demonstrated a high specificity binding to FGF Receptor 3 (FGFR3-Fc). This is very 5 significant, since most FGF ligands bind more than one FGF receptor. EXAMPLE 18 Preparation of Antibodies that Bind PRO]87, PR0533, PR0214, PR0240, PRO21 1. PR0230, PR0261, PR0246 or PRO317 10 This example illustrates preparation of monoclonal antibodies which can specifically bind PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317. Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified PRO]87, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317, fusion proteins containing PRO187, PR0533, PR0214, 15 PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 and cells expressing recombinant PRO 187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation. Mice, such as Balb/c, are immunized with the PROI 87, PR0533, PRO214, PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously 20 or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays 25 to detect anti-PRO 187, anti-PR0533, anti-PRO21 4, anti-PR0240, anti-PRO2 11, anti-PR0230, anti-PR0261, anti PR0246 or anti-PRO317 antibodies. After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are 30 then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU. 1, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non fused cells, myeloma hybrids, and spleen cell hybrids. The hybridoma cells will be screened in an ELISA for reactivity against PRO187, PR0533, PRO214, 35 PR0240, PRO2 11, PR0230, PR026 1, PR0246 or PRO317. Determinationof "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 is within the skill in the art. -149- WO 00/15666 PCT/US99/20594 The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti PR026 1, anti-PR0246 or anti-PRO317 monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be 5 accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed. Deposit of Material: The following materials have been deposited with the American Type Culture Collection, 10801 10 University Blvd., Manassas, VA 20110-2209, USA (ATCC): Material ATCC Deposit No.: Deposit Date DNA27864-1155 209375 10/16/97 DNA49435-1219 209480 11/21/97 DNA32286-l191 209385 10/16/97 15 DNA34387-1138 209260 9/16/97 DNA32292-1131 209258 9/16/97 DNA33223- 1136 209264 9/16/97 DNA33473-1176 209391 10/17/97 DNA35639-1172 209396 10/17/97 20 DNA33461-1199 209367 10/15/97 These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures the maintenance of a viable culture of the deposit for 30 years from the date of deposit. The 25 deposit will be made available by the ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc., and the ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. § 30 122 and the Commissioner's rules pursuant thereto (including 37 C.F.R. § 1.14 with particular reference to 886 OG 638). The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to 35 practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice -150- WO 00/15666 PCTIUS99/20594 the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the 5 invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. -15 1-

Claims (48)

1. An isolated antibody that binds to a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide. 5

2. The antibody of Claim I which specifically binds to said polypeptide.

3. The antibody of Claim I which induces the death of a cell that expresses said polypeptide.

4. The antibody of Claim 3, wherein said cell is a cancer cell that overexpresses said polypeptide as 10 compared to a normal cell of the same tissue type.

5. The antibody of Claim I which is a monoclonal antibody.

6. The antibody of Claim 5 which comprises a non-human complementarity determining region (CDR) 15 or a human framework region (FR).

7. The antibody of Claim 1 which is labeled.

8. The antibody of Claim I which is an antibody fragment or a single-chain antibody. 20

9. A composition of matter which comprises an antibody of Claim I in admixture with a pharmaceutically acceptable carrier.

10. The composition of matter of Claim 9 which comprises a therapeutically effective amount of said 25 antibody.

11. The composition of matter of Claim 9 which further comprises a cytotoxic or a chemotherapeutic agent. 30

12. An isolated nucleic acid molecule that encodes the antibody of Claim 1.

13. A vector comprising the nucleic acid molecule of Claim 12.

14. A host cell comprising the vector of Claim 13. 35 -152- WO 00/15666 PCT/US99/20594

15. A method for producing an antibody that binds to a PRO 187, PR0533, PRO21 4, PR0240, PRO211, PR0230, PR0261, PR0246 or PRO317 polypeptide, said method comprising culturing the host cell of Claim 14 under conditions sufficient to allow expression of said antibody and recovering said antibody from the cell culture. 5

16. An antagonist of a PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide.

17. The antagonist of Claim 16 wherein said antagonist inhibits tumor cell growth. 10

18. An isolated nucleic acid molecule that hybridizes to a nucleic acid sequence that encodes a PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO3 17 polypeptide, or the complement thereof.

19. The isolated nucleic acid molecule of Claim 18 wherein said hybridization is under stringent 15 hybridization and wash conditions.

20. A method for determining the presence of a PRO187, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 polypeptide in a sample suspected of containing said polypeptide, said method comprising exposing the sample to an anti-PRO187, anti-PR0533, anti-PR0214, anti-PR0240, anti 20 PRO2 11, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PRO317 antibody and determining binding of said antibody to a PRO 187, PR0533, PRO214, PR0240, PRO211, PR0230, PRO26 1, PRO246 or PRO317 polypeptide in said sample.

21. The method of Claim 20, wherein said sample comprises a cell suspected of comprising a PRO187, 25 PR0533, PRO214, PRO240, PRO211, PRO230, PRO261, PRO246 or PRO317 polypeptide.

22. The method of Claim 21, wherein said cell is a cancer cell.

23. A method of diagnosing tumor in a mammal, said method comprising detecting the level of 30 expression of a gene encoding a PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test tissue cells were obtained. 35 -153- WO 00/15666 PCT/US99/20594

24. A method of diagnosing tumor in a mammal, said method comprising (a) contacting an anti PRO187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PR021 1. anti-PR0230, anti-PR0261, anti-PR0246 or anti-PRO317 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the anti-PRO187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PRO21 1. anti-PR0230, 5 anti-PR0261, anti-PR0246 or anti-PR0317 antibody and a PRO187, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal.

25. The method of Claim 24, wherein said antibody is detectably labeled. 10

26. The method of Claim 24, wherein said test sample of tissue cells is obtained from an individual suspected of having neoplastic cell growth or proliferation.

27. A cancer diagnostic kit comprising an anti-PRO 187, anti-PR0533, anti-PRO214, anti-PR0240, anti 15 PRO2 11, anti-PR0230, anti-PR026 1, anti-PR0246 or anti-PRO317 antibody and a carrier in suitable packaging.

28. The kit of Claim 27 which further comprises instructions for using said antibody to detect the presenceof a PRO 187, PR0533, PRO214, PRO240, PRO2 11, PRO230, PR026 1, PR0246 or PRO317 polypeptide in a sample suspected of containing the same. 20

29. A method for inhibiting the growth of tumor cells, said method comprising exposing tumor cells that express a PRO187, PR0533, PR0214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 polypeptide to an effective amount of an agent that inhibits a biological activity of a PRO 187, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 polypeptide, wherein growth of said tumor cells is 25 thereby inhibited.

30. The method of Claim 29, wherein said tumor cells overexpress said polypeptide as compared to normal cells of the same tissue type. 30

31. The method of Claim 29, wherein said agent is an anti-PRO187, anti-PR0533, anti-PR0214, anti PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody.

32. The method of Claim 31, wherein said anti-PRO187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody induces cell death. 35

33. The method of Claim 29, wherein said tumor cells are further exposed to radiation treatment, a cytotoxic agent or a chemotherapeutic agent. -154- WO 00/15666 PCT/US99/20594

34. A method for inhibiting the growth of tumor cells, said method comprising exposing tumor cells that express a PRO187, PR0533, PR0214, PR0240, PRO211, PR0230, PR0261, PR0246 or PR0317 polypeptide to an effective amount of an agent that inhibits the expression of a PRO 187, PR0533, PRO214, PR0240, PRO2 11, PRO230, PR0261, PR0246 or PRO317 polypeptide, wherein growth of said tumor cells is 5 thereby inhibited.

35. The method of Claim 34, wherein said tumor cells overexpress said polypeptide as compared to normal cells of the same tissue type. 10

36. The method of Claim 34, wherein said agent is an antisense oligonucleotide that hybridizes to a nucleic acid which encodes the PRO187, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 polypeptide, or the complement thereof.

37. The method of Claim 36, wherein said tumor cells are further exposed to radiation treatment, a 15 cytotoxic agent or a chemotherapeutic agent.

38. An article of manufacture, comprising: a container; a label on the container; and 20 a composition comprising an active agent contained within the container, wherein the composition is effective for inhibiting the growth of tumor cells and wherein the label on the container indicates that the composition is effective for treating conditions characterized by overexpression of a PRO 187, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PR0317 polypeptide in said tumor cells as compared to in normal cells of the same tissue type. 25

39. The article of manufacture of Claim 38, wherein said active agent inhibits a biological activity of and/or the expression of said PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide. 30

40. The article of manufacture of Claim 39, wherein said active agent is an anti-PROl87, anti-PR0533, anti-PRO214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody.

41. The article of manufacture of Claim 39, wherein said active agent is an antisense oligonucleotide. 35 -155- WO 00/15666 PCTIUS99/20594

42. A method of identifying a compound that inhibits a biological or immunological activity of a PRO187, PR0533, PRO214, PR0240, PRO2 1 1,PR0230, PRO261, PR0246 or PRO317 polypeptide, said method comprising contacting a candidate compound with a PRO 187, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 polypeptide under conditions and for a time sufficient to allow the two components 5 to interact and determining whether a biological or immunological activity of said PRO187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PRO317 polypeptide is inhibited.

43. The method of Claim 42, wherein said candidate compound is an anti-PRO187, anti-PR0533, anti PR0214, anti-PR0240, anti-PRO21 1, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody. 10

44. The method of Claim 42, wherein said candidate compound or said PRO 187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide is immobilized on a solid support.

45. The method of Claim 44, wherein the non-immobilized component is detectably labeled. 15

46. A method of identifying a compound that inhibits an activity of a PRO 187, PR0533, PRO214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide, said method comprising the steps of (a) contacting cells and a candidate compound to be screened in the presence of a PRO187, PR0533, PRO214, PR0240, PRO2 11, PRO230, PR026 1, PR0246 or PRO317 polypeptide under conditions suitable for the induction 20 of a cellular response normally induced by a PRO187, PR0533, PR0214, PR0240, PRO211, PRO230, PR0261, PR0246 or PRO317 polypeptide and (b) determining the induction of said cellular response to determine ifthe test compound is an effective antagonist, wherein the lack of induction of said cellular response is indicative of said compound being an effective antagonist. 25

47. A method for identifying a compound that inhibits the expression of a PRO 187, PR0533, PRO214, PR0240, PRO21 1, PRO230, PR0261, PR0246 or PRO317 polypeptide in cells that express said polypeptide, wherein said method comprises contacting said cells with a candidate compound and determining whether expression of said PRO187, PR0533, PR0214, PR0240, PRO21 1, PR0230, PR0261, PR0246 or PR0317 polypeptide is inhibited. 30

48. The method of Claim 47, wherein said candidate compound is an antisense oligonucleotide. -156-