JP2003521229A - Secreted and transmembrane polypeptides and nucleic acids encoding them - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel polypeptides and nucleic acid molecules encoding the polypeptides. Also provided herein are vectors and host cells comprising the nucleic acid molecule, chimeric polypeptide molecules comprising the polypeptide of the invention fused to a heterologous polypeptide sequence, antibodies binding to the polypeptide of the invention, and the invention. Is a method for producing the polypeptide of

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates generally to the identification and isolation of novel DNAs and the recombinant production of novel polypeptides encoded by the DNAs.

BACKGROUND OF THE INVENTION Extracellular proteins play important roles in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, such as proliferation, migration, differentiation or interaction with other cells, is typically governed by information received from other cells and / or the immediate environment. This information is often found in secreted polypeptides (e.g., mitogens,
Survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones), which are then received and interpreted by various cellular receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules normally pass through the cell secretory pathway to reach their site of action in the extracellular milieu. Secreted proteins have a variety of industrial applications including pharmaceuticals, diagnostics, biosensors and bioreactors. Most protein drugs currently available, such as thrombolytic agents, interferons, interleukins, erythropoietin, colony stimulating factors, and various other cytokines, are secreted proteins. Efforts are being made by both industry and academia to identify new native secreted proteins. Much effort has been devoted to the screening of mammalian recombinant DNA libraries to identify the coding sequences of novel secreted proteins. Examples of screening methods and techniques are described in the literature [eg Klein et al., Proc. Natl.
Acad. Sci. 93; 7108-7113 (1996); US Pat. No. 5,536,637].
.

Membrane-bound proteins and receptors play important roles in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, such as proliferation, migration, differentiation or interaction with other cells, is typically governed by information received from other cells and / or the immediate environment. This information is often transmitted by secreted polypeptides (eg, mitogens, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones), which in turn are received by various cellular receptors or membrane-bound proteins. Be interpreted. Such membrane-bound proteins and cellular receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cell adhesion such as selectins and integrins. Contains molecules. For example, the transduction of signals that regulate cell growth and differentiation are regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinase, the enzyme that catalyzes that process, also acts as a growth factor receptor. For example,
Includes fibroblast growth factor and nerve growth factor receptors. Membrane-bound proteins and receptor molecules have various industrial applications, including pharmaceuticals and diagnostics. For example, the receptor immunoadhesin is a receptor-
It can be used as a therapeutic agent that blocks ligand-ligand interactions. Membrane-bound proteins can also be used to screen peptides or small molecule inhibitors for potential related receptor / ligand interactions. Efforts are being made by both industry and academia to identify novel native receptor proteins. Much effort has been devoted to the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel receptor proteins.

1. PRO1800 Hep27 protein is synthesized and accumulated in the nucleus of human hepatoblastoma cells (HepG2 cells) following growth arrest induced by butyrate treatment (Gabrielli et al.,.
Eur. J. Biochem. 232: 473-477 (1995)). Hep27 synthesis is released from the butyrate block and is suppressed in cells that resume DNA synthesis. The Hep27 protein sequence shows significant homology to the known short chain alcohol dehydrogenase (SCAD) family of proteins, suggesting that Hep27 is a novel member of the SCAD family of proteins. Hep2 according to nuclear localization
7 has a region similar to the target sequence of the binucleus, suggesting that there is regulation of Hep27 mRNA expression and protein synthesis at the posttranscriptional level. Here we describe the identification and characterization of a novel polypeptide homologous to the Hep27 protein, herein designated the PRO1800 polypeptide.

2. The development of PRO539 multicellular organisms depends, at least in part, on the mechanisms that identify, direct, or maintain positional information of cell, tissue, or organ patterns. Various secreted signaling molecules, such as transforming growth factor-beta (TGF-
β), Wnt, members of the fibroblast growth factor and hedgehog family, etc., are associated with various cell and structural patterning activities in Drosophila and vertebrates. Perrimon, Cell: 80: 517-520 (1995). Costal-2 is a novel kinesin-related protein in the hedgehog signaling pathway. Hedgehog (Hh) was first identified as a segment polarity gene by genetic screening in Drosophila melanogaster, Nuss
Lein-Volhard et al., Roux. Arch. Dev. Biol. 193: 267-282 (1984), fulfill a wide range of developmental functions. Perrimon, supra. Although only one Drosophila Hh gene has been identified, three human Hh homologues: Sonic Hh (SHh), Desert Hh (
DHh) and Indian Hh (IHh) have been isolated, Echelard et al., Supra; Krauss et al., Cell 75, 1431-44 (1993); Riddle et al., Cell 75: 1401-16 (1993).
SHh is expressed at high levels in the notochord and floor plate of developing vertebrate embryos. The in vitro explant assay as well as the ectopic expression of SHh in transgenic animals indicate that SHh plays a key role in neural tube patterning, Echelard et al., Supra, Krauss et al., Cell 75, 1431. -44 (1993), Riddle et al., Cell 75
: 1401-16 (1993), Roelink et al., Cell 81: 445-5 (1995). In vitro explant assay as well as ectopic expression of SHh in transgenic animals indicates that SHh plays a key role in neural tube patterning, Echelard et al.
(1993), supra, Ericson et al., Cell 81: 747-56 (1995); Marti et al., Nature 375: 3
22-5 (1995); Roelink et al. (1995), supra; Hynes et al., Neuron 19: 15-26 (1997).
Also, Hh is a limb (Krauss et al., Cel 75: 1431-44 (1993); Laufer et al., Cell 79,
993-1003 (1994)), somites (Fan and Tessier-Lavigne, Cell 79, 1175-86 (1994)
); Johnson et al., Cell 79: 1165-73 (1994)), lung (Bellusci et al., Develop. 124: 5).
3-63 (1997)) and skin (Oro et al., Science 276: 817-21 (1997)). Similarly, IHh and DHh are associated with bone, intestinal and germ cell development, Apeqvist et al., Curr. Biol. 7: 801-4 (1997); Bellusci et al., Development 124:
55-63 (1997); Bitgood et al., Curr. Biol. 6: 298-304 (1996); Roberts et al., Devel.
opment 121: 3163-74 (1995). SHh knockout mice further strengthened the notion that SHh is important in many aspects of vertebrate development, Chiang et al., Nature 383:
407-13 (1996). These mice show abnormalities in intermediate structures such as the notochord and floor plate, the absence of ventral cell cell types in the neural tube, the absence in terminal limb structures, monocular disease, and the absence in the spinal column and most ribs.

At the cell surface, the Hh signal is associated with the 12-transmembrane domain protein Patc.
hed (Ptch) [Hooper and Scott, Cell 59: 751-65 (1989); Nakano et al., N.
ature 341: 508-13 (1989)] and G-protein coupled like receptor Smooth.
ened (Smo) [Alcedo et al., Cell 86: 221-232 (1996); van den Heuval and Ingham, Nature 382: 547-551 (1996)]. Genetic and biochemical evidence supports a receptor model in which Ptch and Smo are part of a multi-component receptor complex, Chen and Struhl, Cell 87: 553-63.
(1996); Marigo et al., Nature 384: 176-9 (1996); Stone et al., Nature 384: 129-34.
(1996). Upon binding of Hh to Ptch, the normal inhibitory effect of Ptch on Smo is released, allowing Smo to transduce Hh signals through the cytoplasmic membrane.
Loss of functional mutations in the Ptch gene results in basal cell nevus syndrome (BCNS),
It was identified in patients with a genetic disease characterized by multiple basal cell carcinoma (BCC). Also, dysfunctional Ptch gene mutations were associated with diffuse basal cell carcinoma in a large proportion,
Chidambaram et al., Cancer Research 56: 459-601 (1996); Gailani et al., Nature Gen.
et. 14: 78-81 (1996); Hahn et al., Cell 85: 841-51 (1996); Johnson et al., Science.
272: 1668-71 (1996); Unden et al., Cancer Res. 56: 4561-5 (1996); Wicking et al.
Am. J. Hum. Genet. 60: 21-6 (1997). Loss of Ptch function is thought to cause uncontrolled Smo signaling in basal cell carcinoma. Similarly, activation of the Smo mutation was identified in sporadic BCC tumors (Xie et al., Nature 391: 90-2 (1998)).
, Smo as a signaling subunit in the SHh receptor complex
Emphasizes the role of. However, the precise mechanism by which Ptch regulates Smo has not yet been clarified, and the signal transduction mechanism by which the Hh signal is transmitted from the receptor to the target downstream must be clarified. Gene top analysis in Drosophila identified several segment polarity genes, which were found to function as components of the Hh signaling pathway, Ingham, Curr. Opin. Genet. Dev. 5: 492-8 (1995 ); Perrimon, supra. These are kinesin-like molecules, Costal-2 (Cos-2) [Robbins et al., Cell 90:
225-34 (1997); Sisson et al., Cell 90: 235-45 (1997)], protein-directed fu
sed [Preat et al., Genetics 135: 1047-62 (1993); Therond et al., Proc. Natl. ac
ad. Sci. USA 93: 4224-8 (1996)], a novel molecule with an unknown function-directed fused suppressor [Pham et al., Genetics 140: 587-98 (1995); Preat, Genetics.
132: 725-36 (1992)] and Zn finger protein Ci. [Alexandle et al., Ge
nes De. 10: 2003-12 (1996); Dominguez et al., Science 272: 1621-5 (1996); Ore
nic et al., Genes Dev. 4: 1053-67 (1990)]. Further components involved in Hh signaling are transcription factors CBP [Akimaru et al., Nature 386: 735-738 (1997)],
Negative regulator slimb [Jiang and Struhl, Nature 391: 493-496 (
1998)] and SHh response component COUP-TFII [Krishnan et al., Science 278: 194].
7-1950 (1997)].

Mutations in Cos-2 are embryonic lethal, with central components of each segment and Hh
It presents phenotypes similar to Hh overexpression, including replication of the extended domain of responsive genes. In contrast, the mutant embryos for fused and Ci contained H deletions in each segment and substitutions in the anterior mirror-like copy and substitutions in the anterior mirror-like copy.
h Shows a phenotype similar to loss of function, Busson et al., Roux. Arch. Dev. Biol.
197: 221-230 (1988). The molecular characterization of Ci is that it is Wing
Alexandre et al. (1996), supra; Therond et al., Proc. Natl. Acad. Sc, which suggested that it is a transcription factor that directly activates Hh-responsive genes such as less and Dpp.
i. USA 93: 4224-8 (1996). Consistent with the putative opposite functions of Cos-2 and fused, the fused mutation is suppressed by the Cos-2 mutant and by the suppressor of the fused mutant, Preat et al., Genetics 135: 1047-62 (1993). However, the fused-free mutation and the N-terminal kinase domain mutation were
Although completely suppressed by the d-mutant suppressor, the fused C-terminal mutation displays a strong Cos-2 phenotype in the background of the fused suppressor. This suggests that the fused kinase domain acts as a constitutive activator of SHh signaling in the absence of the fused suppressor. A recent study was the 92 kDa Drosophila fused.
, Cos-2 and Ci were present in microtubules with multiprotein complexes, indicating that Hh signaling leads to dissociation of this complex from microtubules, Robbins et al., Cell.
90: 225-34 (1997); Sisson et al., Cell 90: 235-45 (1997). fused and Co
Both s-2 are phosphorylated in response to Hh treatment, but Robbins et al., supra; Th
erond et al., Genetics 142: 1181-98 (1996), the kinase responsible for this activity remains characterized. To date, the vertebrate homologs known for these components are the Gli protein family (eg, Gli-1, Gli-2 and Gli-2).
li-3) only. These are putative Zn finger transcription factors structurally related to Ci. Among these, Gli-1 has been shown to be a candidate mediator of SHh signals [Hynes et al., Neuron 15: 35-44 (1995), Lee et al., Developme.
nt 124: 2537-52 (1997); Alexandre et al., Genes Dev. 10: 2003-13 (1996)], H
It is suggested that the mechanism of gene activation in response to h is conserved between flies and vertebrates. To determine whether other signaling components in the Hh cascade are evolutionarily conserved and to test the function of fused in Hh signaling at the biochemical level, Applicants have isolated and characterized human fused cDNA. Were determined.
Tissue distribution in mice showed that fused was expressed in SHh-responsive tissues. Biochemical studies have shown that fused is a functional kinase. Functional studies provide evidence that fused is an activator of Gli and a dominant negative form of fused can block SHh signaling in Xenopus embryos. Taken together, these data indicate that both Cos-2 and fused are directly involved in Hh signaling. For further references on Costal-2 proteins, see Simpson et al., Dev. Biol. 122: 201-209 (1987), Grau et al., Dev. Biol. 122: 186-200 (1987), Prea.
et al., Genetics 135: 1047-1062 (1993), Sisson et al., Cell 90: 235-245 (1997) and Rob.
Bins et al., Cell 90: 225-234 (1997). Applicants now identify and describe a cDNA encoding a human Costal-2 homologous polypeptide, herein designated PRO539.

3. PRO982 Efforts are being made by both industry and academia to identify novel native secreted proteins. Much effort has gone into screening mammalian recombinant DNA libraries to identify coding sequences for novel secreted proteins. We describe here the identification and characterization of a novel secreted polypeptide, herein designated the PRO982 polypeptide.

[0009] 4. PRO1434 nel gene has been described as a protein expressed in chicken neurons
(Watanabe et al., Genomics 38 (3): 273-276 (1996)). Recently, two novel human cDNAs (designated NELL1 and NELL2) encoding polypeptides with homology to those encoded by the chicken nel gene have been isolated and characterized. It contains 6 EGF-like repeats (Watanabe et al., Supra). Given the nerve-specific expression of these genes, it is suggested that they may influence neurodevelopment. Therefore, nel, NELL1 and NE
There is significant interest in identifying and characterizing novel polypeptides with homology to LL2. Here we describe the identification and characterization of a novel polypeptide homologous to the nel protein, herein designated PRO1434.

5. PRO1863 Efforts have been made by both industry and academia to identify novel native transmembrane proteins. Much effort has gone into screening mammalian recombinant DNA libraries to identify coding sequences for novel transmembrane proteins. We describe here the identification and characterization of a novel transmembrane polypeptide, designated herein as the PRO1863 polypeptide.

[0011] 6. The characterization of PRO1917 inositol phosphatase is of interest as it is the basis for understanding the signaling activity that stimulates Ca ²⁺ release from the endoplasmic reticulum. Molecular cloning has provided diverse inositol polyphosphate phosphatases that are highly expressed in kidney and liver (Caxton et al. (1997) Biochem J. 328: 75-81).

[0012] 7. The PRO1868 inflammatory response is complex and is mediated by a variety of signaling molecules produced locally by mast cells, nerve endings, platelets, leukocytes and complement activity. Certain of these signaling molecules cause the expression of selectins, which act as cell surface molecules that make the endothelial cell lining more porous and / or recognize and attract leukocytes through specialized carbohydrate recognition. Stronger leukocyte binding is mediated by integrins and mediates leukocyte migration through the endothelium. In addition, the signaling agent acts as a chemoattractant, causing the bound leukocytes to travel towards the source of the attractant. Other signaling substances produced in the course of the inflammatory reaction pass into the blood and stimulate the bone marrow to produce more white blood cells and release them into the bloodstream. Inflammation is typically initiated by an antigen, which can be almost any molecule capable of initiating an immune response. While under normal physiological conditions these are foreign molecules, the molecules produced by the organism itself are the catalysts known to occur in various disease states. T cell proliferation in mixed lymphocyte cultures or mixed lymphocyte reactions (MLRs) is an established indicator of a compound's ability to stimulate the immune system. In the inflammatory response, the responding leukocytes may be neutrophils, eosinophils, monocytes or lymphocytes. Histological examination of infected tissues provides evidence of immune stimulation or inhibition of the response. Current Protocols in Immun
ology, ed. E. Coligan, 1994, John Wiley and Sons, Inc. Inflammatory bowel disease (IBD), although clearly distinguished as a disease, shares visceral diseases that include both ulcerative colitis (UC) and Crohn's disease that share common features and often share pathologies. A word used to describe collectively. The commonality of diagnostic criteria makes it difficult to accurately determine two diseases. Each illness, however, typically differs in its type and location of injury. UC lesions are characteristically mucosal surface ulcers that appear in the large intestine adjacent to the rectum. CD lesions are characteristically widespread linear fissures, sometimes appearing anywhere in the intestine, including the stomach, esophagus, and duodenum.

Conventional treatment of IBD usually involves administration of anti-inflammatory or immunosuppressive agents such as, for example, sulfasalazine, corticosteroids, 6-mercaptopurine / azathoprine, or cyclospoin. However, these all only provide relief for some of the suffering patients. However, anti-inflammatory /
If immunosuppressive therapy fails, colectomy is the last line of defense. Surgery requires about 30% of CD patients in the first year after diagnosis, and the likelihood of requiring surgery increases about 5% each year. Unfortunately, CD also has a high recurrence rate, about 5
% Patients will need the next surgery one year later. Moreover, UC patients are at significantly increased risk of developing colorectal cancer. Presumably, this is due to repeated cycles of epithelial damage associated with regeneration, which constantly increases the risk of neoplastic transformation. A recently discovered member of the immunoglobulin superfamily, known as the junctional adhesion molecule (JAM), has been identified to be selectively concentrated at the intercellular junctions of endothelial and epithelial cells of different origins. . Martin-Padura, I. et al. J. Cell Biol. 142 (1): 117
-27 (1998). JAM is a type 1 with two extracellular type intrachain disulfide rings of V type
It is a complex membrane protein. JAM shares considerable homology with the A33 antigen (Fig.
.1 or Fig. 18). Monoclonal antibodies against JAM in vitro and spontaneously and chemically induced migration of monocytes across endothelial cell monolayers in vitro.
It was discovered that it was suppressed by o. Martin-Padura listed above. It was recently discovered that JAM expression is increased in the colon of CRF2-4-/-mice with colitis. CRF2-4-/-(IL-10R subunit knockout mice) develop spontaneous colitis mediated by lymphocytes, monocytes and neutrophils. Also, some animals develop colon cancer. As a result, it is likely that the compounds of the invention may be further expressed or associated with high levels of human disease such as inflammatory bowel disease, other intestinal inflammatory diseases and colorectal cancer.

The compounds of the invention also have significant homology with the known colorectal cancer-prognostic marker A33 antigen. The A33 antigen is expressed on normal colon epithelium as well as on over 90% of early or metastatic colon cancers. In carcinomas derived from colonic mucosa, A33
The antigen is uniformly expressed in more than 95% of cases. However, the A33 antigen
It has not been detected in a wide range of other normal tissues, ie its expression appears rather organ-specific. Therefore, the A33 antigen appears to play an important role in the induction of colorectal cancer. Since colon cancer is a widespread disease, early diagnosis and treatment are important medical goals. Diagnosis and treatment of colon cancer can be performed using specific monoclonal antibodies (mAbs) with fluorescent, nuclear magnetic or radioactive tags. MAbs tagged with radiogenes, toxins and / or drugs are in sit in minimal patient characterization
It can be used to treat u. MAbs can also be used for diagnosis during diagnosis and treatment of colon cancer. For example, serum A33 antigen levels are elevated in patients, and low levels after surgery will indicate successful tumor resection. On the other hand, a subsequent increase in serum A33 antigen levels after surgery is indicative of the formation of metastases of the original tumor or the appearance of new early tumors. Monoclonal antibodies may be used in place of or in combination with surgery and / or other chemotherapy. For example, a preclinical analysis and localization study in patients with colorectal carcinoma with a mAb for A33 is described by Welt et al., J. Clin. Oncol. 8: 1894-1906 (
1990) and Welt et al., J. Clin. Oncol. 12: 1561-1571 (1994), and U.S. Pat. And the latter relates to the use of anti-A33 mAb. We describe here the identification and characterization of a novel polypeptide with homology to the A33 antigen protein, herein designated PRO1868 polypeptide.

8. PRO3434 Efforts are being made by both industry and academia to identify novel native secreted proteins. Much effort has gone into screening mammalian recombinant DNA libraries to identify coding sequences for novel secreted proteins. We describe here the identification and characterization of a novel transmembrane polypeptide, designated herein as the PRO3434 polypeptide.

9. The PRO1927 protein is glycosylated by a complex series of reactions mediated by membrane-bound glycosyltransferases. There are a number of different glycosyltransferases that account for the structural diversity of synthesized carbohydrates. N-acetylglucosaminyl transferase proteins include a family of glycosyl transferases that confer a variety of important biological functions in mammalian organisms. As an example, UDP-N-acetylglucosamine: α-3-D-mannoside β-1
2,2-N-acetylglucosaminyltransferase I is a higher mannose-N-
A glycosyltransferase that catalyzes the essential first step in the conversion of glycans to hybrid and complex N-glycans (Sarkar et al., Proc. Natl. Acad. Sci.
USA. 88: 234-238 (1991)). UPD-N-acetylglucosamine: α1,3-D mannoside β1,4-N-acetylglucosaminyltransferase is 3-
And Te4-a key enzyme in the formation of tactile asparagine-linked sugar chains, which has recently been purified from bovine small intestine using cDNA cloning (Minowa et al., J. Bi.
ol. Chem. (1998) 273 (19): 11556-62). It is of interest to identify and characterize additional members of the N-acetylglucosaminyl transferase protein family, and more generally to identify novel glycosyl transferases. Where we are
Described herein is the identification and characterization of a novel polypeptide homologous to the N-acetylglucosaminyl transferase protein, designated PRO1475 polypeptide.

(Outline of the Invention) 1. PRO1800 A cDNA clone (DNA3, which encodes a novel polypeptide named “PRO1800” in the present application and having homology with a nucleic acid encoding Hep27 (DNA3).
5672-2508) have been identified. In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1800 polypeptide. In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO1800 polypeptide having a sequence from about 1 or about 16 to about 278 amino acid residues of Fig2 (SEQ ID NO: 2), or (b) At least about 80% sequence identity to the complement of the DNA molecule of (a), preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about. 95%
DNA having the sequence identity of In another aspect, the invention provides a PRO comprising a DNA that hybridizes to the complement of a nucleic acid between about 36 or about 81 and about 869 nucleotides of Fig1 (SEQ ID NO: 1).
It relates to an isolated nucleic acid molecule encoding a 1800 polypeptide. Preferably,
Hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Deposit No. 203538 (DNA35672-2508), or (b) the complement of the DNA molecule of (a). Against
Comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. It relates to an isolated nucleic acid molecule. In a preferred embodiment, the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203538 (DNA35672-2508).

In yet a further aspect, the invention provides (a) at least about 80% sequence identity with amino acid residue 1 of Fig2 (SEQ ID NO: 2) or the sequence from about 16 to about 278, preferably at least about 85%. DNA encoding a polypeptide having a% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity, or the complement of (b) (a) DNA. To an isolated nucleic acid molecule comprising: In a further aspect, the invention provides a test DNA molecule comprising: (a) PRO1800 having amino acid residue 1 of Fig2 (SEQ ID NO: 2) or the sequence from about 16 to about 278.
Hybridizing with a DNA molecule encoding a polypeptide or (b) the complement of a DNA molecule of (a) under stringent conditions, wherein the DNA molecule is at least about 80% relative to (a) or (b) A test DNA molecule is isolated if it has at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. And an isolated nucleic acid molecule having at least about 230 nucleotides. In a particular embodiment, the invention comprises a DNA encoding a PRO1800 polypeptide with or without an N-terminal signal sequence and / or an initiation methionine, or which is complementary to such an encoding nucleic acid molecule. Provide a nucleic acid molecule that is separated. The signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 15 in the sequence of Figure 2 (SEQ ID NO: 2). In another aspect, the invention provides (a) residue 1 or about 16 of Fig2 (SEQ ID NO: 2).
To at least about 80% positive when compared to the about 278 amino acid sequence,
Preferably at least about 85% positive, more preferably at least about 90%
A DNA encoding a polypeptide positive, most preferably at least about 95% positive, or an isolated nucleic acid molecule comprising the complement of (b) (a) DNA. Another embodiment is a PR that finds use as a hybridization probe.
It concerns a fragment of the O1800 polypeptide coding sequence. Such a nucleic acid fragment has a length of about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides,
It is more preferably about 20 to about 50 nucleotides in length, most preferably about 20 to about 40 nucleotides in length, and can be derived from the nucleic acid sequence shown in Fig1 (SEQ ID NO: 1). In another embodiment, the invention provides an isolated PRO1800 polypeptide encoded by any of the isolated nucleic acid sequences identified above. In a particular aspect, the invention provides an isolated native sequence PRO1800 polypeptide, which in certain embodiments comprises amino acid comprising residue 1 or about 16 to about 278 of Fig2 (SEQ ID NO: 2). Contains an array.

In another aspect, the invention features amino acid residue 1 or about 1 of Fig2 (SEQ ID NO: 2).
6 to about 278 sequences, at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity. It relates to an isolated PRO1800 polypeptide comprising an amino acid sequence having sequence identity. In a further aspect, the invention provides at least about 80% positive, preferably at least about 85% positive, more preferably when compared to the amino acid sequence of residue 1 or about 16 to about 278 of Fig2 (SEQ ID NO: 2), more preferably It relates to an isolated PRO1800 polypeptide comprising an amino acid sequence with a score of at least about 90% positive, and most preferably at least about 95% positive. In yet another aspect, the invention includes amino acid residue 1 of Fig2 (SEQ ID NO: 2) or the sequence from about 16 to about 278, or a fragment thereof sufficient to provide a binding site for an anti-PRO1800 antibody. To an isolated PRO1800 polypeptide consisting of Preferably, the PRO1800 fragment retains the qualitative biological activity of the native PRO1800 polypeptide. In a still further aspect, the invention provides (i) a test DNA molecule, (a) Fig2
PR having a sequence of about 1 or about 16 to about 278 amino acid residues of (SEQ ID NO: 3)
A DNA molecule encoding an O1800 polypeptide, or (b) hybridized to the complement of the DNA molecule of (a) under stringent conditions, wherein the test DNA molecule is at least about (Ii) test if it has 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. DNA
A host cell containing the molecule is cultured under conditions suitable for expression of the polypeptide, and (i
ii) Providing the polypeptide produced by recovering the polypeptide from the cell culture medium. In yet another embodiment, the invention relates to agonists and antagonists of the native PRO1800 polypeptide. In a particular embodiment, the agonist or antagonist is an anti-PRO1800 antibody. In a further embodiment, the invention relates to a method of identifying an agonist or antagonist of a native PRO1800 polypeptide by contacting the native PRO1800 polypeptide with a candidate molecule and monitoring the biological activity mediated by said polypeptide. In a still further embodiment, the invention relates to a composition comprising a PRO1800 polypeptide, or an agonist or antagonist specified above, together with a pharmaceutically acceptable carrier.

2. PRO539 In the present application, a cDNA clone (DNA47, which is named "PRO539"), which encodes a novel polypeptide and has homology with a nucleic acid encoding Costal-2.
465-1561) was identified. In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO539 polypeptide. In one aspect, the isolated nucleic acid comprises (a) a DN encoding a PRO539 polypeptide having a sequence from about 1 to about 830 amino acid residues of Figure4 (SEQ ID NO: 7).
At least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity to the complement of the A molecule, or (b) (a) DNA molecule. Sex, most preferably at least about 95% sequence identity. In another aspect, the invention pertains to an isolated nucleic acid molecule encoding a PRO539 polypeptide that comprises DNA that hybridizes to the complement of the nucleic acid from about nucleotide 186 to about 2675 of Fig3 (SEQ ID NO: 6). Preferably, hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA (DNA47465-1561) of ATCC Deposit No. 203661, or (b) the complement of a nucleic acid molecule of (a). To at least about 80% sequence identity, preferably at least about 85% sequence identity,
More preferably it relates to an isolated nucleic acid molecule comprising DNA having at least about 90% sequence identity, most preferably at least about 95% sequence identity.
In a preferred embodiment, the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203661 (DNA47465-1561).

In a still further aspect, the invention features (a) at least about 80% sequence identity, preferably at least about 85% sequence with the sequence from amino acid residue 1 to about 830 of Fig4 (SEQ ID NO: 7). DNA comprising a polypeptide having identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity, or (b) complement of DNA of (a). To an isolated nucleic acid molecule. In a further aspect, the invention provides a test DNA molecule, (a) a DNA molecule encoding a PRO539 polypeptide having a sequence from amino acid residue 1 to about 830 of Fig4 (SEQ ID NO: 7), or (b) (a). ) Hybridizing to the complement of a DNA molecule under stringent conditions, the DNA molecule having at least about 80% sequence identity to (a) or (b), preferably at least about 85% sequence identity. Sex, more preferably at least about 90% sequence identity, most preferably at least about 95%.
Produced by isolating a test DNA molecule having the sequence identity of
It relates to an isolated nucleic acid molecule having at least 100 nucleotides. In a particular embodiment, the present invention relates to PR with or without initiation methionine.
Provided is an isolated nucleic acid molecule which comprises DNA encoding the O539 polypeptide or which is complementary to such encoding nucleic acid molecule. In another aspect, the invention provides (a) residues 1 to about 83 of Fig4 (SEQ ID NO: 7).
A DNA encoding a polypeptide that scores at least about 80% positive, preferably at least about 85% positive, more preferably at least about 90% positive, and most preferably at least about 95% positive when compared to the amino acid sequence of 0, Or (b) an isolated nucleic acid molecule comprising the complement of the DNA of (a). Another embodiment is a PR that finds use as a hybridization probe.
It relates to a fragment of the O539 polypeptide coding sequence. Such a nucleic acid fragment has about 2
0 to about 80 nucleotides long, preferably about 20 to about 60 nucleotides long, more preferably about 20 to about 50 nucleotides long, and most preferably about 20 to about 4 nucleotides.
It is 0 nucleotides long and can be derived from the nucleotide sequence shown in Figure 3 (SEQ ID NO: 6). In another embodiment, the invention provides an isolated PRO539 polypeptide encoded by any of the isolated nucleic acid sequences identified above.

In a particular aspect, the invention provides an isolated native sequence PRO539 polypeptide, which in certain embodiments comprises residues 1 to about 8 of Fig4 (SEQ ID NO: 7).
It contains an amino acid sequence containing 30 amino acids. In another aspect, the invention features amino acid residues 1 to about 8 of Fig4 (SEQ ID NO: 7).
At least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity to the 30 sequences. To an isolated PRO539 polypeptide comprising an amino acid sequence having: In a further aspect, the invention features residues 1 to about 830 of Fig4 (SEQ ID NO: 7).
At least about 80% positive, preferably at least about 85% positive, more preferably at least about 90% positive, when compared to the amino acid sequence of
Most preferably it relates to an isolated PRO539 polypeptide which comprises an amino acid sequence with a score of at least about 95% positive. In yet another aspect, the invention comprises a sequence of amino acid residues 1 to about 830 of Fig4 (SEQ ID NO: 7), or a fragment thereof sufficient to provide a binding site for an anti-PRO539 antibody, It relates to an isolated PRO539 polypeptide. Preferably, the PRO539 fragment retains the qualitative biological activity of the native PRO539 polypeptide. In a still further aspect, the invention provides (i) a test DNA molecule, (a) Fig4
A DNA molecule encoding a PRO539 polypeptide having a sequence of about 1 to about 830 amino acid residues of (SEQ ID NO: 7), or (b) a complement of the DNA molecule of (a),
Hybridized under stringent conditions, the test DNA molecule has at least about 80% sequence identity to (a) or (b), preferably at least about 85% sequence identity, more preferably at least about 90%. (Ii) culturing the host cell containing the test DNA molecule under conditions suitable for the expression of the polypeptide, and (iii) if it has a% sequence identity, most preferably at least about 95% sequence identity; Provided is a polypeptide produced by recovering the polypeptide from a cell culture medium. In yet another embodiment, the invention relates to agonists and antagonists of native PRO539 polypeptides. In a particular embodiment, the agonist or antagonist is an anti-PRO539 antibody. In a further embodiment, the invention relates to a method of identifying an agonist or antagonist of a native PRO539 polypeptide by contacting the native PRO539 polypeptide with a candidate molecule and monitoring the biological activity mediated by said polypeptide. In a more preferred embodiment, the biological activity is microtubi
le) or the ability to combine fusion and cubitus interruptus. In a still further embodiment, the invention relates to a composition comprising a PRO539 polypeptide, or an agonist or antagonist identified above, together with a pharmaceutically acceptable carrier.

In yet another embodiment, the present invention provides compounds and methods that result in agonists and antagonists that promote PRO539 regulation of hedgehog signaling. In particular, vertebrate PRO539 antagonists that inhibit, prevent, suppress and / or neutralize the normal function of PRO539 in the SH signaling pathway include both bioorganic chemical small molecules and antisense nucleic acids. In yet another embodiment, the invention provides alternative splice variants of human PRO539. In yet a further embodiment, the invention provides screening and assay methods for identifying molecules that modulate PRO539 hedgehog signaling regulation. Preferably, the molecule prevents the interaction of PRO539 with its bound complex protein (eg, fusion or cubitus interruptus) or prevents or inhibits complex degradation. The assay involves incubating a mixture containing PRO539 and substrate with a candidate molecule and detecting the ability of the candidate molecule to modulate PRO539 hedgehog signaling. Preferably the screened molecule is a small molecule drug candidate. In yet another embodiment, a diagnostic method for determining whether a particular disease is regulated by hedgehog signaling: (a) culturing a test cell or tissue, (b) regulated by PRO539. Administering a compound capable of inhibiting Hedgehog signaling regulation; and (c) determining whether Hedgehog signaling is regulated. A method comprising:

3. PRO982 A cDNA clone (DNA57700-1408) encoding a novel secreted polypeptide designated "PRO982" in this application was identified. In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO982 polypeptide. In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO982 polypeptide having amino acid residue 1 of Fig6 (SEQ ID NO: 9) or a sequence of about 22 to about 125, or (b) ( At least about 80% sequence identity to the complement of the DNA molecule of a), preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95%. It comprises DNA with% sequence identity. In another aspect, the invention features residues 89 to about 400 of Figure 5 (SEQ ID NO: 8).
Nucleic acid molecule encoding a PRO982 polypeptide that comprises DNA that hybridizes to the complement of the nucleic acid of. Preferably, hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA (DNA57700-1408) of ATCC Deposit No. 203583, or (b) the complement of the DNA molecule of (a). Against
Comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. It relates to an isolated nucleic acid molecule. In a preferred embodiment, the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203583 (DNA57700-1408).

In a still further aspect, the invention features (a) at least about 80% sequence identity, preferably at least about 80% sequence identity with the sequence of about 1 or about 22 to about 125 amino acid residues of Fig6 (SEQ ID NO: 9). 85% sequence identity, more preferably at least about 90%
To a DNA encoding a polypeptide having sequence identity of, most preferably at least about 95% sequence identity, or (b) an isolated nucleic acid molecule comprising the complement of DNA of (a). In a further aspect, the invention provides a test DNA molecule comprising: (a) PRO982 having a sequence from about 1 or about 22 to about 125 amino acid residues of Fig6 (SEQ ID NO: 9).
Hybridizing with a DNA molecule encoding a polypeptide, or (b) the complement of a DNA molecule of (a) under stringent conditions, wherein the DNA molecule is at least about 80% relative to (a) or (b) A test DNA molecule is isolated if it has at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. And an isolated nucleic acid molecule having at least about 50 nucleotides, preferably at least about 100 nucleotides. In a particular embodiment, the invention comprises a DNA encoding a PRO982 polypeptide with or without an N-terminal signal sequence and / or an initiation methionine, or which is complementary to such an encoding nucleic acid molecule. Provide a nucleic acid molecule that is separated. The signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 21 in the sequence of Figure 6 (SEQ ID NO: 9). In another aspect, the invention provides (a) residue 1 or about 22 of Fig6 (SEQ ID NO: 9).
To at least about 80% positive when compared to about 125 amino acid sequences,
Preferably at least about 85% positive, more preferably at least about 90%
A DNA encoding a polypeptide positive, most preferably at least about 95% positive, or an isolated nucleic acid molecule comprising the complement of (b) (a) DNA. Another embodiment is a PR that finds use as a hybridization probe.
It relates to a fragment of the O982 polypeptide coding sequence. Such a nucleic acid fragment has about 2
0 to about 80 nucleotides long, preferably about 20 to about 60 nucleotides long, more preferably about 20 to about 50 nucleotides long, and most preferably about 20 to about 4 nucleotides.
It is 0 nucleotides long. In another embodiment, the invention provides an isolated PRO982 polypeptide encoded by any of the isolated nucleic acid sequences identified above. In a specific aspect, the invention provides an isolated native sequence PRO982 polypeptide, which in certain embodiments is residue 1 or about 2 of Figure 6 (SEQ ID NO: 9).
It contains an amino acid sequence containing 2 to 125.

In another aspect, the invention features amino acid residue 1 or about 2 of Figure 6 (SEQ ID NO: 9).
2 to about 125 sequences, at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. It relates to an isolated PRO982 polypeptide comprising an amino acid sequence having sequence identity. In a further aspect, the invention provides at least about 80% positive, preferably at least about 85% positive, more preferably at least about 80% positive when compared to the amino acid sequence of residue 1 or about 22 to 125 of Figure 6 (SEQ ID NO: 9). It relates to an isolated PRO982 polypeptide which comprises an amino acid sequence with a score of about 90% positive, most preferably at least about 95% positive. In yet another aspect, the invention includes amino acid residue 1 of Fig6 (SEQ ID NO: 9) or the sequence from about 22 to about 125, or a fragment thereof sufficient to provide a binding site for an anti-PRO982 antibody. To an isolated PRO982 polypeptide consisting of Preferably, the PRO982 fragment retains the qualitative biological activity of the native PRO982 polypeptide. In a still further aspect, the invention provides (i) a test DNA molecule, (a) Fig6
PRO having amino acid residue 1 of (SEQ ID NO: 9) or a sequence of about 22 to about 125
982 polypeptide-encoding DNA molecule, or (b) hybridized to the complement of the DNA molecule of (a) under stringent conditions, and the test DNA molecule is (a) or (
at least about 80% sequence identity to b), preferably at least about 85
(Ii) a host cell containing the test DNA molecule for expression of the polypeptide if it has a sequence identity of%, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. Culture under suitable conditions, and (iii
) Providing a polypeptide produced by recovering the polypeptide from a cell culture medium.

[0027] 4. PRO1434 A cDNA clone (DNA68818-2536) has been identified in this application, designated "PRO1434", that encodes a novel polypeptide and has homology to a nucleic acid that encodes a nel protein. In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1434 polypeptide. In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO1434 polypeptide having the sequence from about 1 or about 28 to about 325 amino acid residues of Figure 8 (SEQ ID NO: 11), or (b) At least about 80% sequence identity to the complement of the DNA molecule of (a), preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about. 95
It comprises DNA with% sequence identity. In another aspect, the invention features about nucleotide 581 of Figure 7 (SEQ ID NO: 10).
Or an isolated nucleic acid molecule encoding a PRO1434 polypeptide that comprises DNA that hybridizes to the complement of about 662 to about 1555 nucleic acids. Preferably, hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA (DNA68818-2536) of ATCC Deposit No. 203657, or (b) the complement of a nucleic acid molecule of (a). To at least about 80% sequence identity, preferably at least about 85% sequence identity,
More preferably it relates to an isolated nucleic acid molecule comprising DNA having at least about 90% sequence identity, most preferably at least about 95% sequence identity.
In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203657 (DNA68818-2536).

In a still further aspect, the invention provides (a) at least about 80% sequence identity with amino acid residue 1 of Fig8 (SEQ ID NO: 11) or the sequence from about 28 to about 325, preferably at least about 85%. % Sequence identity, more preferably at least about 90%
To a DNA encoding a polypeptide having sequence identity of, most preferably at least about 95% sequence identity, or (b) an isolated nucleic acid molecule comprising the complement of DNA of (a). In a further aspect, the invention provides a test DNA molecule, (a) PRO143 having amino acid residue 1 of Fig8 (SEQ ID NO: 11) or a sequence from about 28 to about 325.
At least about 80 relative to (a) or (b) by hybridizing under stringent conditions with a DNA molecule encoding a 4 polypeptide, or (b) the complement of the DNA molecule of (a). Isolate a test DNA molecule if it has a% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. To an isolated nucleic acid molecule having at least 65 nucleotides. In a particular aspect, the invention comprises DNA encoding a PRO1434 polypeptide with or without an N-terminal signal sequence and / or an initiation methionine, and its soluble, ie transmembrane, domain is deleted or deleted. Activated variants, or isolated nucleic acid molecules that are complementary to such encoding nucleic acid molecules are provided. The signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 27 in the sequence of Figure 8 (SEQ ID NO: 11). The transmembrane domain has been tentatively identified as extending from about amino acid position 11 to about amino acid position 30 of the PRO1434 amino acid sequence (Fig8, SEQ ID NO: 11). In another aspect, the invention provides (a) residue 1 or about 2 of Fig8 (SEQ ID NO: 11).
At least about 80% positive, preferably at least about 85% positive, more preferably at least about 90 when compared to the amino acid sequence of 8 to about 325.
% Nucleic acid, and most preferably at least about 95% positive DNA that encodes a polypeptide, or an isolated nucleic acid molecule comprising the complement of (b) (a) DNA. Another embodiment is a PR that finds use as a hybridization probe.
A fragment of the O1434 polypeptide coding sequence. Such a nucleic acid fragment has a length of about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides,
More preferably about 20 to about 50 nucleotides long, most preferably about 20 to about 40 nucleotides long and can be derived from the nucleotide sequence shown in Figure 7 (SEQ ID NO: 10). In another embodiment, the invention provides an isolated PRO1434 polypeptide encoded by any of the isolated nucleic acid sequences identified above.

In a particular aspect, the invention provides an isolated native sequence PRO1434 polypeptide, which in certain embodiments is residue 1 or about 28 to about 325 of Figure 8 (SEQ ID NO: 11). It contains an amino acid sequence containing. In another aspect, the invention features at least about 80% sequence identity, preferably at least about 85% sequence identity to amino acid residue 1 of Fig8 (SEQ ID NO: 11) or the sequence from about 28 to about 325. It relates to an isolated PRO1434 polypeptide comprising an amino acid sequence having identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. In a further aspect, the invention features residue 1 or about 28 of Figure 8 (SEQ ID NO: 11).
To at least about 80% positive when compared to about 325 amino acid sequences,
Preferably at least about 85% positive, more preferably at least about 90%
It relates to an isolated PRO1434 polypeptide which comprises an amino acid sequence that scores positive, most preferably at least about 95% positive. In yet another aspect, the invention includes amino acid residue 1 of Fig8 (SEQ ID NO: 11) or the sequence from about 28 to about 325, or a fragment thereof sufficient to provide a binding site for an anti-PRO1434 antibody. To an isolated PRO1434 polypeptide consisting of Preferably, the PRO1434 fragment retains the qualitative biological activity of the native PRO1434 polypeptide. In a still further aspect, the invention provides (i) a test DNA molecule, (a) Fig8
P having the sequence of about 1 or about 28 to about 325 amino acid residues of (SEQ ID NO: 11)
DNA molecule encoding RO1434 polypeptide or (b) (a) DNA
The test DNA molecule is hybridized to the complement of the molecule under stringent conditions,
Or to (b) at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity. (Ii) test DN if
A host cell containing the A molecule is cultured under conditions suitable for expression of the polypeptide, and (
iii) providing a polypeptide produced by recovering the polypeptide from the cell culture medium. In yet another embodiment, the invention relates to agonists and antagonists of the native PRO1434 polypeptide. In a particular embodiment, the agonist or antagonist is an anti-PRO1434 antibody. In a further embodiment, the invention relates to a method of identifying an agonist or antagonist of a native PRO1434 polypeptide by contacting the native PRO1434 polypeptide with a candidate molecule and monitoring the biological activity mediated by said polypeptide. In a still further embodiment, the invention relates to a composition comprising a PRO1434 polypeptide, or an agonist or antagonist specified above, together with a pharmaceutically acceptable carrier.

5. PRO1863 A cDNA clone (DNA59847-2510) encoding a novel transmembrane polypeptide designated "PRO1863" in this application was identified. In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1863 polypeptide. In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO1863 polypeptide having the sequence from about 1 or about 16 to about 437 amino acid residues of Figure 10 (SEQ ID NO: 16), or (b) At least about 80% sequence identity to the complement of the DNA molecule of (a), preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about. 9
It comprises DNA with 5% sequence identity. In another aspect, the invention provides a PR comprising a DNA that hybridizes to the complement of a nucleic acid from about 17 or about 62 to about 1327 nucleotides of Figure 9 (SEQ ID NO: 15).
It relates to an isolated nucleic acid molecule encoding the O1863 polypeptide. Preferably, hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA (DNA59847-2510) of ATCC Deposit No. 203576, or (b) the complement of the DNA molecule of (a). Against
Comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. It relates to an isolated nucleic acid molecule. In a preferred embodiment, the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203576 (DNA59847-2510).

In a yet further aspect, the invention provides (a) at least about 80% sequence identity, preferably at least about 80% sequence identity with the sequence of about 1 or about 16 to about 437 amino acid residues of Fig10 (SEQ ID NO: 16). 85% sequence identity, more preferably at least about 9
An isolated nucleic acid molecule comprising a DNA encoding a polypeptide having 0% sequence identity, most preferably at least about 95% sequence identity, or (b) a complement of the DNA of (a). . In a further aspect, the invention provides a test DNA molecule comprising:
Hybridized with a DNA molecule encoding the 863 polypeptide or (b) the complement of the DNA molecule of (a) under stringent conditions, whereby the DNA molecule (a) or (b)
To at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. To an isolated nucleic acid molecule having at least 345 nucleotides produced by isolating a test DNA molecule. In a particular embodiment, the invention comprises a DNA encoding a PRO1863 polypeptide with or without an N-terminal signal sequence and / or an initiation methionine, and wherein the soluble, ie transmembrane domain is deleted or Inactivated variants, or isolated nucleic acid molecules that are complementary to such encoding nucleic acid molecules are provided. The signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 17 in the sequence of Figure 10 (SEQ ID NO: 16). The transmembrane domain has a PRO1863 amino acid sequence (Fig10, SEQ ID NO:
It has been tentatively identified as extending from about amino acid position 243 to about amino acid position 260 in 16). In another aspect, the invention provides (a) at least about 80% positive, preferably at least about 85% positive when compared to the amino acid sequence of residues about 1 or about 16 to about 437 of Figure 10 (SEQ ID NO: 16). A DNA encoding a polypeptide with a positive, more preferably at least about 90% positive, most preferably at least about 95% positive score, or an isolated nucleic acid molecule comprising the complement of (b) (a) DNA. . Another embodiment is a PR that finds use as a hybridization probe.
A fragment of the O1863 polypeptide coding sequence. Such a nucleic acid fragment has a length of about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides,
It is more preferably about 20 to about 50 nucleotides in length, most preferably about 20 to about 40 nucleotides in length, and can be derived to the nucleotide sequence shown in Figure 9 (SEQ ID NO: 15). In another embodiment, the invention provides an isolated PRO1863 polypeptide encoded by any of the isolated nucleic acid sequences identified above. In a particular aspect, the invention provides an isolated native sequence PRO1863 polypeptide, which in certain embodiments comprises about 1 residue of Figure 10 (SEQ ID NO: 16).
Or an amino acid sequence comprising about 16 to about 437.

In another aspect, the invention features at least about 80% sequence identity, preferably at least about 85%, to amino acid residue 1 of Figure 10 (SEQ ID NO: 16) or the sequence from about 16 to about 437. It relates to an isolated PRO1863 polypeptide comprising an amino acid sequence having a% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. In a further aspect, the invention features residue 1 or about 1 of Figure 10 (SEQ ID NO: 16).
At least about 80% positive, preferably at least about 85% positive, more preferably at least about 90 when compared to the 6 to about 437 amino acid sequence.
It relates to an isolated PRO1863 polypeptide which comprises an amino acid sequence with a score of% positive, most preferably at least about 95% positive. In yet another aspect, the invention provides amino acid residue 1 of Figure 10 (SEQ ID NO: 16).
Or about 16 to about 437, or an isolated PRO1863 polypeptide comprising sufficient sequence thereof to provide a binding site for an anti-PRO1863 antibody. Preferably, the PRO1863 fragment retains the qualitative biological activity of the native PRO1863 polypeptide. In a still further aspect, the invention provides (i) a test DNA molecule, (a) Fig1.
A DNA molecule encoding a PRO1863 polypeptide having a sequence of about 1 or about 16 to about 437 amino acid residues of 0 (SEQ ID NO: 16), or (b) (a) DN
Hybridization with the complement of the A molecule under stringent conditions results in the test DNA molecule (a
) Or (b), at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity. If there is identity, then (ii) test D
A polypeptide produced by culturing a host cell containing an NA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture medium is provided. In yet another embodiment, the invention relates to agonists and antagonists of native PRO1863 polypeptides. In a particular embodiment, the agonist or antagonist is an anti-PRO1863 antibody. In a further embodiment, the invention relates to a method of identifying an agonist or antagonist of a native PRO1863 polypeptide by contacting the native PRO1863 polypeptide with a candidate molecule and monitoring the biological activity mediated by said polypeptide. In a still further embodiment, the invention relates to a composition comprising a PRO1863 polypeptide, or an agonist or antagonist identified above, together with a pharmaceutically acceptable carrier.

6. PRO1917 In the present application, designated as "PRO1917", a cDNA clone (DNA7) encoding a novel polypeptide having homology with inositol phosphatase.
6400-2528) was identified. In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1917 polypeptide. In one aspect, the isolated nucleic acid is (a) a DNA molecule that encodes a PRO1917 polypeptide having amino acid residue 1 of Figure 12 (SEQ ID NO: 18) or a sequence of about 31 to about 487, or (b) ( At least about 80% sequence identity to the complement of the DNA molecule of a), preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95%.
It comprises DNA with% sequence identity. In another aspect, the invention features residues 96 to about 1 of Figure 11 (SEQ ID NO: 17).
It relates to an isolated nucleic acid molecule encoding a PRO1917 polypeptide that comprises DNA that hybridizes to the complement of 466 nucleic acids. Preferably, hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA (DNA76400-2528) of ATCC Deposit No. 203573, or (b) the complement of the DNA molecule of (a). Against
Comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. It relates to an isolated nucleic acid molecule. In a preferred embodiment, the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203573 (DNA76400-2528). In a still further aspect, the invention provides (a) at least about 80% sequence identity with amino acid residue 1 of Figure 12 (SEQ ID NO: 18) or the sequence from about 31 to about 487,
Preferably at least about 85% sequence identity, more preferably at least about 90.
It relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide having a% sequence identity, most preferably at least about 95% sequence identity, or the complement of the DNA of (a).

In a further aspect, the invention provides for a test DNA molecule to be (a) PRO19 having amino acid residue 1 of Fig12 (SEQ ID NO: 18) or a sequence from about 31 to about 487.
Hybridizing with a DNA molecule encoding a 17 polypeptide or (b) the complement of the DNA molecule of (a) under stringent conditions such that the DNA molecule has at least about 80 relative to (a) or (b). Isolate a test DNA molecule if it has a% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. At least about 50 nucleotides, preferably at least about 10
It relates to an isolated nucleic acid molecule having 0 nucleotides. In a particular embodiment, the present invention comprises a DNA encoding a PRO1917 polypeptide with or without an N-terminal signal sequence and / or an initiation methionine, or a DNA that is complementary to such an encoding nucleic acid molecule. Provide a nucleic acid molecule that is separated. The signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 30 in the sequence of Figure 12 (SEQ ID NO: 18). In another aspect, the invention provides (a) at least about 80% positive, preferably at least about 85% positive when compared to the amino acid sequence of residue 1 or about 31 to about 487 of Figure 12 (SEQ ID NO: 18). , More preferably at least about 9
It relates to an isolated nucleic acid molecule comprising 0% positive, most preferably at least about 95% positive score DNA encoding a polypeptide, or (b) (a) the complement of DNA. Another embodiment is a PR that finds use as a hybridization probe.
It relates to a fragment of the O1917 polypeptide coding sequence. Such a nucleic acid fragment has a length of about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides,
More preferably about 20 to about 50 nucleotides in length, and most preferably about 20 to about 40 nucleotides in length.

In another embodiment, the invention provides an isolated PRO1917 polypeptide encoded by any of the isolated nucleic acid sequences identified above. In a particular aspect, the invention provides an isolated native sequence PRO1917 polypeptide, which in certain embodiments comprises residue 1 of Figure 12 (SEQ ID NO: 18) or an amino acid comprising from about 31 to about 487. Contains an array. In another aspect, the invention features at least about 80% sequence identity, preferably at least about 85% sequence, to amino acid residue 1 of Fig12 (SEQ ID NO: 18) or the sequence from about 31 to about 487. It relates to an isolated PRO1917 polypeptide comprising an amino acid sequence having identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. In a further aspect, the invention features residue 1 or about 3 of Figure 12 (SEQ ID NO: 18).
At least about 80% positive, preferably at least about 85% positive, more preferably at least about 90 when compared to the 1 to about 487 amino acid sequence.
It relates to an isolated PRO1917 polypeptide which comprises an amino acid sequence with a score of% positive, most preferably at least about 95% positive. In yet another aspect, the invention features amino acid residue 1 of Figure 12 (SEQ ID NO: 18).
Or about 31 to about 487 sequences, or a fragment thereof sufficient to provide a binding site for an anti-PRO1917 antibody. Preferably, the PRO1917 fragment retains the qualitative biological activity of the native PRO1917 polypeptide. In a still further aspect, the invention provides (i) a test DNA molecule, (a) Fig1.
P having the amino acid residue 1 of 2 (SEQ ID NO: 18) or the sequence of about 31 to about 487
DNA molecule encoding RO1917 polypeptide, or DNA of (b) (a)
The test DNA molecule is hybridized to the complement of the molecule under stringent conditions,
Or to (b) at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity. (Ii) test DN if
A host cell containing the A molecule is cultured under conditions suitable for expression of the polypeptide, and (
iii) providing a polypeptide produced by recovering the polypeptide from the cell culture medium. In yet another embodiment, the invention relates to agonists and antagonists of the native PRO1917 polypeptide. In a particular embodiment, the agonist or antagonist is an anti-PRO1917 antibody. In a further embodiment, the present invention relates to a method of identifying an agonist or antagonist of a native PRO1917 polypeptide by contacting the native PRO1917 polypeptide with a candidate molecule and monitoring the biological activity mediated by said polypeptide. In a still further embodiment, the invention relates to a composition comprising a PRO1917 polypeptide, or an agonist or antagonist identified above, together with a pharmaceutically acceptable carrier.

7. PRO1868 The present invention relates to compositions and methods for the diagnosis and treatment of inflammatory diseases in mammals, including humans. The present invention is based on the identification of proteins, including agonist and antagonist antibodies, that promote or suppress immune responses in mammals. Inflammatory disorders can be treated by calming the inflammatory response. Molecules that enhance the inflammatory response promote or enable the immune response of the antigen. If sedation of the inflammatory response is beneficial, molecules that promote the inflammatory response may be suppressed. Molecules that promote inflammatory response can be used therapeutically if enhanced inflammatory response is beneficial. Also, such facilitating molecules may be suppressed if sedation of the inflammatory response is useful. Neutralizing antibodies are examples of molecules that suppress molecules with immunostimulatory activity and are useful in treating inflammatory diseases. Also, molecules that suppress the inflammatory response can be used to suppress the inflammatory response (either directly to the protein or through the use of antibody agonists), thus improving the inflammatory disease. Therefore, the protein of the present invention is useful for diagnosis and / or treatment (including prevention) of immune-related diseases. Antibodies that bind to the facilitating protein can be used to mitigate the inflammatory response. Antibodies that bind to suppressor proteins can be used to promote the inflammatory response and immune system. In addition, the protein and antibody of the present invention can be used for preparing a drug or a drug for treating inflammation or immune-related diseases. In one embodiment, the invention relates to agonists and antagonists of PRO1868 polypeptides that suppress one or more functions or activities of PRO1868 polypeptides. In another embodiment, the invention determines the presence of PRO1868 polypeptide comprising exposing cells suspected of containing the polypeptide to anti-PRO1868 antibody and measuring the binding of the antibody to the cells. Regarding the method. In yet another embodiment, the invention provides a PRO1868 polypeptide in (a) a test sample of tissue cells obtained from a mammal and (b) a control sample of known normal tissue cells of the same cell type. A method of diagnosing an inflammatory-related disease in a mammal, wherein high expression level in a test sample means the presence of an inflammatory disease in the mammal, comprising detecting the expression level of the encoding gene.

In another embodiment, (a) contacting a test sample of tissue culture cells obtained from a mammal with anti-PRO1868 antibody and (b) detecting the formation of a complex of the antibody and PRO1868 polypeptide. The present invention relates to a method of diagnosing an inflammatory response in a mammal. The detection may be qualitative or quantitative and may be performed relative to monitoring formation of known normal tissue cells of the same cell type in a control sample. A higher amount of the large amount of complex formed in the test sample is indicative of the presence of tumor in the mammal from which the test sample was obtained. Suitably the antibody carries a detection label. Complex formation can be observed, for example, by light microscopy, flow cytometry, fluorescence, or other techniques known in the art. Test samples are usually taken from individuals suspected of having a defect or abnormality associated with an inflammatory response. In another embodiment, the invention provides an anti-PRO1868 antibody in a suitable package (
It relates to a diagnostic kit comprising a carrier (eg a buffer). Preferably the kit includes instructions for using the antibody to detect PRO1868 polypeptides. In a further embodiment, the invention also relates to an article of manufacture comprising: a container; a label on the container; and a composition comprising an active agent contained within the container, the composition comprising promoting a inflammatory response in a mammal. Alternatively, the label on the container indicates that the composition can be used for the treatment of inflammatory diseases, and the active agent in the composition is the expression of PRO1868 polypeptide and / or
Alternatively, it is a drug that promotes or suppresses the activity. In a preferred embodiment, the active agent is PRO1.
868 polypeptide or anti-PRO1868 antibody. A further embodiment provides for the candidate compound to be a PRO1868 polypeptide, these 2
A method of identifying a compound capable of suppressing the expression and / or activity of a PRO1868 polypeptide by contacting them for a time and under conditions sufficient to allow the two compounds to interact with each other. In certain aspects, either the candidate compound or the PRO1868 polypeptide is immobilized on a solid support. In other embodiments, the non-immobilized component carries a detection label.

In yet a further aspect, the invention provides: Inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory myopathy. Dermatomyositis, polymyositis), Sjogren's syndrome, systemic vaculitis, sarcoidosis, autoimmune hemolytic anemia (immunoaplastic anemia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic) Thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves disease, Hashimoto thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes, immune-mediated renal disease (
Glomerulonephritis, tubular interstitial nephritis), demyelinating diseases of the central and peripheral nervous system such as multiple sclerosis, idiopathic polyneuropathy, such as infectious hepatitis (A, B,
Hepatitis C, D, E and other nonhepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and hepatobiliary such as sclerosing cholangitis Diseases, inflammatory and fibrotic lung diseases (eg cystic fibrosis, eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonia), gluten irritable bowel disease, Whipple's disease, bullous skin disease, many Autoimmune or immune-mediated skin disorders, including erythema erythematosus and contact dermatitis, psoriasis, eg allergic disorders of the lung, such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonia, eg rejection and grafts. The present invention relates to a method for treating an inflammatory disease by administering a therapeutically effective amount of a PRO1868 antagonist to a patient in need thereof for the treatment of a disease selected from transplantation-related diseases including host disease. In a further embodiment, the invention provides a method of diagnosing a tumor in a mammal, the method comprising:
Detecting the expression level of a gene encoding a PRO1868 polypeptide in (a) a test sample of tissue cells obtained from a mammal and (b) a control sample of known normal tissue cells of the same cell type. Comprising a method in which a high expression level in the test sample indicates the presence of a tumor in the mammal from which the test tissue cells were harvested.

In another embodiment, the invention provides a method of diagnosing a tumor in a mammal, the method comprising:
) Contacting an anti-PRO1868 antibody with a test sample of tissue cells obtained from a mammal, and (b) detecting the formation of a complex between the anti-PRO1868 and PRO1868 polypeptides in the test sample. provide. The detection may be qualitative or quantitative and may be performed relative to monitoring complex formation in a control sample of known normal tissue cells of the same cell type. The formation of more complex than the test sample indicates the presence of tumor in the mammal from which the test tissue cells were collected. Suitably the antibody carries a detection label. Complex formation can be observed, for example, by light microscopy, flow cytometry, fluorescence, or other techniques known in the art. Suitably, the test sample is taken from an individual mammal suspected of having neoplastic cell growth or proliferation (eg, cancer cells). In another embodiment, the invention provides an anti-PRO1868 antibody in a suitable package (
A cancer diagnostic kit comprising a carrier (eg, a buffer) is provided. Preferably the kit comprises instructions for using the antibody to detect PRO1868 polypeptide. In yet another embodiment, the invention inhibits the growth of cancer cells comprising exposing cells that overexpress PRO1868 to an effective amount of an agent that inhibits the expression and / or activity of PRO1868 polypeptides. Provide a way. Preferably,
The drug is an anti-PRO1868 polypeptide, small organic and inorganic peptides, phosphorylated peptides, antisense or ribozyme molecules, or triple helix molecules. In a particular embodiment, an agent such as an anti-PRO1868 antibody induces cell death. In a further aspect, the tumor cells are further exposed to radiotherapy and / or toxic or chemotherapeutic agents. In a further embodiment, the invention also relates to an article of manufacture comprising: a container; a label on the container; and a composition comprising the active agent contained in the container, the composition being for inhibiting the growth of cancer cells. Effective, the label on the container indicates that the composition can be used for the treatment of conditions characterized by overexpression of PRO1868 polypeptide, wherein the active agent in the composition is PRO18
It is a drug that suppresses the expression and / or activity of 68 polypeptide. In a preferred embodiment, the active agent is anti-PRO1868 antibody.

A cDNA clone (DNA, designated “PRO1868” in the present application, encoding a novel polypeptide and having homology with a nucleic acid encoding A33 antigen.
77624-2515) was identified. In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1868 polypeptide. In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO1868 polypeptide having a sequence from about amino acid residue 1 or about 31 to about 310 of Figure 14 (SEQ ID NO: 20), or (b) At least about 80% sequence identity to the complement of the DNA molecule of (a), preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about. 9
It comprises DNA with 5% sequence identity. In another aspect, the invention features about 51 nucleotides of Figure 13 (SEQ ID NO: 19).
Or an isolated nucleic acid molecule encoding a PRO1868 polypeptide that comprises DNA that hybridizes to the complement of between about 141 and about 980 nucleic acids. Preferably, hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA (DNA77624-2515) of ATCC Deposit No. 203553, or (b) the complement of a nucleic acid molecule of (a). To at least about 80% sequence identity, preferably at least about 85% sequence identity,
More preferably it relates to an isolated nucleic acid molecule comprising DNA having at least about 90% sequence identity, most preferably at least about 95% sequence identity.
In a preferred embodiment, the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203553 (DNA77624-2515).

In yet a further aspect, the invention provides (a) at least about 80% sequence identity with amino acid residue 1 of Figure 14 (SEQ ID NO: 20) or the sequence from about 31 to about 310,
Preferably at least about 85% sequence identity, more preferably at least about 90.
It relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide having a% sequence identity, most preferably at least about 95% sequence identity, or (b) the complement of the DNA of (a). In a further aspect, the invention provides a test DNA molecule comprising: (a) PRO18 having amino acid residue 1 of Fig14 (SEQ ID NO: 20) or a sequence of about 31 to about 310.
At least about 80 relative to (a) or (b) by hybridizing under stringent conditions with a DNA molecule encoding the 68 polypeptide, or (b) the complement of the DNA molecule of (a). Isolating a test DNA molecule when it has a% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. To an isolated nucleic acid molecule having at least 390 nucleotides. In a particular aspect, the invention comprises a DNA encoding a PRO1868 polypeptide with or without an N-terminal signal sequence and / or an initiation methionine, and wherein the soluble, ie transmembrane domain is deleted or Inactivated variants, or isolated nucleic acid molecules that are complementary to such encoding nucleic acid molecules are provided. The signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 30 in the sequence of Figure 14 (SEQ ID NO: 20). The transmembrane domain has a PRO1868 amino acid sequence (Figure 14, SEQ ID NO :).
It has been tentatively identified as extending from about amino acid position 243 to about amino acid position 263 in 20). In another aspect, the invention provides (a) at least about 80% positive, preferably at least about 85% positive when compared to the amino acid sequence of residue 1 or about 31 to about 310 of Figure 14 (SEQ ID NO: 20). , More preferably at least about 9
It relates to an isolated nucleic acid molecule comprising 0% positive, most preferably at least about 95% positive score DNA encoding a polypeptide, or (b) (a) the complement of DNA. Another embodiment is a PR that finds use as a hybridization probe.
A fragment of the O1868 polypeptide coding sequence. Such a nucleic acid fragment has a length of about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides,
More preferably about 20 to about 50 nucleotides long, most preferably about 20 to about 40 nucleotides long and can be derived from the nucleotide sequence shown in Figure 13 (SEQ ID NO: 19). In another embodiment, the invention provides an isolated PRO1868 polypeptide encoded by any of the isolated nucleic acid sequences identified above.

In a particular embodiment, the invention provides an isolated native sequence PRO1868 polypeptide, which in certain embodiments is residue 1 or about 31 to about 310 of Figure 14 (SEQ ID NO: 20). It contains an amino acid sequence containing. In another aspect, the invention features at least about 80% sequence identity, preferably at least about 85% sequence, to amino acid residue 1 of Figure 14 (SEQ ID NO: 20) or the sequence from about 31 to about 310. It relates to an isolated PRO1868 polypeptide comprising an amino acid sequence having identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. In a further aspect, the invention features residue 1 or about 3 of Figure 14 (SEQ ID NO: 20).
At least about 80% positive, preferably at least about 85% positive, more preferably at least about 90 when compared to the amino acid sequence of 1 to about 310.
It relates to an isolated PRO1868 polypeptide which comprises an amino acid sequence with a score of% positive, most preferably at least about 95% positive. In yet another aspect, the invention provides amino acid residue 1 of Figure 14 (SEQ ID NO: 20).
Or about 31 to about 310 sequences, or a fragment thereof sufficient to provide a binding site for an anti-PRO1868 antibody. Preferably, the PRO1868 fragment retains the qualitative biological activity of the native PRO1868 polypeptide. In a still further aspect, the invention provides (i) a test DNA molecule, (a) Fig1.
4 (SEQ ID NO: 20), a DNA molecule encoding a PRO1868 polypeptide having the sequence of about 1 or about 31 to about 310 of amino acid residues, or (b) (a) DN
Hybridization with the complement of the A molecule under stringent conditions results in the test DNA molecule (a
) Or (b), at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity. If there is identity, then (ii) test D
A polypeptide produced by culturing a host cell containing an NA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture medium is provided. In yet another embodiment, the invention relates to agonists and antagonists of native PRO1868 polypeptides. In a particular embodiment, the agonist or antagonist is an anti-PRO1868 antibody. In a further embodiment, the invention relates to a method of identifying an agonist or antagonist of a native PRO1868 polypeptide by contacting the native PRO1868 polypeptide with a candidate molecule and monitoring the biological activity mediated by said polypeptide. In a still further embodiment, the invention relates to a composition comprising a PRO1868 polypeptide, or an agonist or antagonist identified above, together with a pharmaceutically acceptable carrier.

In another embodiment, the invention provides a composition comprising a PRO1868 polypeptide or agonist or antagonist antibody in combination with a carrier or excipient. In one aspect, the composition comprises a therapeutically effective amount of the peptide or antibody. In another aspect,
When the composition comprises a pro-inflammatory molecule, the composition: (a) increases the entry of inflammatory cells into the tissues of the mammal in need thereof, and (b) promotes an immune response in the mammal in need thereof or It serves to enhance or (c) increase T-lymphocyte proliferation in a mammal in need thereof in response to an antigen. In a further aspect, when the composition comprises an anti-inflammatory molecule, the composition: (a) reduces the infiltration of inflammatory cells into the tissues of the mammal in need thereof, and (b) immunizes in the mammal in need thereof. It serves to suppress or reduce the response, or (c) to reduce the proliferation of T-lymphocytes in the mammal in need thereof in response to an antigen. In other aspects, the composition comprises an additional active ingredient, which may be, for example, an additional antibody or cytotoxic or chemotherapeutic agent. Preferably the composition is disinfected. In a further embodiment, the invention provides a nucleic acid encoding an anti-PRO1868 antibody,
And a vector or a recombinant host cell constituting such a nucleic acid. In a still further embodiment, the present invention provides that, for example, by culturing a host cell transformed with a nucleic acid encoding the antibody under conditions such that the antibody is expressed and recovering the antibody from the cell medium, To a method for producing such an antibody.

8. PRO3434 A cDNA clone (DNA77631-2537) encoding a novel polypeptide designated "PRO3434" in this application was identified. In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO3434 polypeptide. In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO3434 polypeptide having amino acid residue 1 of Figure 16 (SEQ ID NO: 22) or a sequence of about 17 to about 1029, or (b) ( At least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 9 to the complement of the DNA molecule of a).
It comprises DNA with 5% sequence identity. In another aspect, the invention features residues about 46 or about 9 of Figure 15 (SEQ ID NO: 21).
PRO34 containing DNA that hybridizes to the complement of 4 to about 3132 nucleic acids
34 polypeptide isolated nucleic acid molecule. Preferably, hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Deposit No. 203651 (DNA77631-2537), or (b) the complement of the DNA molecule of (a). Against
Comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. It relates to an isolated nucleic acid molecule. In a preferred embodiment, the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA in ATCC Deposit No. 203651 (DNA77631-2537).

In a still further aspect, the invention features (a) at least about 80% sequence identity with amino acid residue 1 of Figure 16 (SEQ ID NO: 22) or the sequence from about 17 to about 1029, preferably at least about 85%. % Sequence identity, more preferably at least about 9
An isolated nucleic acid molecule comprising a DNA encoding a polypeptide having 0% sequence identity, most preferably at least about 95% sequence identity, or (b) a complement of the DNA of (a). . In a further aspect, the invention provides a test DNA molecule comprising (a) PRO3 having amino acid residue 1 of Fig16 (SEQ ID NO: 22) or a sequence from about 17 to about 1029.
434 polypeptide-encoding DNA molecule, or (b) hybridized to the complement of the DNA molecule of (a) under stringent conditions, whereby the DNA molecule (a) or (b)
To at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. To an isolated nucleic acid molecule having at least about 460 nucleotides produced by isolating a test DNA molecule. In a particular embodiment, the invention comprises a DNA encoding a PRO3434 polypeptide with or without an N-terminal signal sequence and / or an initiation methionine, or which is complementary to such an encoding nucleic acid molecule. Provide a nucleic acid molecule that is separated. The signal peptide has been tentatively identified as extending from amino acid position 1 to about amino acid position 16 in the sequence of Figure 16 (SEQ ID NO: 22). In another aspect, the invention provides (a) at least about 80% positive, preferably at least about 85% positive when compared to the amino acid sequence of residue 1 or about 17 to about 1029 of Figure 16 (SEQ ID NO: 22). , More preferably at least about 90% positive, most preferably at least about 95% positive, DNA encoding a polypeptide, or (b) an isolated nucleic acid molecule comprising the complement of DNA of (a). Another embodiment is a PR that finds use as a hybridization probe.
It relates to a fragment of the O3434 polypeptide coding sequence. Such a nucleic acid fragment has a length of about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides,
More preferably about 20 to about 50 nucleotides in length, and most preferably about 20 to about 40 nucleotides in length. In another embodiment, the invention provides an isolated PRO3434 polypeptide encoded by any of the isolated nucleic acid sequences identified above. In a particular aspect, the invention provides an isolated native sequence PRO3434 polypeptide, which in certain embodiments comprises amino acid residues 1 or about 17 to about 1029 of Figure 16 (SEQ ID NO: 22). Contains an array.

In another aspect, the invention features at least about 80% sequence identity, preferably at least about 85%, to amino acid residue 1 of Figure 16 (SEQ ID NO: 22) or the sequence from about 17 to about 1029. It relates to an isolated PRO3434 polypeptide comprising an amino acid sequence having a% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. In a further aspect, the invention features residue 1 or about 1 of Figure 16 (SEQ ID NO: 22).
At least about 80% positive, preferably at least about 85% positive, more preferably at least about 90 when compared to the amino acid sequence from 7 to 1029.
% PROPOSITIVE, most preferably at least about 95% positive score, with respect to an isolated PRO3434 polypeptide. In yet another aspect, the invention provides amino acid residue 1 of Figure 16 (SEQ ID NO: 22).
Or about 17 to about 1029 sequences, or a fragment thereof sufficient to provide a binding site for an anti-PRO3434 antibody. Preferably, the PRO982 fragment retains the qualitative biological activity of the native PRO3434 polypeptide. In a still further aspect, the invention provides (i) a test DNA molecule, (a) Fig1.
A DNA molecule encoding a PRO3434 polypeptide having the sequence of about 1 or about 17 to about 1029 amino acid residues of SEQ ID NO: 6 (SEQ ID NO: 22), or D of (b) (a)
Hybridization with the complement of the NA molecule under stringent conditions allows the test DNA molecule to
at least about 80% sequence identity to a) or (b), preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity,
Most preferably, (ii) culturing a host cell containing a test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) culturing the polypeptide from the cell culture medium, provided that it has at least about 95% sequence identity. A polypeptide produced by recovering is provided. In yet another embodiment, the invention relates to agonists and antagonists of the native PRO3434 polypeptide. In a particular embodiment, the agonist or antagonist is an anti-PRO3434 antibody. In a further embodiment, the invention relates to a method of identifying an agonist or antagonist of a native PRO3434 polypeptide by contacting the native PRO3434 polypeptide with a candidate molecule and monitoring the biological activity mediated by said polypeptide. In a yet further embodiment, the invention relates to a composition comprising a PRO3434 polypeptide, or an agonist or antagonist identified above, together with a pharmaceutically acceptable carrier.

9. PRO1927 In the present application, designated as "PRO1927", is a cDNA clone encoding a novel polypeptide having sequence identity with glycosyltransferase (D
NA82307-2531) was identified. In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1927 polypeptide. In one aspect, the isolated nucleic acid is (a) a DNA molecule that encodes a PRO1927 polypeptide having amino acid residue 1 of Figure 18 (SEQ ID NO: 24) or a sequence of about 24 to about 548, or (b) ( At least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% to the complement of the DNA molecule of a).
It comprises DNA with% sequence identity. In another aspect, the invention features PRO1927 that comprises a DNA that hybridizes to the complement of a nucleic acid between residues about 120 or about 1694 of Figure 17 (SEQ ID NO: 23).
It relates to an isolated nucleic acid molecule encoding a polypeptide. Preferably, hybridization occurs under stringent hybridization and wash conditions. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA (DNA82307-2531) of ATCC Deposit No. 203537, or (b) the complement of the DNA molecule of (a). Against
Comprises DNA having at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. It relates to an isolated nucleic acid molecule. In a preferred embodiment, the nucleic acid comprises a DNA encoding the same mature polypeptide encoded by the human protein cDNA (DNA82307-2531) of ATCC Deposit No. 203537. In a still further aspect, the invention provides (a) at least about 80% sequence identity, preferably at least about 85% sequence identity with the sequence of about 1 or about 24 to about 548 amino acid residues of Figure 18 (SEQ ID NO: 24). Sequence identity, more preferably at least about 9
It relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide having 0% sequence identity, most preferably at least about 95% sequence identity, or the complement of DNA of (a).

In a further aspect, the invention provides a test DNA molecule comprising: (a) PRO19 having amino acid residue 1 of Fig18 (SEQ ID NO: 24) or a sequence from about 24 to about 548.
A DNA molecule encoding a 27 polypeptide, or (b) a complement of the DNA molecule of (a), under hybridizing conditions, wherein the DNA molecule is at least about 80 relative to (a) or (b). Isolating a test DNA molecule when it has a% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. At least about 50 nucleotides, preferably at least about 10
It relates to an isolated nucleic acid molecule having 0 nucleotides. In a particular embodiment, the invention provides a PRO1927 polypeptide with or without an N-terminal signal sequence and / or an initiation methionine, and soluble variants thereof (ie transmembrane domains deleted or inactivated). An isolated nucleic acid molecule comprising DNA encoding, or complementary to such an encoding nucleic acid molecule. The signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 23 in the sequence of Figure 18 (SEQ ID NO: 24). I
The type I transmembrane domain has a PRO1927 amino acid sequence (Fig18, SEQ ID NO:
It has been tentatively identified as extending from about amino acid position 6 to about amino acid position 25 in 24). In another aspect, the invention provides (a) at least about 80% positive, preferably at least about 85% positive when compared to the amino acid sequence of residue 1 or about 24 to about 548 of Figure 18 (SEQ ID NO: 24). , More preferably at least about 9
It relates to an isolated nucleic acid molecule comprising 0% positive, most preferably at least about 95% positive score DNA encoding a polypeptide, or (b) (a) the complement of DNA. Another embodiment is a PR that finds use as a hybridization probe.
O1927 polypeptide coding sequence fragment. Such a nucleic acid fragment has a length of about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides,
More preferably about 20 to about 50 nucleotides in length, and most preferably about 20 to about 40 nucleotides in length. In another embodiment, the invention provides an isolated PRO1927 polypeptide encoded by any of the isolated nucleic acid sequences identified above. In a particular aspect, the invention provides an isolated native sequence PRO1927 polypeptide, which in certain embodiments comprises residue 1 of Figure 18 (SEQ ID NO: 24) or an amino acid comprising from about 24 to about 548. Contains an array. In another aspect, the invention features at least about 80% sequence identity, preferably at least about 85% sequence to amino acid residue 1 of Figure 18 (SEQ ID NO: 24) or the sequence from about 24 to about 548. It relates to an isolated PRO1927 polypeptide comprising an amino acid sequence having identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity. In a further aspect, the invention features residue 1 or about 2 of Figure 18 (SEQ ID NO: 24).
At least about 80% positive, preferably at least about 85% positive, more preferably at least about 90 when compared to the amino acid sequence from 4 to about 548.
It relates to an isolated PRO1927 polypeptide which comprises an amino acid sequence with a score of% positive, most preferably at least about 95% positive. In yet another aspect, the invention provides amino acid residue 1 of Figure 18 (SEQ ID NO: 24).
Or about 24 to about 548 sequences, or a fragment thereof sufficient to provide a binding site for an anti-PRO1927 antibody. Preferably, the PRO1927 fragment retains the qualitative biological activity of the native PRO1927 polypeptide.

In a still further aspect, the invention provides (i) a test DNA molecule, (a) Fig1.
P having the amino acid residue 1 of 8 (SEQ ID NO: 24) or a sequence of about 24 to about 548
DNA molecule encoding RO1927 polypeptide, or DNA of (b) (a)
The test DNA molecule is hybridized to the complement of the molecule under stringent conditions,
Or to (b) at least about 80% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity. (Ii) test DN if
A host cell containing the A molecule is cultured under conditions suitable for expression of the polypeptide, and (
iii) providing a polypeptide produced by recovering the polypeptide from the cell culture medium. In yet another embodiment, the invention relates to agonists and antagonists of native PRO1927 polypeptides. In a particular embodiment, the agonist or antagonist is an anti-PRO1927 antibody. In a further embodiment, the invention relates to a method of identifying an agonist or antagonist of a native PRO1927 polypeptide by contacting the native PRO1927 polypeptide with a candidate molecule and monitoring the biological activity mediated by said polypeptide. In a still further embodiment, the invention relates to a composition comprising a PRO1927 polypeptide, or an agonist or antagonist identified above, together with a pharmaceutically acceptable carrier.

10. Further Embodiments In another embodiment of the present invention, the invention provides a vector comprising DNA encoding any of the polypeptides described herein. Host cells comprising such vectors are also provided. For example, the host cell is a CHO cell,
It can be E. coli or yeast. Further provided is a method for producing any of the polypeptides described herein, which comprises culturing a host cell under conditions suitable for expression of the desired polypeptide, and removing the desired polypeptide from the cell culture. Comprising recovering. In another embodiment, the invention provides a chimeric molecule comprising any of the polypeptides described herein fused to a heterologous polypeptide or amino acid sequence.
Examples of such chimeric molecules include any of the polypeptides described herein fused to an immunoglobulin Fc region or epitope tag sequence. In another embodiment, the invention provides an antibody that specifically binds to any of the above or below described polypeptides. In some cases, the antibody is a monoclonal antibody,
It is a humanized antibody, an antibody fragment or a single chain antibody. In yet another embodiment, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences, wherein the probes are derived from any of the above or below nucleotide sequences. sell. In another embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide. In one aspect, the isolated nucleic acid molecule is (a) a full length amino acid sequence disclosed herein, an amino acid sequence lacking a signal peptide disclosed herein, or a cell of a transmembrane protein disclosed herein with or without a signal peptide. A DNA molecule encoding a PRO polypeptide having an ectodomain, or (b) at least about 80% sequence identity to the complement of the DNA molecule of (a), preferably at least about 81% sequence identity; Even more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84%.
Sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably at least about 89% sequence identity, even more preferably at least about 90% acid sequence identity, even more preferably at least about 91% sequence identity, even more preferably. Is at least about 92% sequence identity, even more preferably at least about 93% sequence identity, even more preferably at least about 94% sequence identity, even more preferably at least about 95% sequence identity, More preferably at least about 96% sequence identity, and even more preferably at least about 9
It comprises a nucleotide sequence having 7% sequence identity, even more preferably at least about 98% sequence identity, and even more preferably at least about 99% sequence identity.

In another aspect, the isolated nucleic acid molecule comprises (a) a coding sequence for a full-length PRO polypeptide cDNA disclosed herein, a coding sequence for a PRO polypeptide lacking the signal peptide disclosed herein. Or at least about 80 relative to the coding sequence of the extracellular domain of a transmembrane PRO polypeptide disclosed herein with or without a signal peptide, or (b) (a) the complement of a DNA molecule. %
Sequence identity, preferably at least about 81% sequence identity, even more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84%. Of sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 8
6% sequence identity, even more preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably at least about 89% sequence identity, even more preferably At least about 90% acid sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity, even more preferably at least about 93% sequence identity, More preferably at least about 94% sequence identity, even more preferably at least about 95% sequence identity, even more preferably at least about 96.
Nucleotides having a% sequence identity, even more preferably at least about 97% sequence identity, even more preferably at least about 98% sequence identity, even more preferably at least about 99% sequence identity. Comprises an array. In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATTC disclosed herein, or (b) of (a) At least about 80% sequence identity to the complement of the DNA molecule, preferably at least about 81% sequence identity, even more preferably at least about 82% sequence identity, even more preferably at least about 83%. Of sequence identity, even more preferably at least about 84% sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably at least about 8 % Sequence identity, even more preferably at least about 90
% Acid sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity, even more preferably at least about 93% sequence identity, even more preferably At least about 94% sequence identity, even more preferably at least about 95% sequence identity, even more preferably at least about 96% sequence identity, even more preferably at least about 97% sequence identity, even more Preferably it relates to an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 98% sequence identity, and even more preferably at least about 99% sequence identity. Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide having a transmembrane domain deleted or transmembrane domain inactivated, or Disclosed herein are the transmembrane domains of such polypeptides, which are complementary to such encoding nucleotide sequences. Therefore, the soluble extracellular domain of the PRO polypeptides described herein is considered.

Another embodiment relates to a PRO polypeptide coding sequence, or the complement thereof, which encodes a PRO polypeptide, which for example may optionally code for a polypeptide that constitutes the binding site of the anti-PRO antibody. The fragment may find use as a hybridization probe or antisense oligonucleotide probe. Such nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, even more preferably at least about 50 nucleotides in length, even more preferably at least about 30 nucleotides in length. 60 nucleotides in length, even more preferably at least about 70 nucleotides in length, even more preferably at least about 80 nucleotides in length, even more preferably at least about 90 nucleotides in length, even more preferably at least about 100 nucleotides in length, More preferably at least about 1
10 nucleotides long, even more preferably at least about 120 nucleotides long, even more preferably at least about 130 nucleotides long, even more preferably at least about 140 nucleotides long, and even more preferably at least about 1 nucleotide long.
50 nucleotides long, even more preferably at least about 160 nucleotides long, even more preferably at least about 170 nucleotides long, even more preferably at least about 180 nucleotides long, and even more preferably at least about 1 nucleotide long.
90 nucleotides long, even more preferably at least about 200 nucleotides long, even more preferably at least about 250 nucleotides long, even more preferably at least about 300 nucleotides long, and even more preferably at least about 3 nucleotides long.
50 nucleotides long, even more preferably at least about 400 nucleotides long, even more preferably at least about 450 nucleotides long, even more preferably at least about 500 nucleotides long, and even more preferably at least about 6 nucleotides long.
00 nucleotides in length, even more preferably at least about 700 nucleotides in length, even more preferably at least about 800 nucleotides in length, even more preferably at least about 900 nucleotides in length, even more preferably at least about 1 nucleotides in length.
000 nucleotides in length, where the term "about" means a reference nucleotide sequence length that is plus or minus 10% of its reference length. Novel fragments of PRO polypeptide-encoding nucleotide sequences can be prepared by aligning a PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs to determine which PRO It is noted that the peptide-encoding nucleotide sequence fragment can be routinely determined by determining whether it is novel. All such PRO polypeptide-encoding nucleotide sequences are considered herein. Also considered are PRO polypeptide fragments encoded by these nucleotide molecule fragments,
Preferred is a PRO polypeptide fragment comprising a binding site for anti-PRO antibody. In another embodiment, the invention provides an isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences identified above.

In one aspect, the invention provides a full-length amino acid sequence disclosed herein, a full-length amino acid sequence lacking a signal peptide disclosed herein, or an extracellular domain of a transmembrane protein disclosed herein with or without a signal peptide. Having at least about 80% sequence identity, preferably at least about 81% sequence identity, even more preferably at least about 82% sequence identity, and even more preferably at least about 83%. Sequence identity, even more preferably at least about 84
% Sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity, even more preferably at least About 88% sequence identity,
Even more preferably at least about 89% sequence identity, even more preferably at least about 90% acid sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity. Identity, even more preferably at least about 93% sequence identity, even more preferably at least about 94%.
Of sequence identity, even more preferably at least about 95% sequence identity, even more preferably at least about 96% sequence identity, even more preferably at least about 97% sequence identity, even more preferably at least about It relates to an isolated PRO polypeptide comprising an amino acid sequence having 98% sequence identity, and even more preferably at least about 99% sequence identity. In a further aspect, the invention provides at least about 80% sequence identity, preferably at least about 81%, to the amino acid sequence encoded by any of the human protein cDNAs deposited with the ATTC disclosed herein. Of sequence identity, even more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84% sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably At least about 89% sequence identity, and even more preferably at least about 90
% Acid sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity, even more preferably at least about 93% sequence identity, even more preferably At least about 94% sequence identity, even more preferably at least about 95% sequence identity, even more preferably at least about 96% sequence identity, even more preferably at least about 97% sequence identity, even more Preferably it relates to an isolated PRO polypeptide comprising an amino acid sequence having at least about 98% sequence identity, and even more preferably at least about 99% sequence identity.

In a further aspect, the invention provides a full-length amino acid sequence disclosed herein, a full-length amino acid sequence lacking a signal peptide disclosed herein, or the extracellular domain of a transmembrane protein disclosed herein with or without a signal peptide. At least about 80% positive, preferably at least about 81% positive, more preferably at least about 82% positive, even more preferably at least about 83% positive, when compared to the amino acid sequence of a PRO polypeptide having: Even more preferably at least about 84% positive, even more preferably at least about 85%.
% Positive, even more preferably at least about 86% positive, even more preferably at least about 87% positive, even more preferably at least about 88% positive, even more preferably at least about 89% positive,
Even more preferably at least about 90% positive, even more preferably at least about 91% positive, even more preferably at least about 92% positive, even more preferably at least about 93% positive, even more preferably at least about. 94% positive, even more preferably at least about 95% positive, even more preferably at least about 96% positive, even more preferably at least about 97% positive, even more preferably at least about 9%.
It relates to an isolated PRO polypeptide comprising an amino acid sequence showing a score of 8% positive, and even more preferably at least about 99% positive. In a particular aspect, the invention provides an isolated PRO polypeptide lacking an N-terminal signal sequence and / or an initiation methionine, which is encoded by a nucleotide sequence encoding such an amino acid sequence described above. Methods of producing this are also described herein, wherein these methods culture host cells comprising a vector containing a suitable encoding nucleic acid molecule under conditions suitable for the expression of the PRO polypeptide, the cells Comprising recovering the PRO polypeptide from the medium.

Another aspect of the invention provides an isolated PRO polypeptide having a transmembrane domain deleted or transmembrane domain inactivated. Also described herein are methods of producing this, wherein the methods culture host cells comprising a vector containing a suitable encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide,
Comprising recovering the PRO polypeptide from the cell culture medium. In yet another embodiment, the present invention relates to agonists and antagonists of the native PRO polypeptides identified herein. In a particular embodiment, the agonist or antagonist is an anti-PRO antibody or small molecule. In a further embodiment, the invention provides a method of identifying an agonist or antagonist to a PRO polypeptide, comprising contacting the PRO polypeptide with a candidate molecule and monitoring the biological activity mediated by said PRO polypeptide. Regarding Preferably, the PRO polypeptide is a native PRO polypeptide. In a still further embodiment, the invention relates to a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as described herein, or an anti-PRO antibody together with a carrier. In some cases, the carrier is a pharmaceutically acceptable carrier. Another embodiment of the invention is a PRO polypeptide, or agonist thereof, for the preparation of a medicament useful in the treatment of a condition responsive to a PRO polypeptide, agonist or antagonist thereof or anti-PRO antibody as described above. It relates to the use of antagonists or anti-PRO antibodies.

Detailed Description of the Preferred Embodiments I. Definitions The terms "PRO polypeptide" and "PRO" as used herein refer to
Immediately after is a numerical symbol, which refers to various polypeptides, including the complete symbol (eg,
(PRO / number) refers to the particular polypeptide sequence described herein. As used herein, the terms “PRO / number polypeptide” and “PRO / number”, where “number” is given as the actual numerical symbol used herein, refer to native sequence polypeptides and variants (here Will be defined in more detail in). P described here
RO polypeptides may be isolated from a variety of sources, such as human tissue types or other sources, and may be prepared by recombinant or synthetic methods. A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide of natural origin. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term “native sequence PRO polypeptide” specifically refers to naturally occurring truncated or secreted forms of a particular PRO polypeptide (eg,
Extracellular domain sequences), naturally-occurring mutant forms (eg, alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In various embodiments of the invention, the native sequence PRO polypeptide is a mature or full length native sequence polypeptide comprising the full length amino acid sequence shown in the accompanying figures. Start and stop codons are shown in bold or underlined in the figure. However, the PRO polypeptide disclosed in the accompanying figures is shown to begin with a methionine residue, designated herein as amino acid position 1 in the figures, although it may be located upstream or downstream of amino acid position 1 in the figures. It is conceivable and possible to use the methionine residue of E. coli as the starting amino acid residue of the PRO polypeptide. The PRO polypeptide "extracellular domain" or "ECD" means a form of the PRO polypeptide that is substantially free of transmembrane and cytoplasmic domains. Usually PR
O polypeptides ECD have less than 1% of their transmembrane and / or cytoplasmic domains, preferably less than 0.5% of such domains. PRO of the present invention
It will be appreciated that any transmembrane domain identified for a polypeptide will be identified according to the criteria routinely used in the art to identify that type of hydrophobic domain. The exact boundaries of the transmembrane domain may vary, but are likely not to exceed about 5 amino acids from either end of the first identified domain. Thus, a PRO polypeptide extracellular domain optionally comprises no more than about 5 amino acids from either side of the transmembrane domain and / or extracellular domain boundaries as identified in the Examples or specification. However, those polypeptides and their encoding nucleic acids, with or without a signal peptide, are contemplated by the present invention.

The approximate location of the "signal peptides" of the various PRO polypeptides disclosed herein are shown in the accompanying figures. However, as noted, the C-terminal boundary of the signal peptide may vary, but is most likely to be less than about 5 amino acids on either side of the signal peptide C-terminal boundary as originally defined herein. High, the C-terminal boundary of the signal peptide can be identified according to the criteria routinely used to identify such types of amino acid sequence components (eg, Nielsen et al., Prot. Eng. 10: 1-
6 (1997) and von Heinje et al., Nucl. Acids. Res. 14: 4683-4690 (1986)). Furthermore, it is also recognized that in some cases cleavage of the signal peptide from the secreted polypeptide is not completely homogeneous, resulting in one or more secreted species. These mature polypeptides, and the polynucleotides encoding them, in which the signal peptide is cleaved within less than about 5 amino acids on either side of the C-terminal border of the signal peptide as defined herein, are contemplated by the present invention. It "PRO polypeptide variant" refers to a full length native sequence PRO polypeptide disclosed herein, a full length native sequence PRO polypeptide sequence lacking the signal peptide disclosed herein, a signal, as defined above or below. Active PR having at least about 80% amino acid sequence identity with the extracellular domain of PRO disclosed herein with or without peptides or other fragments of the full-length PRO polypeptides disclosed herein.
O polypeptide. Such PRO polypeptide variants include, for example, PRO polypeptides in which one or more amino acid residues have been added or deleted at the N- or C-terminus of the full-length native amino acid sequence. Generally, a PRO polypeptide variant is a full length native amino acid sequence disclosed herein, a full length native sequence PRO polypeptide sequence lacking the signal peptide disclosed herein, the extracellular domain of a PRO disclosed herein with or without a signal peptide. Or the full length PR disclosed herein
At least about 80% amino acid sequence identity with the other fragment of the O polypeptide, 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 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 Is at least about 91% amino acid sequence identity, more preferred Or 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 more preferably at least about 99%. Have amino acid sequence identity. Generally, a PRO variant polypeptide is at least about 10 amino acids in length, more at least about 20 amino acids in length, more at least about 30 amino acids in length, more at least about 40 amino acids in length, more at least about 50 amino acids in length. Long, more at least about 60 amino acids in length, more at least about 70 amino acids in length, more at least about 80 amino acids in length,
More than at least about 90 amino acids long, more than at least about 100 amino acids long, more at least about 150 amino acids long, and more at least about 20 amino acids long.
It is 0 amino acids long, more than at least about 300 amino acids long, or more.

“Percent (%) amino acid sequence identity”, as identified herein, relative to a PRO polypeptide as defined herein refers to the alignment of the sequences and if necessary to obtain maximum percent sequence identity. It is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the PRO polypeptide, with the introduction of gaps and without considering any conservative substitutions as part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be made by a variety of methods within the skill of the art, including publicly available BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. This can be achieved by using computer software. One of ordinary skill in the art can determine the appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes herein,% amino acid sequence identity values can also be obtained using the sequence comparison program ALIGN-2, the complete source code for which is given in Table 1 below. To be ALI
The GN-2 sequence comparison computer program was written by Genentech, Inc., and the source code shown in Table 1 below has been submitted to the US Copyright Office, Washington DC, 20559 with user documentation and is under US Copyright Registration Number TXU510087. It is registered in. The ALIGN-2 program is suitably available from Genentech, Inc., South San Francisco, California and may be compiled from the source code given in Table 1 below. The ALIGN-2 program is compiled for use with a UNIX® operating system, preferably Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. In the situation where ALIGN-2 is used for amino acid sequence comparisons, the% amino acid sequence identity of a given amino acid sequence A with, or with respect to a given amino acid sequence B (or with a given amino acid sequence B, or It can also be said to be a given amino acid sequence A which has or contains a certain percentage of amino acid sequence identity).
Is calculated as: 100 times the fraction X / Y, where X is the number of amino acid residues in the score that are matched by the alignment of A and B of the sequence alignment program ALIGN-2. , Y is the total number of amino acid residues of B. It will be appreciated that when the length of amino acid sequence A differs from the length of amino acid sequence B, the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A. As an example of calculating% amino acid sequence identity using this method, Tables 2 and 3 show the calculation of% amino acid sequence identity of the amino acid sequence referred to as "comparative protein" to the amino acid sequence referred to as "PRO". In the method, “PRO” means the amino acid sequence of the provisional PRO polypeptide of interest, and “comparative protein” means the amino acid of the polypeptide factor of the “PRO” polypeptide of interest. Means an array, "X", "Y",
“Z” means different temporary amino acid sequences.

Unless otherwise stated, all% amino acid sequence identity values used herein are obtained using the WU-BLAST-2 computer program as described in the immediately preceding paragraph. However, the% amino acid sequence identity value is, as described below,
WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:
460-480 (1996)). Most WU-BLAST-2 search parameters are set to default values. Not set to default values, ie adjustable parameters set to the following values: overlap span = 1, overlap fraction = 0.
125, word threshold (T) = 11, and scoring matrix = BLOSUM62. WU
-When using BLAST-2, the% amino acid sequence identity value is (a) an amino acid sequence of a PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and a comparative amino acid sequence of interest (ie, , A sequence which may be a PRO polypeptide variant with which the subject PRO polypeptide is compared), the number of matching identical amino acid residues is (b) the amino acid residue of the subject PRO polypeptide. It is determined by the quotient divided by the total number of radicals. For example, "Amino acid sequence B and at least 80
In the description, "an isolated amino acid sequence containing an amino acid sequence having a% amino acid sequence identity," amino acid sequence A is a target comparative amino acid sequence and amino acid sequence B is the target PRO polypeptide amino acid sequence. . In addition,% amino acid sequence identity is determined by the sequence comparison program NCBI-BLAST2 (Altschul
Et al., Nucleic Acids Res. 25: 3389-3402 (1997)). NCB
The I-BLAST2 sequence comparison program can be downloaded from http://ww.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, all of which are set to initial values, eg unmask = ok, chains = all, expected occurrences = 10, minimum low compound length = 15/5 , Multipath e-value = 0.01, multipath constant = 25, final gap alignment dropoff = 25, and scoring matrix = BLOSUM62. In the situation where NCBI-BLAST2 is used for amino acid sequence comparisons, the% amino acid sequence identity of a given amino acid sequence A with or to a given amino acid sequence B (or with a given amino acid sequence B, or On the other hand, a certain percentage
A given amino acid sequence A with or containing amino acid sequence identity may also be calculated) as follows: 100 times the fraction X / Y, where X is A in the sequence alignment program NCBI-BLAST2. And the number of amino acid residues in the score that were matched by the alignment of B to be identical, where Y is B
Is the total number of amino acid residues. It will be appreciated that when the length of amino acid sequence A differs from the length of amino acid sequence B, the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A.

A “PRO variant polynucleotide” or “PRO variant nucleic acid sequence” is a nucleic acid molecule that encodes an active PRO polypeptide, as defined below, which is a full-length native sequence PRO poly disclosed herein. A peptide sequence, a full length native sequence PRO polypeptide sequence lacking the signal peptide disclosed herein, an extracellular domain of a PRO polypeptide disclosed herein with or without a signal peptide, or any other of the full length PRO polypeptide sequences disclosed herein. A nucleic acid sequence encoding a fragment of at least 8
It has 0% sequence identity. Generally, a PRO variant polypeptide nucleotide is a full length native sequence PRO polypeptide sequence disclosed herein, a full length native sequence PRO polypeptide sequence lacking the signal peptide disclosed herein, a PRO polypeptide disclosed herein with or without a signal peptide. At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity with the nucleic acid sequence encoding the extracellular domain of the peptide, or any other fragment of the full-length PRO polypeptide disclosed herein.
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. Sex, 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 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 It has at least about 98% nucleic acid sequence identity, and more preferably at least about 99% nucleic acid sequence identity. Variants do not include native nucleotide sequences. Generally, a PRO variant polynucleotide is at least about 30 nucleotides long, more at least about 60 nucleotides long, more at least about 90 nucleotides long, more at least about 120 nucleotides long, more at least about 150 nucleotides long. Long, more often at least about 180 nucleotides, more at least about 210 nucleotides, and more at least about 24
0 nucleotides, more at least about 270 nucleotides, more at least about 300 nucleotides, more at least about 450 nucleotides, more at least about 600 nucleotides, more at least about 900 nucleotides, or More than that.

“Percent (%) nucleic acid sequence identity” to a PRO-encoded nucleic acid sequence identified herein means that the PRO sequences are aligned, introducing gaps if necessary to obtain maximal percent sequence identity. Defined as the percentage of nucleotides in the candidate sequence that are identical to the nucleotides in the sequence. Alignments for the purpose of determining percent nucleic acid sequence identity can be performed by a variety of methods within the purview of those skilled in the art, such as BLAS.
All% nucleic acid sequence identity as used herein can be achieved using publicly available computer software such as T, BLAST-2, ALIGN or Megalign (DNASTAR) software unless otherwise stated. Values are obtained using the WU-BLAST-2 computer program as described in the paragraph immediately above. However, the% nucleic acid sequence identity values are shown in the sequence comparison program provided with the complete source code for the ALIGN-2 program in Figure 248A-Q, as described below.
It can also be obtained using ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code shown in Figure 248A-Q is submitted to the US Copyright Office, Washington DC, 20559 with user documentation and under US Copyright Registration No. TXU510087. It is registered in. The ALIGN-2 program is conveniently available from Genentech, Inc., South San Francisco, California and Fi
It may be compiled from the source code given to g248A-Q. The ALIGN-2 program is compiled for use with a UNIX operating system, preferably Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. In the context where ALIGN-2 is used for nucleic acid sequence comparisons, the% nucleic acid sequence identity of a given nucleic acid sequence C to, or to a given nucleic acid sequence D (or with a given nucleic acid sequence D, or A given nucleic acid sequence C with or to which it has a certain% nucleic acid sequence identity can also be calculated) as: 100 times the fraction W / Z, where W is The number of nucleotides in the score that were matched as identical by the C and D alignment of the sequence alignment program ALIGN-2, and Z is the total number of nucleotides in D. When the length of the nucleic acid sequence C is different from the length of the nucleic acid sequence D, C
It will be appreciated that the% nucleic acid sequence identity of D to C is different from the% nucleic acid sequence identity of D to C. As an example of calculating% nucleic acid sequence identity, Tables 4 and 5 show
A method for calculating the% nucleic acid sequence identity of a nucleic acid sequence referred to as “comparative DNA” with a nucleic acid sequence referred to as “PRO-DNA” is shown, where “PRO-DNA” is the provisional target By "comparative DNA" is meant a PRO-encoding nucleic acid sequence, and by "comparative DNA" is meant the nucleotide sequence of a nucleic acid molecule factor that is compared to the "PRO-DNA" nucleic acid molecule of interest, "N", "L" and "V". "Means different temporary nucleotide sequences.

Unless otherwise specified, all% amino acid sequence identity values used herein are obtained using the WU-BLAST-2 computer program as described in the immediately preceding paragraph. However, the% amino acid sequence identity value is, as described below,
WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:
460-480 (1996)). Most WU-BLAST-2 search parameters are set to default values. Not set to default values, ie adjustable parameters set to the following values: overlap span = 1, overlap fraction = 0.
125, word threshold (T) = 11, and scoring matrix = BLOSUM62. WU
-When using BLAST-2, the% nucleic acid sequence identity value is: (a) the nucleic acid sequence of the PRO polypeptide encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide encoding nucleic acid, Comparative nucleic acid sequence of interest (ie PR of interest)
The sequence of identical nucleotides between the O polypeptide-encoding nucleic acid molecule and the sequence that may be the mutated PRO polypeptide with which it is compared (b) the P of interest.
Determined by the quotient divided by the total number of residues of the RO polypeptide. For example, in the description "an isolated nucleic acid sequence comprising a nucleic acid sequence having at least 80% nucleic acid sequence identity with nucleic acid sequence B", nucleic acid sequence A is the target comparison nucleic acid sequence and nucleic acid sequence B is the target nucleic acid sequence. Is a PRO polypeptide-encoding nucleic acid sequence. In addition,% nucleic acid sequence identity is determined by the sequence comparison program NCBI-BLAST2 (Altschul et al., N.
Nucleic Acids Res. 25: 3389-3402 (1997)). NCBI-BLA
The ST2 sequence comparison program can be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, all of which are set to initial values, eg unmask = ok, chains = all, expected occurrence = 1
Includes 0, minimum low composite length = 15/5, multipath e-value = 0.01, multipath constant = 25, final gap alignment dropoff = 25, and scoring matrix = BLOSUM62. In the context where NCBI-BLAST2 is used for nucleic acid sequence comparisons, the% nucleic acid sequence identity of a given nucleic acid sequence C with or to a given nucleic acid sequence D (or,
A given nucleic acid sequence D, or also a given nucleic acid sequence C with or containing some% nucleic acid sequence identity thereto) can be calculated as follows:
Fraction W / Z 100 times where W is the number of nucleic acid residues with a score that is consistent with the C and D alignments of the sequence alignment program NCBI-BLAST2, and Z is the total nucleic acid residues of D. Is a number. When the length of the nucleic acid sequence C is different from the length of the nucleic acid sequence D, D of C
It will be appreciated that the% nucleic acid sequence identity to C is different from the% nucleic acid sequence identity of D to C. In another embodiment, the PRO variant polypeptide nucleotide encodes an active PRO polypeptide, preferably under stringent hybridization and wash conditions.
Nucleic acid molecules that hybridize to the nucleotide sequences encoding the full-length PRO polypeptides disclosed herein. The PRO variant polypeptide may be one encoded by the PRO variant polynucleotide.

The term “positive” refers to residues in sequence comparisons performed as described above that are not identical but have similar properties in the compared sequences (eg, conservative). As a result of the substitution, see Table 6 below). For purposes herein, the% positive values are (a) the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparative amino acid sequence of interest (
That is, the amino acid sequence with which the PRO polypeptide sequence is compared)
The number of amino acid residues with a positive value in the BLOSUM62 matrix of -2,
(B) Determined by the quotient divided by the total number of amino acid residues in the PRO polypeptide of interest. Unless otherwise stated, positive% values are calculated as described in the paragraph immediately above. Obtained using the WU-BLAST-2 computer program. However, the amino acid sequence identities performed as described above for ALIGN-2 and NCBI-BLAST2 include amino acid residues in sequences that have similar as well as similar properties.
Amino acid residues that are positive for the target amino acid residue are the same as the target amino acid residue or are preferred substitutions of the target amino acid residue (defined in Table 6 below). It is a thing. In the amino acid sequence comparison using ALIGN-2 or NCBI-BLAST2, the positive% value of the given amino acid sequence A with or against the given amino acid sequence B (or with the given amino acid sequence B, Or a given amino acid sequence A which has or contains some% amino acid sequence identity thereto) is calculated as: 100 times the fraction X / Y, where X Is A and B of the sequence alignment program ALIGN-2 or NCBI-BLAST2
Is the number of amino acid residues in the score that are considered positive by the alignment, and Y is the total number of amino acid residues in B. It will be appreciated that if the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, then the% positive for A to B is different from the% positive for B to A.

“Isolated,” when used to describe the various polypeptides disclosed herein, refers to polypeptides that have been identified, separated and / or recovered from components of their natural environment. means. Contaminant components of its natural environment are materials that typically interfere with diagnostic or therapeutic uses for the polypeptide, and include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide is (1) sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a spinning cup sequenator, or (2) Coomassie blue or It is preferably purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using silver staining. Isolated polypeptide includes protein in situ within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step. An "isolated" PRO polypeptide-encoding nucleic acid is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule that is normally associated with the natural source of the nucleic acid encoding the PRO polypeptide. An isolated PRO polypeptide-encoding nucleic acid molecule is in a form or setting other than that found in nature. Thus, an isolated PRO polypeptide-encoding nucleic acid molecule can be the PRO that is present in native cells.
Distinct from the polypeptide-encoding nucleic acid molecule. However, an isolated PRO polypeptide-encoding nucleic acid molecule includes, for example, a PRO polypeptide nucleic acid molecule contained in a cell that normally expresses the PRO polypeptide in a chromosomal location where the nucleic acid molecule differs from that of the native cell. . The expression "control sequences" refers to DNA sequences necessary to express an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, 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 sequence. For example, if the DNA of the presequence or secretory leader is expressed as a preprotein that participates in the secretion of the polypeptide, the DNA of that polypeptide
Is operably linked to; a promoter or enhancer is operably linked to the coding sequence if it affects the transcription of the sequence; or the ribosome binding site is such that it facilitates translation. Is operably linked to the coding sequence. Generally, "operably linked" means that D is bound to
It means that the NA sequences are contiguous and, in the case of a secretory leader, contiguous and in the reading phase. However, enhancers do not necessarily have to be in close proximity. Binding is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adapters or linkers are used in accord with conventional practice.

The term “antibody” is used in the broadest sense, eg, a single anti-P
RO polypeptide monoclonal antibody (including agonist, antagonist, and neutralizing antibody), anti-PRO antibody composition with multi-epitope specificity, single chain anti-PR
O antibodies, and fragments of anti-PRO antibodies are included (see below). The term "monoclonal antibody" as used herein is a substantially homogeneous population of antibodies, ie, the individual antibodies that comprise them, are identical except for the naturally occurring mutations that may be present in small amounts. Refers to the antibody obtained from the population. The "stringency" of a hybridization reaction is readily determined by one of ordinary skill in the art and is an empirical calculation that generally depends on probe length, wash temperature, and salt concentration. In general, longer probes have higher temperatures for proper annealing and shorter probes have lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment near, but below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures make the reaction conditions more stringent, while lower temperatures reduce stringency.
Moreover, stringency is inversely proportional to salt concentration. Further details and explanations of the stringency of the hybridization reaction can be found in Ausubel et al., Current Protocols in Molecular Biology, Wiley.
See Interscience Publishers, (1995). As defined herein, "stringent conditions" or "high stringency conditions" means (1) low ionic strength and high temperature for washing, eg, 0.015M sodium chloride / 0.0015M at 50 ° C. Using sodium citrate / 0.1% sodium dodecyl sulphate; (2) Denaturants such as formamide during hybridization, eg 50% (v / v) formamide and 0.1% bovine serum albumin at 42 ° C. /0.1% Ficoll / 0.1% Polyvinylpyrrolidone / 50 mM pH 6
． 5 sodium phosphate buffer, and 750 mM sodium chloride, 75 m
With M sodium citrate; (3) 50% formamide at 42 ° C., 5 × SSC (0.75M NaCl, 0.075M sodium citrate),
50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1%
High stringency consisting of SDS and 0.1% SSC with 10% dextran sulfate, 0.2x SSC (sodium chloride / sodium citrate) wash at 42 ° C and 50% formamide at 55 ° C, then EDTA at 55 ° C. Identified by those using sexual wash. “Moderate stringency conditions” are described in Sambrook et al., Molecular Cloning: A Laboratory.
Manual (Identified as described in New York: Cold Spring Harbor Laboratory Press, 1989, and involves the use of less stringent wash solutions and hybridization conditions (eg, temperature, ionic strength and% SDS) as described above. Moderate stringency conditions were: 20% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / mL. Denatured sheared salmon sperm DNA
Incubation in a solution containing at 37 ° C. overnight followed by 37-in 1 × SSC.
The condition is that the filter is washed at 50 ° C. One of ordinary skill in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

The term “epitope tag” as used herein refers to a PRO polypeptide fused to a “tag polypeptide”, or a chimeric polypeptide comprising a domain sequence thereof. The tag polypeptide has a sufficient number of residues to provide an epitope for which the antibody can be produced, or an epitope identifiable by some other reagent, but whose length is of the PRO polypeptide of interest. Short enough not to interfere with the activity of the peptide. Also, the tag polypeptide is preferably fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues, usually about 8 to about 50 amino acid residues (preferably about 10 to about 20 residues). The term "immunoadhesin" as used herein refers to antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with an immunoglobulin constant domain. Structurally, an immunoadhesin contains a fusion of an immunoglobulin constant domain sequence with an amino acid sequence that has the desired binding specificity and is other than the antigen recognition and binding site of an antibody (ie, "heterologous"). . The adhesin portion of the immunoadhesin molecule is typically a contiguous amino acid sequence containing at least the binding site for the receptor or ligand. The immunoglobulin constant domain sequence of the immunoadhesin is Ig
It can be obtained from any immunoglobulin such as G-1, IgG-2, IgG-3 or IgG-4 subtype, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. As used herein, “active” and “activity” refer to forms of PRO that retain the biological and / or immunological activity of the native or naturally occurring PRO polypeptide and are “biological”. By activity is meant a biological function (inhibitory or stimulatory) produced by a natural or naturally occurring PRO that excludes the ability of the natural or naturally occurring PRO to generate antibodies against the antigenic epitopes. However, "immunological" activity refers to the ability to generate antibodies against the antigenic epitopes of natural or naturally occurring PRO. The term "antagonist" is used in its broadest sense and refers to any molecule that blocks, inhibits, or neutralizes the biological activity of a native PRO polypeptide disclosed herein. Similarly, the term "agonist" is used in its broadest sense and refers to any molecule that mimics the biological activity of a native PRO polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PRO polypeptides, peptides, small organic molecules, and the like. A method of identifying an agonist or antagonist of a PRO polypeptide can include contacting the PRO polypeptide with a candidate antagonist or agonist and measuring a change in one or more biological activities normally associated with the PRO polypeptide.

As used herein, “treatment” refers to both curative treatment, prophylactic therapy, and preventative therapy, in which a patient prevents or reduces (reduces) the targeted pathological condition or disease. To be Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder has been prevented. By "chronic" administration is meant administration of the drug in a continuous manner as opposed to an acute manner to maintain the initial therapeutic effect (activity) over an extended period of time. An "intermittent" administration is a treatment that is cyclical in nature, rather than continuous without interruption. "Mammal" for treatment is classified as a mammal, including humans, domestic and agricultural animals, zoos, sports, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, rabbits, etc. Means any animal. Preferably the mammal is a human. Administration "in combination with" one or more therapeutic agents includes simultaneous (concurrent) and sequential administration in any order. As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer and is nontoxic to cells or mammals exposed to them at the dosages and concentrations employed. . The physiologically acceptable carrier is often an aqueous pH buffered solution. Examples of physiologically acceptable carriers are phosphate, citrate, and other organic acid salt buffers; antioxidants including ascorbic acid; low molecular weight (about 1
<0 residues) polypeptide; protein such as serum albumin, gelatin, or immunoglobulin; hydrophobic polymer such as polyvinylpyrrolidone; amino acid,
E. Glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextran; E
Chelating agents such as DTA; sugar alcohols such as mannitol or proto-rubitol; salt forming counterions such as sodium; and / or nonionic surfactants such as TWEEN (trade name), polyethylene glycol (PEG), and PLURONICS First name) is included. An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments are Fab, Fab ′, F (ab ′) ₂ and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8 (10): 1
057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antibody-binding fragments, called "Fab" fragments, each of which has a single antigen-binding site and the rest "Fc", reflecting its ability to crystallize readily. Named Fragment. Pepsin treatment yields an F (ab ′) ₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen. "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable region in close, non-covalent association. In this arrangement the three CDRs of each domain interact to determine the antigen binding site on the surface of the _VH - _VL monomer. Correctly, the six CDRs have the antigen binding specificity for the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has a lower affinity than the entire binding site, but has the ability to recognize and bind antigen . The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH
Also includes 1). Fab fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Here, Fab′-SH represents Fab ′ in which the cysteine residue of the constant domain has a free thiol group. F (ab ′) ₂ 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. The "light chains" of antibodies (immunoglobulins) from any vertebrate species are classified into one of two distinct types called kappa and lambda, 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 classified into different classes. Five major immunoglobulin classes: IgA, Ig
D, IgE, IgG and IgM, some of which are further subclasses (isotypes), eg IgG1, IgG2, IgG3, IgG4, IgA and Ig.
It is classified into A2.

“Single-chain Fv” or “sFv” antibody fragments include antibody fragments that comprise the V _H and V _L domains of an antibody, these domains being present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains, which enables the sFV to form the desired structure for antigen binding.
For an overview of scFv, see The Pharmacology of Monoclonal Antibodies, vol.
113, Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (199
See 4) Pluckthun. The term "diabodies (diabodies)" refers to small antibody fragments with two antigen-binding sites, which fragments bind to the light chain variable domain (V _L) in the same polypeptide chain (V H _{-V L)} Heavy chain variable domain ( _VH ). By using a linker that is too short to pair between two domains on the same chain, the domains are forced to pair with the complementary domains on the other chain, creating two antigen binding sites. Diabodies are described, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90: 6444-6448 (1993). An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with its diagnostic or therapeutic use for the antibody, including enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is (1) Lori method.
More than 95% antibody, most preferably 99% by weight when measured by (Lowry method)
By using (2) Spinning cup sequenator until
Enough to obtain an N-terminal or internal amino acid sequence of at least 15 residues,
Alternatively, (3) it is purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. 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 term "label" as used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody to produce a "labeled" antibody. The label may itself be detectable (eg a radioactive or fluorescent label) or, in the case of an enzymatic label, may catalyze the chemical conversion of a detectable substrate compound or composition. By "solid phase" is meant a non-aqueous matrix to which the antibodies of the invention can adhere. Examples of solid phases contemplated herein include those formed partly or wholly from glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol and silicone. In certain embodiments, depending on the context, the solid phase can comprise the wells of an assay plate; in others it can be a purification column (eg, an affinity chromatography column). The term also includes a discontinuous solid phase of discrete particles, as described in US Pat. No. 4,275,149. A “liposome” is a small vesicle composed of various types of lipids, phospholipids, and / or surfactants, and is a drug for mammals (such as PRO polypeptide or its antibody).
Useful for transportation. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. A "small molecule" is defined herein as having a molecular weight of less than about 500 Daltons.

[0069]

[0070]

[0071]

II. Compositions and Methods of the Invention A. Full-Length PRO Polypeptide The present invention provides newly identified and isolated nucleic acid sequences that encode a polypeptide designated herein as PRO polypeptide. In particular, cDNAs encoding various PRO polypeptides have been identified and isolated, as described in further detail in the Examples below. Note that although proteins produced in separate expression rounds are given different PRO numbers, the UNQ numbers are unique to all given DNA and encoding proteins and do not change. However, for the sake of simplicity, herein, the proteins encoded by the full-length natural nucleic acid molecules disclosed herein, as well as additional natural homologues and variants included within the definition of PRO above, are referred to in their source or preparative form. Regardless of this, it is referred to as “PRO / number”. Various cDNA clones have been deposited with the ATCC, as disclosed in the Examples below. The exact nucleotide sequences of these clones can be readily determined by sequencing the deposited clones using methods routine in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the PRO polypeptides and encoding nucleic acids described herein,
Applicants have identified what is believed to be the reading frame that best matches the currently available sequence information.

1. Full length PRO1800 polypeptide WU-BLAST2 sequence alignment program was used to obtain full length native sequence PRO18.
00 (shown in FIG. 2 and SEQ ID NO: 2) is partially human Hep27 protein (
HE27_HUMAN) with some amino acid sequence identity. Therefore, at present, PRO1800 disclosed in the present application is newly identified as Hep2.
It is a 7-homolog and is believed to have typical activity for the protein.
2. Full-length PRO539 polypeptide WU-BLAST2 sequence alignment program was used to generate full-length native sequence PRO53.
A portion of 9 (shown in Figure 4 and SEQ ID NO: 7) was found to have some amino acid sequence identity with a portion of the kinesin-related protein from Drosophila melanogaster (AF19250_1). Therefore, at present, P disclosed in the present application
RO539 is a newly identified member of the hedgehog signaling pathway protein family and is believed to have activity typical of the Drosophila Costal-2 protein. 3. Full Length PRO982 Polypeptide To the best knowledge, the DNA57700-1480 sequence encodes a novel secretory factor, herein designated PRO982. Thus, using the WU-BLAST2 sequence alignment computer program, it was revealed to have some sequence identity with known proteins. 4. Full length PRO1434 polypeptide WU-BLAST2 sequence alignment program was used to obtain full length native sequence PRO14.
A portion of 34 (shown in Figure 10 and in SEQ ID NO: 13) was found to have some amino acid sequence identity with the mouse nel protein precursor. Therefore, PRO1434 disclosed in the present application is now believed to be a newly identified nel homolog, with activity typical of this nel protein family.

5. The full-length PRO1863 polypeptide DNA59847-2510 was isolated from a human prostate library. To the best of our knowledge, the DNA59847-2510 sequence encodes a novel factor designated herein as PRO1863; using the WU-BLAST2 sequence alignment computer program, there may be no significant sequence identity with any known protein. It was revealed. 6. Full length PRO1917 polypeptide WU-BLAST2 sequence alignment program was used to obtain the full length native sequence PRO19
Amino acids 41 to 487 of 17 (shown in FIG. 14 and SEQ ID NO: 20) are Da
It was found to have some degree of amino acid sequence identity with the inositol phosphatase designated "AF012714_1" in the yhoff database. So now,
PRO1917 disclosed in this application is a newly identified member of the inositol phosphatase family and is believed to have the enzymatic activity typical of inositol phosphatases. 7. Full length PRO1868 polypeptide WU-BLAST2 sequence alignment program was used to obtain full length native sequence PRO18
A portion of 68 (shown in Figure 16 and SEQ ID NO: 28) was found to have some amino acid sequence identity with the human A33 antigen protein (P_W14146). Therefore, PRO1868 disclosed in the present application is now believed to be a newly identified A33 antigen homolog, with an activity and / or expression pattern typical of A33 antigen proteins. It has also been found that PRO1868 polypeptides can be used in the therapeutic treatment of inflammatory diseases and colorectal cancers described above. 8. The full-length PRO3434 polypeptide DNA77631-2537 clone was isolated from a human aortic tissue library using a capture technique that selects for nucleotide sequences encoding secreted proteins. To the best of our knowledge, the DNA77631-2537 sequence encodes a novel factor designated herein as PRO3434; using the WU-BLAST2 sequence alignment computer program, lacking significant sequence identity with any known protein. Became clear. 9. Full-length PRO1927 polypeptide WU-BLAST2 sequence alignment program was used to obtain full-length native sequence PRO19.
27 (shown in FIG. 20 and in SEQ ID NO: 26) was “AB0” in the Dayhoff database.
It was found to have some degree of amino acid sequence identity with the amino acid sequence of the protein designated "00628_1". Therefore, at present, PRO1 disclosed in the present application
927 is a newly identified member of the glycosyltransferase family of proteins and is believed to have glycosylation activity.

B. PRO Variants In addition to the full-length native sequence PRO polypeptides described herein, PRO variants could also be prepared. PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO polypeptide DNA, or by synthesizing the desired PRO polypeptide. Those skilled in the art will appreciate that amino acid changes, such as changes in the number or position of glycosylation sites or changes in membrane anchoring properties, can alter post-translational processes of PRO polypeptides. Mutations in the native full-length sequence PRO or in various domains of the PRO polypeptides described herein may be performed using any of the techniques and guidelines for conservative and non-conservative mutations described, for example, in US Pat. No. 5,364,934. You can do it.
A mutation may be a substitution, deletion or insertion of one or more codons encoding a PRO polypeptide that results in a change in the amino acid sequence of the PRO polypeptide as compared to the native sequence PRO. In some cases, the mutation is at least one amino acid P
Substitution of any one or more domains of the RO polypeptide by any other amino acid. A guideline for which amino acid residues are to be inserted, substituted or deleted without adversely affecting the desired activity is to compare the sequence of the PRO polypeptide with that of a protein molecule of known homology and It is found by minimizing the amino acid sequence changes made within the high region of Amino acid substitutions can be the result of substitutions of one amino acid for another amino acid with similar structural and / or chemical properties, eg, replacement of leucine with serine, a conservative amino acid substitution. Insertions and deletions
In some cases it can be in the range of 1 to 5 amino acids. Permissible mutations are determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting mutants for activity exhibited by the full-length or mature native protein. PRO 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 to the full length native protein. Certain fragments lack amino acid residues that are not essential for the desired biological activity of the PRO polypeptide. PRO fragments may be prepared by any of many conventional techniques. The desired peptide fragment may be chemically synthesized. An alternative method is enzymatic digestion, for example by treating the protein with an enzyme known to cleave the protein at sites determined by specific amino acid residues, or by digesting the DNA with appropriate restriction enzymes. Including the generation of the PRO fragment by isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding the desired polypeptide fragment by polymerase chain reaction (PCR). Oligonucleotides that determine the desired ends of the DNA fragments are used in the 5'and 3'primers of PCR.
Preferably, PRO polypeptide fragments share at least one biological and / or immunological activity with the native PRO polypeptide disclosed herein.

In a particular embodiment, the conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such a substitution results in a change in biological activity, the product is screened for by introducing a more substitutional change, named exemplary substitutions in Table 6 or further described below in the amino acid taxonomy. To be done. Table 6 Original residues Exemplary substitution Preferred substitution Ala (A) val; Leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe Norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala ; tyr leu Pro (P) ala ala 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; ala; norleucine leu

Substantial modification of the function or immunological identity of a PRO polypeptide can be accomplished by (a) a structure of the polypeptide backbone of the substitution region, such as a sheet or helical arrangement, (b) charge or hydrophobicity of the target site, Alternatively, (c) is achieved by selecting substituents that are substantially different in their effect while maintaining the bulk of the side chains. Naturally occurring residues can be divided into groups based on common side chain properties: (1) Hydrophobicity: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilicity: cys, ser. , thr; (3) Acidity: asp, glu; (4) Basicity: asn, gln, his, lys, arg; (5) Residues affecting 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. Residues so substituted may also be introduced at conservative substitution sites, preferably left-over (non-conserved) sites. Mutations include oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR 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)], restricted selective mutagenesis [Wells.
Et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)] or other known techniques performed on cloned DNA. Then, PRO mutant DNA can be prepared. Also, scanning amino acid analysis can be used to identify one or more amino acids along a flanking sequence. The preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine,
Contains serine and cysteine. Alanine is typically the preferred scanning amino acid within this group because it excludes side chains above the beta carbon and does not alter the backbone structure of the mutant [Cuningham and Wells, Science, 244: 1081- 1085 (1
989)]. Also, alanine is typically preferred because it is the most common amino acid. Moreover, it is often found in both buried and exposed locations [Cr
eighton, The Proteins, (WH Freeman &amp; Co., NY); Chothia, J. Mol.
Biol., 150: 1 (1976)]. If alanine substitution does not yield a sufficient amount of variant,
Isoteric amino acids can be used.

C. Modifications of PRO Covalent modifications of PRO polypeptides are included within the scope of this invention. One type of covalent modification is to react a targeted amino acid residue of the PRO polypeptide with an organic derivatizing reagent capable of reacting with a selected side chain or N- or C-terminal residue of the PRO polypeptide. . Derivatization with bifunctional reagents is useful, for example, to crosslink PRO to a water-insoluble support matrix or surface for use in a method of purifying anti-PRO antibodies or vice versa. The cross-linking agent usually used is, for example, 1,1
-Including bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide ester such as 4-azidosalicylic acid, and disuccinimidyl ester such as 3,3'-dithiobis (succinimidylpropionate) Includes homobifunctional imide esters, bifunctional maleimides such as bis-N-maleimido-1,8-octane, and reagents such as methyl-3-[(p-azidophenyl) -dithio] propioimidate. Other modifications include deamination of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, lysine, arginine, and histidine side chains. Methylation of α-amino groups of [TE Creighton, Proteins: Structur
e and Molecular Properties, WH Freeman &amp;amp; Co., San Francisco, p
p.79-86 (1983)], acetylation of N-terminal amines, and amidation of any C-terminal carboxyl groups.

Other types of covalent modifications of the PRO polypeptide included within the scope of this invention are:
Includes alteration of the native glycosylation pattern of the polypeptide. By "altering the native glycosylation pattern" is intended herein a deletion of one or more carbohydrate moieties found in the native sequence PRO (elimination of existing glycosylation sites or chemical and / or chemical
Or by the removal of glycosylation by enzymatic means) and / or the addition of one or more glycosylation sites not present in the native sequence PRO. In addition, this clause includes qualitative changes in glycosylation of native proteins, including changes in the properties and properties of the various carbohydrate moieties present. Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence. This modification may be made, for example, by the addition or substitution of one or more serine or threonine residues to the native sequence PRO (O-linked glycosylation site). The PRO amino acid sequence is optionally altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases to produce codons that are translated into the desired amino acid. Good.

Another means of increasing the number of carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, for example WO 87/05330, published September 11, 1987, and Aplin.
And Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981). Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues presented as targets for glycosylation. Chemical deglycosylation techniques are known in the art, eg Hakimuddin et al., Arch. Biochem. Biophys., 259: 5.
2 (1987) and by Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides is described by Thotakura et al., Meth.
This is accomplished by using various endo and exoglycosidases, as described in Enzymol. 138: 350 (1987). Another type of covalent modification of PRO of the present invention is US Pat. No. 4,640,835 to PRO polypeptides to one of a variety of non-proteinaceous polymers such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes. ； 4th, 4th
96,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PRO polypeptides of the present invention may also be modified in a manner to form chimeric molecules containing the PRO polypeptide fused to other heterologous polypeptides or amino acid sequences. In one embodiment, such a chimeric molecule comprises a fusion of a PRO polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind.
The epitope tag is generally placed at the amino- or carboxyl-terminus of the PRO polypeptide. The presence of such epitope-tagged forms of PRO polypeptides is
It can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO polypeptide to be 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; flu HA-tagged polypeptides and their antibodies 12
CA5 [Field et al., Mol. Cell. Biol., 8: 2159-2165 (1988)]; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.
Molecular and Cellular Biology, 5: 3610-3616 (1985)]; and herpes simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineer.
ing, 3 (6): 547-553 (1990)]. Other tag polypeptides include flag peptides [Hopp et al., BioTechnology, 6: 1204-1210 (1988)]; KT3 epitope peptides [Martin et al., Science, 255: 192-194 (1992)]; α-tubulin epitope peptides. [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991)]; and T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci.
USA, 87: 6393-6397 (1990)]. In an alternative embodiment, the chimeric molecule may comprise a fusion of PRO with an immunoglobulin or a particular region of an immunoglobulin. For the bivalent form of the chimeric molecule (also referred to as the "immunoadhesin"), such a fusion may be the Fc region of an IgG1 molecule. Ig fusions preferably include a soluble (transmembrane domain deleted or inactivated) form of the PRO polypeptide in place of at least one variable region within the Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion is IgG
It comprises the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of the molecule. See US Pat. No. 5,428,130 issued Jun. 27, 1995 for the preparation of immunoglobulin fusions.

D. Preparation of PRO The following description primarily relates to methods of producing PRO by culturing cells transformed or transfected with a vector containing PRO nucleic acid. Of course, it is contemplated that PRO may be prepared using other methods well known in the art. For example, the PRO sequence, or a portion thereof, may be produced by direct peptide synthesis using solid phase techniques [eg, Stewart et al., Solid-Phase Pepti.
de Synthesis, WH Freeman Co., San Francisco, CA (1969); Merrifield, J.
Am. Chem. Soc., 85: 2149-2154 (1963)]. In vitro protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. Various portions of PRO may be chemically synthesized separately and combined using chemical or enzymatic methods to produce full-length PRO.

1. Isolation of PRO-encoding DNA PRO-encoding DNA can be obtained from cDNA libraries prepared from tissues that carry PRO mRNA and are suspected of expressing it at detectable levels. Therefore, human PRODNA can be conveniently obtained from a cDNA library prepared from human tissue, as described in the Examples. PRO-encoding genes can also be obtained from genomic libraries or by known synthetic methods (eg, automated nucleic acid synthesis). Libraries can be screened with probes (such as antibodies to PRO or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by the gene.
Screening a cDNA or genomic library with selected probes
For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold
It can be carried out using standard procedures described in Spring Harbor Laboratory Press, 1989). Another method for isolating the gene encoding the desired PRO polypeptide is to use the PCR method [Sambrook et al., Supra; Dieffenba.
ch et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Pres
s, 1995)].

The following examples describe techniques for screening a cDNA library. The oligonucleotide sequence selected as a probe should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably detectably labeled upon hybridisation with the DNA in the library to be screened. Methods of labeling are well known in the art and include the use of radiolabels such as ³² P labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate and high stringency, are given in Sambrook et al., Supra. Sequences identified in such library screening methods are Genban
It can be compared and aligned with known sequences that have been deposited in public databases such as k or private sequence databases and are available to the public. Within a defined region of the molecule or over its entire length (either at the amino acid or nucleic acid level)
Sequence identity can be determined using methods known in the art and described herein. Nucleic acids having protein-encoding sequences are used for the first time using the deduced amino acid sequences disclosed herein and, if necessary, to detect intermediates and precursors of mRNA that have not been reverse transcribed into cDNA, Sambrook et al., Supra. It can be obtained by screening the selected cDNA or genomic library using conventional primer extension methods as described in.

2. Selection and Transformation of Host Cells Host cells are transfected or transformed with the expression or cloning vectors for PRO production described herein to induce promoters, select transformants, or encode desired sequences. The cells are cultured in a conventional nutrient medium which has been appropriately modified to amplify the gene. Culture conditions such as medium, temperature, pH, etc. can be selected by those skilled in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity are described in Mammalian Cell Biotechnology: a
Practical Approach, edited by M. Butler (IRL Press, 1991) and Sambrook et al., Supra. Methods of prokaryotic and eukaryotic cell transfection, such as CaCl ₂ , CaPO ₄ , liposome-mediated and electroporation are known to those of skill in the art. Depending on the host cell used, transformation is done using standard methods appropriate to the cell. Calcium treatment with calcium chloride or electroporation as described in Sambrook et al., Supra, is generally used for prokaryotes. Infection by Agrobacterium tumefaciens was reported by Shaw et al., Ge
ne, 23: 315 (1983) and WO 89/05859 published June 29, 1989 and used for the transformation of certain plant cells. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52: 456-457 (1978) is preferred. General aspects of mammalian cell host system transformations have been described in US Pat. No. 4,399,216. Transformation into yeast is typically performed by Van solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Nat.
l. Acad. Sci. USA, 76: 3829 (1979). However, other methods of introducing DNA into cells, such as nuclear microinjection,
Electroporation, intact cells, or polycations such as polybrene,
Bacterial protoplast fusion, such as with polyornithine, can also be used.
For various techniques for transforming mammalian cells, see Keown et al., Methods i.
n Enzymology, 185: 527-537 (1990) and Mansour et al., Nature, 336: 348-352 (198).
See 8).

Suitable host cells for cloning or expressing the DNA in the vectors described herein are 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, for example E. coli K12 strain MM294 (ATCC31,446); E. coli X1776.
(ATCC31,537); E. coli strains W3110 (ATCC27,325) and K5772 (ATCC53,6)
35). Other preferred prokaryotic host cells are E. coli, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcesans ( Serratia marcescans), and Shigella, and bacilli, such as B. subtilis and B. licheniformis.
) (Eg, Bacillus licheniformis 41P described in DD 266,710 issued Apr. 12, 1989), Pseudomonas, for example, Pseudomonas aeruginosa and Enterobacteriaceae such as Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host as it is a common host strain for recombinant DNA production and publishing. Preferably, the host cell secretes a minimal amount of proteolytic enzyme. For example, strain W3110 may be modified to make a genetic mutation in a gene encoding a protein that is foreign to the cell, and examples of such hosts include E. coli W3110 strain 1A2 with the complete genotype tonA; Genotype ton
E. coli W3110 strain 9E4 carrying A ptr3; complete genotype tonA p
rt3 phoA E15 (argF-lac) 169 degP ompTk
E. coli W3110 strain 27C7 (ATCC 55,244); complete genotype t
onA ptr3 phoA E15 (algF-lac) 169 degP
E. coli W3110 strain 37D6 having ompT rbs7ilvGkan; E. coli W3110 strain 4 which is 37D6 strain having a non-kanamycin resistant degP deletion mutation
0B4; and E. coli strains having a mutant periplasmic protease disclosed in US Pat. No. 4,946,783 issued Aug. 7, 1990. Alternatively, in vitro methods of cloning such as PCR or other nucleic acid polymerase polymerase reactions are preferred.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. In addition, Schizosaccharomyces prombe (Beach and Nur
se, Nature, 290: 140 [1981]; EP 139,383, issued May 2, 1985; Kluveromyces hosts (US Pat. No. 4,943,529; Fleer et al., Bio / Tech).
nology, 9: 968-975 (1991)), for example K. lactis (MW98-8C, CBS
683, CBS4574; Louvencourt et al., J. Bacteriol. 737 [1983]), Kefragiris (
K. fragilis) (ATCC 12,424), K. bulgaricus (ATCC 16,04)
5), K. wickeramii (ATCC 24,178), K. walamii (K. walamii)
tii) (ATCC 56,500), K. drosophilarum (ATCC 36,906
; Van den Berg et al., Bio / Technology, 8: 135 (1990)), Katemoto Tolerance (K.
the motolerans) and K. marxianus; yarrowia
) (EP 402,226); Pichia pastoris (EP 183,070; Sheekr
ishna et al., J. Basic Microbiol, 28: 265-278 [1988]); Candida; Trichoder Malaysia (reesia) (EP 244,234); Panax mold (Case et al., Proc. Natl. Acad)
Sci. USA, 76: 5259-5263 [1979]); schwanniomyces,
For example, Schwanniomyces occidentalis (EP 394,538, published October 31, 1990); and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium (published January 10, 1991) WO 91/00357); and Koji bacterium, such as pseudo-nest Koji bacterium (Ballance et al., Biochem. Biophys. Res. Commun.
, 112: 284-289 [1983]; Tilburn et al., Gene, 26: 205-221 [1983]; Yelton et al., Pr.
oc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and black mold (Kelly and H.
ynes, EMBO J., 4: 475-479 [1985]). Preferred methylotropic yeasts here include, but are not limited to, Hansenula,
Includes a methanol-growth yeast selected from the genus consisting of Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this yeast class is set forth in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982). Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms.
Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodaspera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese Hamster Ovary (CHO) and COS cells. A more detailed example is
Monkey kidney CV1 strain transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney strain (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells /
-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (19
80)); Sertoli cells of mouse (TM4, Mather, Biol. Reprod., 23: 243-251 (1980).
) Human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse breast tumor cells (MMT 060562, ATTC CCL51). Selection of an appropriate host cell is
Within the common general knowledge of this field.

3. Selection and Use of Replicable Vectors A nucleic acid encoding PRO (eg, cDNA or genomic DNA) is inserted into a replicable vector for cloning (amplification of DNA) or expression. Various vectors are publicly available. The vector can be in the form of, for example, a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence is
It is inserted into the vector by various techniques. Generally, the DNA is inserted into the appropriate restriction endonuclease sites using techniques well known in the art. Vector components generally include, but are not limited to, one or more signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those of ordinary skill in the art. PRO is not only directly produced by recombinant techniques, but also as a fusion peptide with a heterologous polypeptide which is a signal sequence or a mature protein or another polypeptide having a specific cleavage site at the N-terminus of the polypeptide. Is also produced. Generally, the signal sequence is a component of the vector or part of the PRO-encoding DNA that is inserted into the vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II leaders. For yeast secretion, the signal sequences are yeast invertase leader, alpha factor leader (
Includes yeast (Saccharomyces) and Kluyveromyces α factor leaders, the latter of which are described in US Pat. (EP362179, published April 4, 1990), or the signals described in WO 90/13646 published November 15, 1990. In mammalian cell expression, the mammalian signal sequence is
It may be used for direct secretion of proteins 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 common to many bacteria,
It is well known for yeasts and viruses. The origin of replication derived from plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are mammalian. It is useful as a cloning vector in animal cells. Expression and cloning vectors typically contain a selection gene, also termed a selectable marker. Typical selection genes are (a) ampicillin, neomycin,
Not resistant to antibiotics or other toxins such as methotrexate or tetracycline, (b) supplementing auxotrophic defects, or (c) obtained from complex media such as the gene code D-alanine racemase for Bacillus It encodes a protein that supplies important nutrients. Examples of suitable selectable markers for mammalian cells are those that can identify cellular components capable of incorporating PRO-encoding nucleic acid, such as DHFR or thymidine kinase. Suitable host cells using wild type DHFR are:
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 preferred selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282: 39 (1979); Kingman et al.,
Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980)]. The trp1 gene provides a selectable marker for mutant strains of yeast lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. Expression and cloning vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to control mRNA synthesis. Suitable promoters recognized by a variety of possible host cells are known. Suitable promoters for use in prokaryotic hosts include the β-lactamase and lactose promoter systems [Cahng
Et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)], alkaline phosphatase, tryptophan (trp) promoter system [Goeddel, Nucleic Ac
ids Res., 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1
983)] is included. Promoters used in bacterial systems also have a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding PRO.

Examples of suitable promoter sequences for use with yeast hosts include 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, Biochem.
istry, 17: 4900 (1987)], for example, enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, Pyruvate kinase, trioselate isomerase, phosphoglucose isomerase, and glucokinase are included. Other yeast promoters are inducible promoters that have the additional effect of transcription being regulated by growth conditions, such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glycerin. There are promoter regions for aldehyde-3-phosphate dehydrogenase and enzymes that control the utilization of maltose and galactose. Vectors and promoters suitable for expression in yeast are further described in EP 73,657. PRO transcription from a vector in mammalian host cells can be performed, for example, by polyomavirus, infectious epithelioma virus (UK 2,211,504 published July 5, 1989), adenovirus (eg adenovirus 2), bovine papilloma virus, avian virus. Sarcoma virus,
Cytomegalovirus, retrovirus, hepatitis B virus and simian virus 4
0 (SV40), a promoter derived from the genome of a virus, a heterologous mammalian promoter such as an actin promoter or an immunoglobulin promoter,
And promoters derived from heat shock promoters, as long as such promoters are compatible with the host cell line.

Transcription of the DNA encoding the desired PRO by higher eukaryotes can be enhanced by inserting an enhancer sequence into the vector. Enhancers, usually about 10 to 300 base pairs, are cis-acting elements of DNA that act on promoters to enhance their transcription. Many enhancer sequences from mammalian genes are currently known (globin, elastase, albumin, α-fetoprotein and insulin). However, typically enhancers from eukaryotic viruses will be used. Examples include the SV40 enhancer on the late side of the replication origin (100-270 base pairs), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer. The enhancer may be spliced into the vector at the 5'or 3'positions of the PRO coding sequence, but is preferably located 5'from the promoter. Expression vectors for use in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also include termination of transcription and mRNA.
It also contains the sequences required for the stabilization of Such sequences are used in eukaryotic or viral DNs.
It can be obtained from the A or cDNA, usually 5'and sometimes 3'untranslated regions. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO. Other methods suitable for adapting the synthesis of PRO in recombinant vertebrate cell culture,
Vectors and host cells are described in Gething et al., Nature, 293: 620-625 (1981); Mantei et al.,
Nature, 281: 40-46 (1979); EP 117,060; and EP 117,058.

4. Detection of Gene Amplification / Expression Amplification and / or expression of a gene can be carried out using appropriately labeled probes based on the sequence provided herein, for example, conventional Southern blotting, Northern quantifying mRNA transcription. Blotting [Thomas, Proc. Natl. Acad. Sci. USA, 77:52
01-5205 (1980)], the dot blot method (DNA analysis), or the in situ hybridization method, and can be directly measured in the sample. Alternatively, DNA
Antibodies capable of recognizing specific double strands, including double strands, RNA double strands and DNA-RNA hybrid double strands or DNA-protein double strands can also be used. The antibody can then be labeled and the assay performed, wherein the duplex is surface bound, such that the antibody bound to the duplex at the time of surface duplex formation. Presence can be detected. Alternatively, gene expression can also be measured by immunological methods that directly quantitate the expression of the gene product, such as immunohistochemical staining of cells or tissue sections and cell culture or body fluid assays. Antibodies useful for immunohistochemical staining and / or assay of sample fluids may be monoclonal or polyclonal and can be prepared in any mammal. Conveniently, the antibody is directed against the native sequence PRO polypeptide, or against a synthetic peptide based on the DNA sequences provided herein, or against an exogenous sequence fused to PRODNA and encoding a specific antibody epitope. Can be prepared.

5. Purification of Polypeptides Forms of PRO can be recovered from culture medium or lysates of host cells. If membrane-bound, it can be detached from the membrane using a suitable wash (eg Triton-X100) or enzymatic cleavage. The cells used to express PRO can be disrupted by various chemical or physical means such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. It may be desirable to purify PRO from recombinant cell proteins or polypeptides. Purified by the following procedure, which is an example of a suitable purification procedure: Fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or cation exchange resins such as DEAE; chromatofocusing; SDS.
-PAGE; ammonium sulfate precipitation; gel filtration using eg Sephadex G-75; protein A Sepharose column to remove contaminants such as IgG; and PR
Metal chelation column that binds the epitope-tagged form of O polypeptides. Known in the art, for example Deutcher, Methodes in Enzymology, 182 (1990).
Scopes, Protein Purification: Principles and Practice, Springer-Verlag
Many of the protein purification methods described in New York (1982) can be used. The purification process chosen will depend, for example, on the production method used and the nature of the particular PRO produced.

E. Uses of PRO Nucleic acid sequences encoding PRO (or their complements) may be used in a variety of areas in molecular biology, including use as hybridization probes, in chromosome and gene mapping, and in the production of antisense RNA and DNA. Has uses. PRO nucleic acids will also be useful in the preparation of PRO polypeptides by the recombinant techniques described herein. The full-length native sequence PRO gene, or a portion thereof, may be the isolation of full-length PRO cDNA or other genes with the desired sequence identity to the PRO sequences disclosed herein (eg, naturally occurring variants of PRO or other species (Encoding PRO from E. coli) can be used as a hybridization probe for a cDNA library.
In some cases, the length of the probe is about 20 to about 50 bases. Hybridization probes may be derived, at least in part, from novel regions of the full-length native nucleotide sequence, which regions, without undue experimentation, produce the native sequence PRO.
Can be derived from a genomic sequence containing the promoter, enhancer component and intron. For example, screening methods include isolating the coding region of the PRO polypeptide gene using known DNA sequences to synthesize a selected probe of about 40 bases. Hybridization probes can be labeled with a variety of labels, including radioactive nucleotides such as ³² P or ³⁵ S, or enzyme labels such as alkaline phosphatase attached to the probe via the avidin / biotin binding system. P of the present invention
A labeled probe having a sequence complementary to the RO polypeptide gene is human c
It can be used to screen a library of DNA, genomic DNA or mRNA and determine which members of the library hybridize to the probe. Hybridization techniques are described in further detail in the examples below.

Any of the ESTs disclosed in this application can be used in the methods described herein, as can probes. Other useful fragments of PRO nucleic acid are target PRO mRNA (sense) or PROD
An antisense or sense oligonucleotide comprising an antisense or sense oligonucleotide comprising a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to an NA (antisense) sequence is according to the invention encoded by PRODNA. Contains region fragments. Such fragments generally contain at least about 14 nucleotides, preferably about 14 to 30 nucleotides. The possibility of controlling antisense or sense oligonucleotides based on a cDNA sequence encoding a given protein has been described, for example, by Stein and Cohen (CancerRes. 48: 2659: 1988) and van der Krol et al. (BioTechniques 6: 958, 1988). Binding of the antisense or sense oligonucleotide to the target nucleic acid sequence results in the formation of a duplex, which is one of several methods including promoting duplex degradation, premature termination of transcription or translation, or Other methods prevent transcription or translation of the target sequence. Thus, antisense oligonucleotides are used to block the expression of PRO proteins. The antisense or sense oligonucleotide further comprises an oligonucleotide having a modified sugar-phosphodiester backbone (or other sugar bond, such as those described in WO 91/06629), such sugar bond being resistant to an endogenous nuclease. is there. Oligonucleotides with such resistant sugar linkages are stable in vivo (ie, can withstand enzymatic degradation) but retain the sequence specificity of being able to bind the target nucleotide sequence.

Other examples of sense or antisense oligonucleotides are organic moieties, such as those described in WO 90/10048, and other moieties that enhance the affinity of the oligonucleotide for the target nucleic acid sequence, eg It contains an oligonucleotide covalently linked to poly- (L-lysine). Furthermore, an intercalating agent such as ellipticine, an alkylating agent, or a metal working substance may be bound to the sense or antisense oligonucleotide to modify the binding specificity of the antisense or sense oligonucleotide to the target nucleotide sequence. Antisense or sense oligonucleotides can be prepared, for example, by CaPO ₄ -mediated D
By any gene conversion method including NA transfection, electroporation, or by using a gene conversion vector such as Epstein-Barr virus
It is introduced into cells containing the target nucleic acid sequence. In a preferred method, the antisense or sense oligonucleotide is inserted into a suitable retroviral vector. Cells containing the target nucleic acid sequence are contacted with the recombinant retroviral vector in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (retrovirus derived from M-MuLV), or the double named DCT5A, DCT5B and DCT5C. Includes a copy vector (see WO 90/13641).

Sense or antisense oligonucleotides may also be introduced into cells containing the target sequence by forming a complex with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the complexing of the ligand-binding molecule substantially limits the ability of the ligand-binding molecule to bind to its corresponding molecule or receptor, or prevent the sense or antisense oligonucleotide or complex thereof from entering the cell. Do not block. Alternatively, sense or antisense oligonucleotides may be introduced into cells containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably degraded intracellularly by endogenous lipase.

The probes can also be used in PCR techniques to generate a pool of sequences for the identification of closely related PRO coding sequences. The PRO-encoding nucleic acid sequence can also be used to create hybridization probes for mapping the PRO-encoding gene and for genetic analysis of individuals with genetic disorders. The nucleic acid sequences provided herein can be mapped to chromosomes and specific regions of chromosomes using well-known techniques such as in situ hybridization, binding analysis for known chromosomal markers, and hybridization screening in libraries. . If the PRO coding sequence encodes a protein that binds to another protein (eg, PRO is a receptor), PRO can be used in an assay to identify its ligand. In this way, inhibitors of receptor / ligand binding interactions can be identified. Proteins involved in such binding interactions can also be used to screen for peptides or small molecule inhibitors or agonists of binding interactions. The receptor PRO can also be used to isolate related ligands. The screening assay is a natural PRO
Or designed for the discovery of lead compounds that mimic the biological activity of the PRO receptor. Such screening assays are also used in high throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules considered include synthetic organic or inorganic compounds. The assays are performed in a variety of formats, including protein-protein binding assays, biological screening assays, immunoassays and cell-based assays that are well known and characterized in the art.

Nucleic acids encoding PRO or any modified forms thereof can also be used to produce transgenic or "knockout" animals, which are useful in the development and screening of therapeutically useful reagents. is there. A transgenic animal (eg, mouse or rat) is an animal that has cells that contain the transgene introduced prenatally, eg, at the embryonic stage, into the animal or an ancestor of the animal. What is a transgene?
It is DNA that has been integrated into the genome of the cell in which a transgenic animal develops.
In one embodiment, the PRO-encoding cDNA is prepared by established techniques.
, And the genomic sequence can be used to produce transgenic animals having cells expressing PRO-encoding DNA. Methods for producing transgenic animals, especially certain animals such as mice or rats, are routine in the art and are described, for example, in US Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells are targeted for the introduction of the PRO transgene with tissue-specific enhancers. Transgenic animals containing a copy of the PRO-encoded transgene introduced into the germline of the animal at the embryonic stage can be used to investigate the effects of increased expression of DNA encoding PRO. Such animals can be used, for example, as tester animals for reagents that would provide protection against pathological conditions associated with their overexpression. In this aspect of the invention, treatment of an animal with a reagent and a lower incidence of the pathological condition as compared to untreated animals carrying the transgene indicates a potential therapeutic treatment for the pathological condition. .

Alternatively, a non-human homologue of PRO is obtained by homologous recombination between a modified genomic DNA encoding PRO introduced into an animal embryonic cell and an endogenous gene encoding PRO. It can be used to generate PRO "knockout" animals with the coding defect or altered gene. For example, the cD encoding PRO
NA can be used for cloning genomic DNA encoding PRO according to established techniques. A portion of the genomic DNA encoding PRO may be deleted or replaced with another gene, such as the gene encoding the selectable marker used to monitor integration. Typically, the vector has a constant flanking D
It contains several kilobases of NA (both at the 5'and 3'ends) [see, for example, Thomas and Capecchi, Cell, 51: 503 (1987) for homologous recombination vectors]. The vector is introduced into embryonic stem cells (eg, by electroporation), and cells in which the introduced DNA is homologously recombined with the endogenous DNA are selected [eg, Li etc.
, Cell, 69: 915 (1992)]. The selected cells are then injected into the blastocyst of an animal (eg mouse or rat) to form an assembled chimera [eg Bradley, Terato.
carcinomas and Embryonic Stem Cells: A Practical Approach, EJ Roberts
on, ed. (IRL, Oxford, 1987), pp. 113-152]. The chimeric embryos are then transplanted into a suitable pseudopregnant female nanny and at intervals, "knockout" animals are created. Offspring with DNA that has been homologously recombined into germ cells are identified by standard techniques and can be used to breed animals in which all cells of the animal contain homologously recombined DNA. . Knockout animals are characterized by the ability to protect against certain pathological conditions and the progression of that pathological condition due to the absence of PRO polypeptide.

Nucleic acids encoding PRO polypeptides can also be used in gene therapy. In gene therapy applications, for example, to replace a defective gene, the gene is introduced to achieve in vivo synthesis of a therapeutically effective amount of the gene product. "Gene therapy"
The term includes both conventional gene therapy in which a single treatment achieves a continuous effect and administration of a gene therapeutic agent including single or repeated administration of therapeutically effective DNA or mRNA. Antisense RNA and DNA can be used as therapeutic agents to block the in vivo expression of certain genes. It has already been shown that a short antisense oligonucleotide can be transferred into cells where it acts as an inhibitor despite low intracellular concentrations due to limited uptake by the cell membrane (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146 [1986]). Oligonucleotides may be modified to facilitate uptake by replacing their negatively charged phosphodiester groups with uncharged groups. There are various techniques for introducing nucleic acids into viable cells. These techniques will vary depending on whether the nucleic acid is transferred to cultured cells in vitro or in vivo in the cells of the intended host. Suitable methods for transferring nucleic acids into mammalian cells in vitro include liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation and the like. Currently preferred in vivo gene transfer techniques are transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends, in Biotechnology 11, 205-219). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cell, such as a cell surface membrane protein or an antibody specific for the target cell, a ligand for a receptor on the target cell. When using liposomes, proteins that bind to cell surface membrane proteins with endocytosis, for example, capsid proteins or fragments thereof that are tropic for certain cell types, antibodies to proteins that undergo internalization in the cycle, intracellular localization A protein that targets
Used for targeting and / or promoting uptake. Receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-2232 (1987); and Wa.
Gner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). Anderson for a review of gene creation and gene therapy protocols
Et al., Science 256, 808-813 (1992).

The PRO polypeptides described herein may be used as molecular weight markers for protein electrophoresis purposes. Nucleic acid molecules encoding the PRO polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there is a need for the identification of novel chromosomal markers, as chromosomal marking reagents based on actual sequences are scarcely available. Each PRO nucleic acid molecule of the present invention can be used as a chromosomal marker. The PRO polypeptides and nucleic acid molecules of the invention can also be used for tissue typing,
The PRO polypeptides of the invention are differentially expressed in one tissue as compared to the other.
PRO nucleic acid molecules will find use for generating probes for PCR, Northern, Southern and Western analyses. The PRO polypeptides described herein may be used as therapeutic agents. P of the present invention
The RO polypeptide is formulated according to known methods for preparing pharmaceutically useful compositions, whereby the PRO product is mixed with a pharmaceutically acceptable carrier medium. Therapeutic formulations may be prepared by mixing the active ingredient with the desired degree of purification with any pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or aqueous solution ( Remington's Pharmaceutical Sciences, 16th edition,
A. Osol, Ed., [1980]), prepared and stored. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used, and buffers such as phosphoric acid, citric acid and other organic acids; antioxidants including ascorbic acid. Agents; low molecular weight (less than 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; glucose, Monosaccharides such as mannose or dextrin, disaccharides or other carbohydrates, chelating agents such as EDTA, saccharides such as mannitol or sorbitol, salt forming counterions such as sodium; and / or TWEEN (trade name), PLURONICS (trade name) Or PEG
Etc. non-ionic surfactants.

The formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filter membranes, either before or after lyophilization and reconstitution. Here, the pharmaceutical composition of the present invention is generally placed in a container having a sterile access port, for example, an intravenous bag or a vial having a stopper pierceable by a hypodermic injection needle. The route of administration is by well-known methods, such as injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes, local administration, or sustained release system. The dosage and desired drug concentration of the pharmaceutical compositions of the present invention will vary depending on the particular intended use. Determination of the proper dosage or route of administration is within the skill of the ordinary physician. Animal studies provide reliable guidance on the determination of effective doses for human therapy. Toxicokinetics and New Dru
g Development, Yacobi et al., ed., Pergamon Press, New York 1989, pp. 42-96, M.
ordenti, J. and chappell, W. `` The use of interspecies scaling in toxico
kinetics ”.

When in vivo administration of the PRO polypeptide or agonist or antagonist thereof is used, the normal dosage is 1 per mammal body weight, depending on the route of administration.
From about 10 ng / kg to 100 mg / kg daily, preferably about 1 μg / kg / day
It is 10 mg / kg / day from the day. Guidance on specific doses and delivery methods is given in the literature; eg, US Pat. Nos. 4,657,760, 5,206,344, or 5,225,21.
See No. 2. That different formulations are effective for different therapeutic compounds and different diseases,
For example, it is expected that targeted administration to one organ or tissue will require delivery in a different manner than other organs or tissues. Microencapsulation of PRO polypeptide is contemplated when sustained release of PRO polypeptide is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder that requires administration of PRO polypeptide. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon (rhIFN), interleukin-2, and MNrgp120. Johnson et al., Nat. Med., 2: 795-799 (1996); Yasuda, Biomed. The
r., 27: 1221-1223 (1993); Hora et al., Bio / Technology, 8: 755-758 (1990); Cle.
land, `` Design and Production of Single Immunization Vaccines Using Poly
actide Polyglycolide Microsphere Systems '' Vaccine Design: The Subunit an
d Adjuvant Approach, Powell and Newman, (Plenum Press: New York, 19
95), p. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.
No. 654,010.

Sustained release formulations of these proteins include poly-lactic-coglycolic acid (PLGA
) Polymers, based on their biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic acid and glycolic acid, are immediately cleared within the human body. Furthermore, the degradability of this polymer can be adjusted from months to years depending on the molecular weight and composition. Lewis, `` Controlled release of bioactive age
nts from lactide / glycolide polymer '': M. Chasin and R. Langer (ed.), Biod
egradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 19
90), pp. 1-41. The present invention also includes methods of screening compounds to identify those that are similar to PRO polypeptides (agonists) or that inhibit the effects of PRO polypeptides (antagonists). The screening assay for antagonist candidates is
Designed to identify compounds that bind or complex with the PRO polypeptide encoded by the genes identified herein, or otherwise inhibit the interaction of the encoded polypeptide with other cellular proteins . Such screening assays include those applicable to high throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. The assay is performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, including those known in the art. All assays for antagonists require that they contact the candidate drug with the PRO polypeptide encoded by the nucleic acid identified herein under conditions and for a time sufficient for these two components to interact. Is common in doing.

In binding assays, the interaction is binding and the complex formed is isolated or detected in the reaction mixture. In a particular embodiment, the PRO polypeptide or candidate drug encoded by a gene identified herein is immobilized covalently or non-covalently on a solid phase, eg, a microtiter plate. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying.
Alternatively, an immobilized antibody specific for the PRO polypeptide to be immobilized, eg a monoclonal antibody, can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled with a detectable label, to the immobilized component, eg, the coated surface containing the anchored component. When the reaction is complete, unreacted components are removed, for example by washing, and the complex adhered to the solid surface is detected. When the first non-immobilized component carries a detectable label, detection of the label immobilized on the surface indicates that complex formation has occurred. If the first non-immobilized component has no label, complex formation can be detected, for example, by a labeled antibody that specifically binds to the immobilized complex. Specific PRs that interact with candidate compounds but are encoded by the gene identified here
If it does not bind to O-polypeptide, its interaction with the polypeptide can be assayed by well-known methods for detecting protein-protein interactions. Such assays include traditional techniques such as cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions have been demonstrated by Fields and coworkers [Fiels and Song, Natur.
e (London) 340, 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88, 95.
78-9582 (1991)] by using the yeast-based gene system described in Ch.
It can be monitored using the yeast-based system disclosed in evray and Nathans [Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991)]. Many transcriptional activators, such as yeast GAL4, consist of two physically distinct modular domains, one acting as a DNA binding domain and the other functioning as a transcriptional activation domain. Yeast expression system described in previous literature (generally "2-hybrid system"
Takes advantage of this property and uses two hybrid proteins, one in which the target protein is fused to the DNA binding domain of GAL4 and the other in which the candidate activating protein is fused to the activation domain. is doing. GAL1
Expression of the -lacZ reporter gene under the control of the GAL4-activated promoter depends on the reconstitution of GAL4 activity via protein-protein interactions.
Colonies containing interacting polypeptides are detected with a chromogenic substance for β-galactosidase. Complete kit for identifying protein-protein interactions between two specific proteins using 2-hybrid technology (MATCH
MAKER) is commercially available from Clontech. This system can be extended to the mapping of protein domains involved in a particular protein interaction, as well as the identification of amino acid residues important for this interaction.

Compounds that inhibit the interaction of the PRO polypeptide-encoding gene identified herein with intracellular or extracellular components can be tested as follows: Usually, the reaction mixture is the gene product and extracellular components. Alternatively, the intracellular component is prepared under conditions and at times when the two products interact and bind. To test the ability of a candidate compound to inhibit binding, the reaction is performed in the absence and presence of the test compound.
In addition, placebo may be added to the third reaction mixture to provide a positive control. The binding (complex formation) of the test compound present in the mixture with intracellular or extracellular components is monitored as described above. The formation of the complex in the control reaction but not in the reaction mixture containing the test compound indicates that the test compound inhibits the interaction of the test compound with its binding partner. To assay an antagonist, a PRO polypeptide may be added with a compound that is screened for a particular activity, and the ability of the compound to inhibit the activity of interest in the presence of the PRO polypeptide is such that the compound is an antagonist of the PRO polypeptide. Indicates that there is. Alternatively, the antagonist may be detected by binding the PRO polypeptide and a potential antagonist with the membrane-bound PRO polypeptide receptor or recombinant receptor under conditions suitable for competitive inhibition assays. PRO polypeptides can be labeled, such as radioactively, and the number of PRO polypeptides bound to the receptor can be used to determine the efficacy of a potential antagonist. The gene encoding the receptor can be identified by many methods known to those of skill in the art, such as ligand panning and FACS sorting. Coligan, etc.,
Current Protocols in Immun., 1 (2): Chapter 5 (1991). Preferably, expression cloning is used, polyadenylated RNA is prepared from cells responsive to PRO polypeptide, and the cDNA library generated from this RNA is distributed into pools, COS cells or other non-PRO polypeptide responsive. Used for transfection of cells. Transfected cells grown on glass slides are exposed to labeled PRO polypeptide. PRO polypeptides can be labeled by a variety of means including iodination or the inclusion of recognition sites for site-specific protein kinases. After fixation and incubation, slides are subjected to autoradiographic analysis. Positive pools are identified and subpools are prepared and retransfected using an interactive subpooling and rescreening step, resulting in the generation of a single clone encoding the putative receptor.

As an alternative method for receptor identification, labeled PRO polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. The crosslinked material is dissolved in PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excited, separated into peptide fragments and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing is used to design a set of degradable oligonucleotide probes that screen a cDNA library to identify genes encoding putative receptors. In another assay for antagonists, mammalian cells expressing the receptor or a membrane preparation are incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction is then measured. More particular examples of potential antagonists are oligonucleotides that bind to fusions of immunoglobulins and PRO polypeptides, particularly but not limited to polypeptide- and monoclonal antibodies and antibody fragments, single chain antibodies, anti-antibodies. -Idiotypic antibodies, and chimeric or humanized forms of these antibodies or fragments, as well as antibodies, including human antibodies and antibody fragments. Alternatively, the potential antagonist recognizes closely related proteins, eg, receptors, but has no effect, and thus PR
It may be a mutated form of PRO polypeptide that competitively inhibits the action of O polypeptide. Other potential PRO polypeptide antagonists are antisense RNA or DNA constructs prepared using antisense technology, eg, antisense RNA or DNA hybridizes to a target mRNA to interfere with protein translation. This acts to directly block the translation of mRNA. 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 the binding of polypeptide nucleotides to DNA or RNA. For example, the 5'coding portion of the polypeptide nucleotide sequence encoding the mature PRO polypeptide herein is:
It is used to design antisense RNA oligonucleotides of about 10 to 40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription (Triple Helix-Lee et al., Nucl, Acid Res., 6: 3073.
(1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 251: 1360.
(1991)), thereby preventing transcription and production of PRO polypeptides. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block translation of mRNA molecules into PRO polypeptides (antisense-Ok
ano, Neurochem., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhi
bitors of Gene Expression (SRS Press: Boca Raton, FL, 1988)). The oligonucleotides described above can also be delivered to cells to express antisense RNA or DNA in vivo and inhibit the production of PRO polypeptides. If antisense DNA is used, oligodeoxyribonucleotides derived from the translation initiation site, eg, between the -10 and +10 positions of the target gene nucleotide sequence, are preferred.

Potential antagonists are small molecules that bind to the active site, receptor binding site, or growth factor or other related binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide. Including. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptide organic or inorganic compounds. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within potential RNA targets can be identified by known techniques. Further details can be found in, for example, Rossi, Current B
See Biology 4: 469-471 (1994) and PCT publication, number WO 97/33551 (published September 18, 1997). Nucleic acid molecules in triple-helix formation used to inhibit transcription are single-stranded and composed of deoxynucleotides. The basic composition of these oligonucleotides is designed to promote triple helix formation via Hoogsteen base pairing rules,
It generally requires resizable extension of purines or pyrimidines on one strand of the duplex. For further details, see, for example, PCT publication, number WO 97/33551, supra. These small molecules can be identified by any one or more of the screening assays discussed above and / or by any other screening technique well known to those of skill in the art.

F. Anti-PRO Antibody The present invention further provides an anti-PRO antibody. Examples of antibodies include
Included are polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies. 1. Polyclonal Antibodies Anti-PRO antibodies include polyclonal antibodies. Methods of preparing polyclonal antibodies are known to those of skill in the art. Polyclonal antibodies in mammals can be generated by one or more injections of, for example, 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 polypeptide or a fusion protein thereof. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin and soybean trypsin inhibitor. Examples of adjuvants that may be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol will be selected by one of ordinary skill in the art without undue experimentation.

2. Monoclonal Antibody Alternatively, the anti-PRO antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared using the hybridoma method as described by Kohler and Milstein, Nature, 256: 495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are typically immunized with an immunizing agent to produce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Induce. The lymphocytes can also be immunized in vitro. The immunizing agent will typically include the PRO polypeptide of interest or a fusion protein thereof. Generally, when human-derived cells are desired, peripheral blood lymphocytes (“PBL
)) Is used, or if a non-human mammalian source is desired, spleen cells or lymph node cells are used. Then, a lymphocyte is fused with an immortalized cell line using an appropriate fusion agent such as polyethylene glycol to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Pre
ss, (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 used. The hybridoma cells are preferably
Cultured in a suitable medium containing one or more substances that inhibit the survival or growth of unfused, immortalized cells. For example, if the parental cell lacks the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the hybridoma's medium typically contains hypoxatin, aminoputilin and thymidine.
HAT medium "), this substance prevents the growth of HGPRT-deficient cells. Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. A more preferred immortalized cell line is the mouse myeloma line, which is available, for example, from the Salk Institute Cell Distribution Center in San Diego, Calif. And the American Type Culture Collection in Rockville, Md. Human myeloma and mouse-human heterologous myeloma cell lines for producing human monoclonal antibodies have also been disclosed [Kozbor, J. Immunol., 133: 3001.
(1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applic.
ations, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured is then assayed for the presence of monoclonal antibodies against PRO. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is measured by immunoprecipitation or an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunoassay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody is, for example, Munson and Pollard, Anal. Biochem.,
It can be measured by the Scatchard analysis method according to 107: 220 (1980). After the desired hybridoma cells have been identified, clones can be subcloned by a limiting dilution step 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 can be grown in vivo as ascites in a mammal. The monoclonal antibody secreted by the subclone is, for example, protein A
-Isolated or purified from culture medium or ascites fluid by conventional immunoglobulin purification methods such as Sepharose method, hydroxylapatite chromatography method, gel electrophoresis method, dialysis method or affinity chromatography. Monoclonal antibodies are also prepared by recombinant DNA methods, such as US Pat. No. 4,816,567.
It can be prepared by the method described in No. The DNA encoding the monoclonal antibody of the present invention can be easily prepared using a conventional method (for example, using an oligonucleotide probe capable of specifically binding to the genes encoding the heavy and light chains of mouse antibody). Can be isolated and sequenced. The hybridoma cells of the invention are a preferred source of such DNA. Once isolated, DNA
Can be placed in an expression vector, which is transfected into a host cell, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, Monoclonal antibody can be synthesized with. In addition, DNA may be non-immunized with immunoglobulin coding sequences, for example, by substituting coding sequences for human heavy and light chain constant domains in place of homologous mouse sequences [US Pat. No. 4,816,567; Morrison et al., Supra]. It can be modified by covalently linking part or all of the coding sequence for the globulin polypeptide. Such non-immunoglobulin polypeptides can be substituted for the constant domain of the antibodies of the invention or for the variable domain of one of the antigen binding sites of the antibodies of the invention to produce chimeric, bivalent antibodies. The antibody may be a monovalent antibody. 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 residue is replaced or deleted with another amino acid residue to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Generation of fragments thereof, particularly Fab fragments, by digestion of the antibody can be accomplished using conventional techniques known in the art.

3. Human and Humanized Antibodies The anti-PRO antibodies of the present invention further include humanized antibodies or human antibodies. Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′, F (ab ′) ₂ or other antigen-binding subsequences of antibodies). It contains a minimal sequence derived from non-human immunoglobulin. Humanized antibodies have residues in the complementarity determining region (CDR) of the recipient
Includes human immunoglobulins (recipient antibodies) replaced by residues in the CDRs of a non-human species (donor antibody) with the desired specificity, affinity and potency, such as mouse, rat or rabbit. In some examples, human immunoglobulin Fv framework residues are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has at least one, and typically no, at least one, where all or almost all CDR regions correspond to those of a non-human immunoglobulin and all or almost all FR regions are of human immunoglobulin consensus sequences. Contains substantially all of the two variable domains. A humanized antibody optimally comprises 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 Str.
uct. 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 of non-human origin introduced. These non-human amino acid residues are often referred to as "import" residues, which typically result from an "import" variable domain. Humanization is basically done by replacing the corresponding sequence of the human antibody with a rodent CDR or CDR sequences by winter and coworkers [
Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1).
988); Verhoeyen et al., Science, 239: 1534-1536 (1988)] by replacing the rodent CDRs or CDR sequences with the corresponding sequences of human antibodies. Thus, such "humanized" antibodies are chimeric antibodies in which substantially less than the intact human variable domain has been replaced with the corresponding sequence from a non-human species (US Pat.
, 816, 567). In fact, humanized antibodies are typically human antibodies in which some CDR residues and optionally some FR residues have been replaced by residues from similar sites in rodent antibodies. Human antibodies are also available in phage display libraries [Hoogenboom and Winter, J. Mol.
Biol., 227: 381 (1992); Marks et al., J. Mol. Biol., 222: 581 (1991)], and can be prepared using various methods known in the art. The methods of Cole et al. And Boerner et al. Can also be used for the preparation of human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss. P. 77 (1
985) and Boerner et al., J. Immunol., 147 (1): 86-95 (1991)]. Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, eg, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon administration, production of human antibodies is observed that is very similar to that found in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in US Pat. Nos. 5,545,807;
No. 45,806; No. 5,569,825; No. 5,625,126; No. 5,633,425; No. 5,661,0
16 and the following scientific literature: Marks et al., Bio / Technology 10, 779-783 (1992); Lonb
erg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); F
ishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Bio
technology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 136.
5-93 (1995).

4. Bispecific Antibodies Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. In the case of the present invention, one of the binding specificities is for PRO, the other one is any other antigen,
Preferred is for cell surface proteins or receptors or receptor subunits. Methods of making bispecific antibodies are well known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs in which the two heavy chains have different specificities [Milstein and Cuello, Nature,
305: 537-539 (1983)]. Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually accomplished by affinity chromatography steps. A similar procedure is WO 93/08829 published May 13, 1993, and Traunecker et al., EMBO J., 10: 3655-.
3656 (1991). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain, comprising at least the hinge region, and portions of the CH2 and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion, and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and cotransfected into a suitable host organism. For further details on making bispecific antibodies, see, eg, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another method described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the proportion of heterodimers recovered from recombinant cell culture. . The preferred interface comprises at least 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 (eg tyrosine or tryptophan). Complementary "cavities" of the same or similar size as the large side chains are created at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (eg alanine or threonine). This provides a mechanism to increase the yield of heterodimers relative to unwanted other end products such as homodimers. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ′) ₂ bispecific antibodies). Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, chemical coupling can be used to prepare bispecific antibodies. Brennan et al., Science, 229: 81 (1985) describe a procedure for the proteolytic cleavage of intact antibodies to produce 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 produced are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form the bispecific antibody. The produced bispecific antibody can be used as a drug for selective immobilization of an enzyme. The Fab 'fragment can be recovered directly from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225.
(1992) describes the preparation of fully humanized bispecific antibody F (ab ') ₂ molecules. Each Fab 'fragment is separately secreted from E. coli and undergoes directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed is capable of binding to normal human T cells and cells overexpressing the ErbB2 receptor and induces cytolytic activity of human cytotoxic lymphocytes against human breast tumor targets. Become.

Various methods 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). Leucine zipper peptides from the Fos and Jun proteins are linked by gene fusion to the Fab ′ portions of two different antibodies. The antibody homodimer is reduced at the hinge region to form a monomer and then reoxidized to form the antibody heterodimer. This method can also be used for the production of antibody homodimers. Hollinge
The "diabody" technology described by R et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provided another mechanism for making bispecific antibody fragments. Fragments consist of a heavy chain variable domain ( _VH ) linked to a light chain variable domain ( _VL ) with a linker short enough to allow pairing between two domains on the same chain. Thus, the V _H and V _L domains of one fragment are forced to pair with the complementary V _L and V _H domains of the other fragment, forming two antigen binding sites. Single chain Fv (s
Other strategies for producing bispecific antibody fragments by the use of Fv) dimers have also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994). Antibodies with more than two valencies are possible. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991). Exemplary bispecific antibodies can bind to two different epitopes of the PRO polypeptide provided herein. Alternatively, the arm of the anti-PRO polypeptide is
In order to concentrate the cell defense mechanism on a specific PRO polypeptide-expressing cell,
Trigger molecules on leukocytes such as T cell receptor molecules (eg CD2, CD3, CD28, or B7) or FcγRI (CD64), FcγRII (CD32) and Fc
It may bind to an arm that binds the Fc receptor for IgG (FcγR) such as γRIII (CD16). Bispecific antibodies can also be used to localize cytotoxic drugs to cells expressing a particular PRO polypeptide. These antibodies are P
RO binding arm and cytotoxic agent or radioactive chelator such as EOTUBE,
It has an arm that binds to DPTA, DOTA, or TETA. Other bispecific antibodies of interest bind to the PRO polypeptide and further bind tissue factor (
TF).

5. Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies may be used, for example, to target immune system cells to unwanted cells [US Pat. No. 4,676,980] and for the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. Proposed. It is believed that this antibody can be prepared in vitro using known methods in synthetic protein chemistry, including those associated with cross-linking agents. For example, immunotoxins can be made 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 for example US Pat. No. 4,6767,980.
Include those disclosed in the issue. 6. Effector Function Processing It is desirable to modify the antibody of the present invention with respect to effector function, eg to improve the effectiveness of the antibody in treating cancer. For example, cysteine residue (s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus produced has improved internalization capacity and / or
Or it may have increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, BJ I.
See mmunol. 148: 2918-2922 (1992). Allodimeric antibodies with improved anti-tumor activity can also be prepared using heterobifunctional crosslinks as described by Wolff et al., Cancer research 53: 2560-2565 (1993). Alternatively, the antibody can be engineered to have two Fc regions, thereby improving complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989). 7. Immune Conjugates The present invention also relates to chemotherapeutic agents, cytotoxic agents such as toxins (eg, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) or radioisotopes (ie, radioconjugates). It also relates to an immunoconjugate comprising the conjugated antibody. Chemotherapeutic agents useful in the formation of such immune complexes have been described above. Enzymatically active toxins and fragments thereof that can be used are diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modexin.
(modeccin) A chain, alpha-sarcin, Aleurites fo
rdii) protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S)
, Momordica charantia inhibitor, curcin (
curcin, crotin, sapaonaria ofici
nalis) inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin
n) and trichothecene. Various radionucleotides are available for the production of radioconjugated antibodies. As an example, ²¹² Bi, ¹³¹ I, ¹³¹ I
n, ⁹⁰ Y and ¹⁸⁶ Re. Antibodies and cytotoxic drug conjugates can be conjugated to various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), imidoester bifunctional. Derivatives (dimethyl adipimidate HCL, etc.), active esters (disuccinimidyl suberate, etc.), aldehydes (glutaraldehyde, etc.),
Bis-azide compounds (bis (p-azidobenzoyl) hexanediamine, etc.), bis-
Using diazonium derivatives (bis- (p-diazoniumbenzoyl) -ethylenediamine, etc.), diisocyanates (triene 2,6-diisocyanate, etc.), and bis-active fluorine compounds (1,5-difluoro-2,4-dinitrobenzene, etc.) Can be created. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to the antibody. WO 94/11026
reference. In other embodiments, the antibody may be conjugated to a "receptor" (such as streptavidin) for use in tumor pre-targeting, and the antibody-receptor complex is administered to the patient, followed by a clarifier. It is used to remove unbound complex from the circulation and then administer a "ligand" (such as avidin) conjugated to a cytotoxic drug (such as a radionucleotide).

8. Immunoliposomes The antibodies disclosed herein may also be prepared as immunoliposomes. Liposomes containing antibodies are described by 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. Pat.
Prepared by methods known in the art, such as those described in 485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes are phosphatidylcholine, cholesterol and PEG.
-Produced by the reverse phase evaporation method with a lipid composition containing derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a given size to produce liposomes with the desired size. Fab of the antibody of the present invention
The'fragment can be conjugated to liposomes via a disulfide exchange reaction as described by Martin et al., J. Biol. Chem. 257: 286-288 (1982). Chemotherapy (
Doxorubicin, etc.) is optionally contained within the liposome. Gabizon etc.,
See J. National Cancer Inst. 81 (19) 1484 (1989). 9. Pharmaceutical Compositions of Antibodies Antibodies that specifically bind to the PRO polypeptides identified herein, as well as other molecules identified in the screening assays disclosed above, may be used in pharmaceutical compositions to treat various diseases. It can be administered in the form. When the PRO polypeptide is intracellular and whole antibodies are used as inhibitors,
Internalized antibodies are preferred. However, lipofections or liposomes can also be used to introduce the antibody, or antibody fragment, into cells. When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example,
Based on the variable region sequences of antibodies, peptide molecules can be designed that retain the ability to bind to target protein sequences. Such peptides can be chemically synthesized or can be produced by recombinant DNA technology. For example, Marasco et al., Proc. Natl. Acad. S
See ci. USA 90, 7889-7893 (1993). The formulation herein comprises one or more active compounds as necessary for the particular indication being treated,
Those having complementary activities that preferably do not adversely affect each other may also be included. Alternatively, or in addition, the composition may include a cytotoxic drug, cytokine or growth inhibitor. These molecules are suitably present in combination in amounts that are effective for the purpose intended. Also, the active ingredient may be incorporated into a colloidal drug delivery system (eg , Liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules) or microemulsions. These techniques are disclosed in Remington's Pharmaceutical Science, supra. The formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through a sterile filtration membrane. Sustained-release preparations may be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are shaped articles,
For example, in the form of a film or microcapsules. Examples of sustained release matrices are polyester hydrogels (eg poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polyactides (US Pat. No. 3,773,919).
L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT (trade name) (injectable globules of lactic acid-glycolic acid copolymer and leuprolide acetate) Acid Copolymer, Poly- (D)
Contains 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 the encapsulated antibodies remain in the body for a long time, they are denatured or aggregated by exposure to water at 37 ° C, resulting in reduced biological activity and possible altered immunogenicity. . Rational methods can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism was found to be intermolecular SS bond formation through thio-disulfide exchange, stabilization could be modification of sulfhydryl residues, lyophilization from acidic solution, control of water content, appropriate Can be achieved by the addition of various additives and the development of specific polymer matrix compositions.

G. Uses of anti-PRO antibody The anti-PRO antibody of the present invention has various utilities. For example, anti-PRO antibodies are used in diagnostic assays for PRO, eg, detecting its expression in specific cells, tissues, or serum. Competitive binding assays, direct or indirect sandwich assays and heterogeneous or homogeneous phase immunoprecipitation assays [Zola, Monoclonal Antibodie
s: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158], and various diagnostic assay techniques known in the art are used. The antibody used in the diagnostic assay is labeled with a detectable moiety. Detectable site, directly or indirectly,
It must produce a detectable signal. For example, detectable moieties include radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S or 125 I, fluorescent or chemiluminescent compounds such as fluorescein isothiocyanate, rhodamine or luciferin,
Alternatively it may be an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating a detectable site to an antibody can be used, including Hunter et al. Nature, 1
44: 945 (1962); David et al., Biochemistry, 13: 1014 (1974); Pain et al., J. Immuno.
l. Meth., 40: 219 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407.
The method described in (1982) is included. Anti-PRO antibodies are also useful for affinity purification of PRO from recombinant cell culture or natural sources. In this method, antibodies to PRO are immobilized on a suitable support such as Sephadex resin or filter paper using methods well known in the art. Next, after contacting the immobilized antibody with a sample containing PRO to be purified, a support is used with a suitable solvent that removes substantially all substances in the sample other than PRO bound to the immobilized antibody. To wash. Finally, the support is washed with another suitable solvent that will release PRO from the antibody. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. The entirety of all patents and references cited herein are hereby incorporated by reference.

EXAMPLES All commercial reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of cells identified by ATCC Deposit Number below and throughout the specification is American Type Culture Collection, Manassas, VA. Example 1: Extracellular domain homology screen to identify novel polypeptides and cDNAs encoding them Extracellular domain (ECD) sequences from approximately 950 known secreted proteins from the Swiss-Prot public database ( The secretory signal sequence, if any) was used to search the EST database. EST databases include public databases (eg Dayhoff, GenBank) and corporate databases (eg LIFESEQ (trade name), I
ncyte Pharmaceuticals, Palo Alto, CA). The search is performed by the computer program BLAST or BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (19
96)) was used as a comparison with the 6-frame translation of the EST sequence of the ECD protein sequence. Comparables that do not encode a known protein and have a BLAST score of 70 (sometimes 90) or higher can be found in the program “phrap” (Phil Green, U.
Niversity of Washington, Seattle, WA) and assembled into a consensus DNA sequence. This extracellular domain homology screen was used to construct consensus DNA sequences against other identified EST sequences using phrap. Furthermore, the consensus DNA sequences obtained are often (but not always) BLAST or BLAST-2 and
Elongated using repeated cycles of phrap and consensus sequences discussed above E
The ST sequence source was used to extend as much as possible. Based on the consensus sequence obtained as described above, oligonucleotides are then synthesized, and PCR is used to identify a cDNA library containing the sequence of interest and a clone of the full-length coding sequence for the PRO polypeptide is isolated. Used as a probe. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give PCR products of about 100-1000 bp in length. The probe sequence is typically 40-55b
It is p-long. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. To screen several libraries for full-length clones, DNA from the libraries was added to Ausube
Screening by PCR with PCR primer pairs as in I. et al., Current Protocols in Molecular Biology. The positive library was then used to isolate clones encoding the gene of interest using one of the probe oligonucleotides and the primer pair. The cDNA library used for the isolation of the cDNA clone is Invitrogen, San Di
Prepared by standard methods using commercially available reagents such as those from ego, CA. cd
NA was primed with an oligo dT containing a NotI site, blunt-ended with a SalI hemikinase adapter, cut with NotI, roughly sized by gel electrophoresis, and a suitable cloning vector (such as pRK5B or pRK5D; pRK5B is a precursor of pRK5D that does not contain an SfiI site; Holmes et al.,
Science, 253: 1278-1280 (1991)) at the unique XhoI and NotI sites.

Example 2: Isolation of cDNA clones by amylase screening 1. Preparation of oligo dT primed cDNA library mRNA was isolated from human tissues of interest using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2). This RNA is called Life Techn
oligo dT primed cDNA in vector pRK5D using reagents and protocols from Strategy, Gaithersburg, MD (Super Script Plasmid System)
Used to generate A. In this method, the double-stranded cDNA was size-classified to over 1000 bp and the SalI / NotI binding cDNA was cloned into the XhoI / NotI cut vector. pRK5D is sp6 transcription start site followed by S
It was cloned into a vector having a fiI restriction enzyme site and an XhoI / NotI cDNA cloning site. 2. Preparation of Random Primed cDNA Library A secondary cDNA library was created to preferably represent the 5'end of the primary cDNA clone. Sp6 RNA was generated from the primary library (described above) and this RNA was used as the vector pSST-AMY. It was used to generate a random primed cDNA library using reagents and protocols from Life Technologies at 0 (Super Script Plasmid System referenced above). In this method, double-stranded cDNA was sized to 500-1000 bp, blunt-ended with a NotI adapter, cut at the SfiI site, and cloned into a SfiI / NotI cut vector. pSST-AMY. 0 is a cloning vector that has a yeast alcohol dehydrogenase promoter in front of the cDNA cloning site and a mouse amylase sequence (mature sequence without a secretory signal) followed by the yeast alcohol dehydrogenase transcription terminator after the cloning site. That is,
CDNA cloned into this vector fused in frame with an amylase sequence
A will lead to the secretion of amylase from appropriately transfected yeast colonies. 3. Transformation and Detection DNA from the library described in paragraph 2 above was chilled on ice and electrocompetent DH10B bacteria (Life Technoligies, 20 ml) were added. The mixture of bacteria and vector was then electroporated as recommended by the manufacturer. SOC medium (Life Technplogies, 1 ml) was then added and the mixture was incubated at 37 ° C for 30 minutes. The transformants were then plated on 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37 ° C). Positive colonies were discarded from the plate and DNA was isolated from the bacterial pellet using standard methods, eg CsCl-gradient. The purified DNA was then loaded into the following yeast protocol. Yeast methods can be divided into three categories: (1) transformation of yeast with plasmid / cDNA binding vectors; (2) detection and isolation of amylase secreting yeast clones; and (3) direct from yeast colonies. PCR amplification of inserts and DNA purification for sequencing and further analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain has the following genotypes: MAT alpha, ura3-52, leu2-3, leu2-112, h
It has is3-11, his3-15, MAL ⁺ , SUC ⁺ , and GAL ⁺ . Preferably, a yeast mutant having an incomplete post-translational pathway can be used. Such mutants have translocation-deficient alleles at sec71, sec72, and sec62, with truncated sec71 being most preferred. Alternatively, antagonists (including antisense nucleotides and / or ligands) that inhibit the normal manipulation of these genes, other proteins involved in this post-translational pathway (eg, SEC61p, S
EC72p, SEC62p, SEC63p, TDJ1p or SSA1p-4p)
Alternatively, complex formation of these proteins is also preferably used in combination with the amylase-expressing yeast. Transformation was performed based on the protocol outlined in Gietz et al., Nucl. Acid. Res., 20: 1425 (1992). Transformed cells are then agar to YEPD
Seed in complex medium broth (100 ml) and grow overnight at 30 ° C. YEPD Broth, Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, C
Prepared as described in old Spring Harbor, NY, p. 207 (1994). The overnight medium was then diluted to approximately 2x10 ⁶ cells / ml (approximately OD ₆₀₀ = 0.1) in fresh YEPD broth (500 ml) and 1x10 ⁷ cells / ml (approximately OD _{60 0} = 0.4-0. It was regrown until 5). The cells are then harvested, transferred to a GS3 rotor bottle in a Sorval GS3 rotor for 5 minutes at 5,000 rpm, prepared for transformation by discarding the supernatant and then resuspending in sterile water, and in a 50 ml Falcon tube. And re-centrifuged at 3,500 rpm in a Beckman GS-6KR centrifuge. Discard the supernatant and transfer the cells to LiAc / TE (10 ml
, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li ₂ OOCCH ₃ ) followed by a wash and resuspended in LiAc / TE (2.5 ml). For the transformation, the prepared cells (100 μl) were transferred to fresh denatured single-stranded salmon sperm DNA (Lofstrand Labs, Gaitherburg, MD) and transformed DNA in a microtube.
It was caused by mixing with (1 μg vol. <10 μl). The mixture was vortexed briefly and then 40% PEG / TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li ₂ OOCCH ₃ , pH 7.5) was added. The mixture was gently agitated and incubated at 30 ° C. with agitation for 30 minutes. The cells are then heat shocked at 42 ° C for 15 minutes, the reaction vessel is centrifuged in a microtube at 12,000 rpm for 5-10 seconds, decanted and TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5). Was resuspended and then centrifuged. The cells are then T
Diluted in E (1 ml) and aliquots (200 μl) spread on 150 mm growth plates (VWR) in pre-prepared selective medium. Alternatively, the transformations were performed in one large reaction rather than multiple small reactions, but the amount of reagents was scaled up accordingly. The selection medium used was Kaiser et al., Methods in Yeast Genetics, Cold Spring Har.
Uracil-free synthetic complete dextrose agar (SCD-Ura) prepared as described in Bor Press, Cold Spring Harbor, NY, p. 208-210 (1994). Transformants were grown at 30 ° C for 2-3 days. Detection of colonies secreting amylase was performed by the inclusion of red starch in the selective growth medium. Starch was coupled to a red dye (reactive Red-120, Sigma) according to the method described by Biely et al., Anal. Biochem., 172: 176-179 (1988). Bound starch on SCD-Ura agar plates to a final concentration of 0.15% (w / v)
And buffered to pH 7.0 with potassium phosphate (final concentration 50-100 mM). Positive colonies were picked and streaked on fresh selection medium (150 mm plate) to give well isolated and identifiable single colonies. Well-isolated colonies positive for amylase secretion were detected by direct introduction of the red dye group into buffered SCD-Ura agar. Positive colonies were determined by their ability to degrade starch and form visible halos directly around the positive colonies.

4. Isolation of DNA by PCR Amplification When positive colonies were isolated, a portion was picked with a toothpick and diluted in sterile water (30 μl) in a 96-well plate. At this point, positive colonies are either frozen and either stored for subsequent analysis or immediately amplified.
Aliquots of cells (5 μl) into 0.5 μl of Klentaq (Clontech, Palo Alto, CA);
4.0 μl 10 mM dNTP (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clonte
ch); 0.25 μl forward oligo 1; 0.25 μl reverse oligo 2; used as template for PCR reaction in a 25 μl volume containing 12.5 μl distilled water. The sequence of the forward oligonucleotide 1 was: 5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT -3 '(SEQ ID NO: 25). The sequence of the inverted oligonucleotide 2 was: 5'- CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3 '(SEQ ID NO: 26). PCR was then performed as follows: a. Denaturation 92 ° C, 5 minutes b. Next 3 cycles: Denaturation 92 ° C, 30 seconds Annealing 59 ° C, 30 seconds Extension 72 ° C, 60 seconds c. Next 3 cycles: Denaturation 92 ° C, 30 seconds Anneal 57 ° C, 30 seconds Extension 72 ° C, 60 seconds d. Next 25 cycles: denaturation 92 ° C, 30 seconds Anneal 55 ° C, 30 seconds Extension 72 ° C, 60 seconds e. The regions underlined at 4 ° C. are annealed to the ADH promoter region and the amylase region, respectively, and the vector pSST-AMY. Amplify the 307 bp region from 0. Typically, the first 18 nucleotides at the 5'end of these oligonucleotides contained the sequence primer annealing site. That is, the total product of the PCR reaction from the empty vector was 343 bp. However, the signal sequence fusion cDNA resulted in a rather long nucleotide sequence. Following PCR, an aliquot (5 μl) of the reaction was tested by agarose gel electrophoresis using a Tris-borate-EDTA (TBE) buffer system in a 1% agarose gel as described by Sambrook et al., Supra. . Clones yielding single, strong PCR products larger than 400 bp were cloned into a 96 Qiaquick PCR clean column (Qiagen Inc).
, Chatsworth, CA) and further analyzed by DNA sequence.

Example 3: Isolation of cDNA Clones Using Signal Algorithm Analysis Various polypeptide-encoding nucleic acid sequences are described in Genentech, Inc. (South San
The proprietary sequence-finding algorithms developed by Francisco, CA) are publicly (eg, GenBank) and / or private (LIFESEQ®, Incyte Pharmaceuticals
, Inc., Palo Alto, CA) database and identified by applying to the assembled and assembled EST fragments. The signal sequence algorithm calculates a secretory signal score based on the letters of the DNA nucleotides surrounding the first, and optionally the second, methionine codon (ATG) at the 5'-end of the sequence or sequence fragment under consideration. The nucleotides following the first ATG must encode at least 35 unambiguous amino acids with no stop codon.
If the first ATG has the required amino acids, the second is not tested. If neither met the requirements, the candidate sequences were not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA surrounding the ATG codon and the corresponding amino acid sequence were analyzed using a set of seven sensors (evaluation parameters) known to be associated with the secretory signal. Used to score. The use of this algorithm has led to the identification of many polypeptide-encoding nucleic acid sequences.

Example 4: Isolation of a cDNA clone encoding human PRO1800. As described in Example 1 above, conrap was used to assemble consensus DNA sequences for other ESTs. This consensus sequence is now referred to as DNA3093
Name it 4. Based on the DNA30934 consensus sequence, 1) to identify a cDNA library containing the sequence of interest by PCR, and 2) PRO1.
Oligonucleotides were synthesized for use as probes to isolate 800 full length coding sequence clones. PCR primers (forward and reverse) were synthesized: Forward PCR primer (30934.fl) 5'-GCATAATGGATGTCACTGAGG-3 '(SEQ ID NO: 3) Reverse PCR primer (30934.rl) 5'-AGAACAATCCTGCTGAAAGCTAG-3' ( SEQ ID NO: 4) Further, a synthetic oligonucleotide hybridization probe was prepared from the DNA30934 sequence having the following nucleotide sequence: Hybridization probe (30934.pl) 5'-GAAACGAGGAGGCGGCTCAGTGGTGATCGTGTCTTCCATAGCAGCC-3 '(SEQ ID NO: 5) RNA of the structure of the cDNA library was isolated from human fetal lung tissue. By DNA sequencing of the clones isolated as described above, DNA35672-2
Full-length DNA sequence designated 508 (Fig1, SEQ ID NO: 3); and PRO18
00 derived protein sequences were obtained. The complete nucleotide sequence of DNA35672-2508 is shown in Figure 1 (SEQ ID NO: 1). Clone DNA35672-2508 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 36-38 and ending at the apparent stop codon at nucleotide positions 870-872 (Fig1). The predicted polypeptide precursor is 278 amino acids long (Fi
g2). The full-length PRO1800 protein shown in Figure 2 has a predicted molecular weight of approximately 29,537 daltons and a pI of approximately 8.97. Full length PR shown in Fig2
Analysis of the O1800 sequence demonstrates the presence of the following: a signal peptide from about amino acid 1 to about amino acid 15, a potential N-glycosylation site from about amino acid 183 to about amino acid 186, about amino acid 43 to about amino acid 48, From about amino acid 80 to about amino acid 85, about amino acid 191 to about amino acid 196, about amino acid 213 to about amino acid 218 and about amino acid 272 to about amino acid 277, and about amino acid 276 to about amino acid 278. Microbody C-terminal target signal. Clone DNA35672-2508
Was deposited with the ATCC on Dec. 15, 1998, ATCC Deposit No. 20353
8 was given. The Dayhoff database (version 35.45) using the WU-BLAST2 sequence alignment analysis method of the full-length sequence shown in Fig2 (SEQ ID NO: 2).
SwissProt 35) analysis of the PRO1800 amino acid sequence and the following Dayhoff sequences: HE27_HUMAN, CELF36H9_1, CEF54F3_3, A69621, AP000007_227, UCPA_ECOLI.
, F69868, Y4lA_RHISN, DHK2_STRVN and DHG1_BACME were found to have significant homology.

Example 5: Isolation of a cDNA clone encoding human PRO539 As described in Example 1 above, conrap was used to assemble consensus DNA sequences for other ESTs. This consensus sequence is now DNA4188
Name it 2. Based on the DNA41882 consensus sequence, 1) to identify a cDNA library containing the sequence of interest by PCR, and 2) PRO5
For use as a probe to isolate 39 full-length coding sequence clones,
Oligonucleotides were synthesized. RNA of the structure of the cDNA library was isolated from human fetal kidney tissue. DNA sequencing of the clones isolated as described above revealed that DNA47465-1
Full-length DNA sequence designated 561 (Fig3, SEQ ID NO: 6); and PRO53
Nine derived protein sequences were obtained. The complete nucleotide sequence of DNA47465-1561 is shown in Figure 3 (SEQ ID NO: 6). Clone DNA47465-1561 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 186-188, and ending at the stop codon at nucleotide positions 2676-2678 (Fig3). The predicted polypeptide precursor is 830 amino acids long (Fi
g4). The full-length PRO539 protein is shown in Fig4 and has a calculated molecular weight of approximately 95,029 and a pI of approximately 8.26. Analysis of the full-length PRO539 sequence shown in Figure 4 (SEQ ID NO: 7) demonstrated the following: a leucine zipper pattern sequence of about amino acids 557 to about amino acids 578 and about amino acids 794 to about amino acids 815, about amino acids 133 to about amino acids. There are 136 potential N-glycosylation sites and the kinesin-related protein Kif-4 coiled-coil domain from about amino acids 231 to about amino acids 672. Cloned DNA47465-15
61 was deposited with the ATCC on Feb. 9, 1999, ATCC deposit no.
1 was given. Dayhoff database (version 35.45 Swis using the WU-BLAST2 sequence alignment analysis method of the full-length sequence shown in FIG. 4 (SEQ ID NO: 7)).
sProt 35) analysis revealed that the PRO539 amino acid sequence and the following Dayhoff sequence: AF0
19250_1, KIF4_MOUSE, TRHY_HUMAN, A56514, G02520, MYSP_HUMAN, AF041382_1
, A45592, HS125H2_1 and HS68O2_2 were proved to be homologous.

Example 6: Isolation of a cDNA clone encoding human PRO982 Using the signal sequence algorithm described in Example 3 below, In
The identification of a single IncyteEST sequence, designated cyteEST cluster SEQ ID NO: 43715, was revealed. This EST sequence can be found in public EST databases (eg GenBank) and / or in corporate EST DNA databases (LIFESEQ®,
The presence of homology was identified by comparison with various EST databases, including Incyte Pharmaceuticals, Inc., Palo Alto, CA). Homology searches were performed using the computer programs BLAST and BLAST2 (Altshul et al., Methods in E
nzymology 266: 460-480 (1996)). A comparison that does not encode a known protein and has a BLAST score of 70 (sometimes 90) or higher is the program "phrap".
(Phil Green, University of Washington, Seattle, Washington) to construct a consensus DNA sequence. The consensus sequence obtained is DNA
It is named 56095. In view of the sequence homology between the resulting DNA560095 and Merck EST number AA024389, Merck EST clone AA024389 was collected and sequenced. The sequence named DNA57700-1408 (SEQ ID NO: 8) is Fig5.
Shown in. It is the full-length DNA sequence of PRO982. The full-length clone shown in Figure 5 contains a single open reading frame, has an apparent translational initiation site at nucleotide positions 26-28, and ends at the stop codon at nucleotide positions 401-403 (SEQ ID NO: 8). The predicted polypeptide precursor is 125 amino acids long, has an estimated molecular weight of approximately 14,198 daltons and an estimated pI of approximately 9.01. Analysis of the full-length PRO982 sequence shown in Figure 6 (SEQ ID NO: 9) demonstrated the presence of a signal peptide from about amino acids 1 to 21 and a potential anaphylatoxin domain from about amino acids 50 to 59. Dayhoff database (version 35.45 Swis
sProt 35) analysis revealed that the PRO982 amino acid sequence and the following Dayhoff sequence: RNT
MDCV_1; A48151; WAP_RAT; S24596; A53640; MT4_HUMAN; U93486_1; SYNBILGFG_
1; The homology between P_R49917 and P_R41880 was proved. Clone DNA577
00-1408 was deposited with the ATCC on January 12, 1999 and is assigned ATCC deposit no. 203583.

Example 7: Isolation of a cDNA clone encoding human PRO1434 As described in Example 1 above, conrap was used to assemble consensus DNA sequences for other ESTs. This consensus sequence is now DNA5418
Name it 7. Based on the DNA54187 consensus sequence, 1) to identify a cDNA library containing the sequence of interest by PCR, and 2) PRO1.
Oligonucleotides were synthesized for use as probes to isolate 434 full length coding sequence clones. PCR primers (forward and reverse) were synthesized: Forward PCR primer 5'-GAGGTGTCGCTGTGAAGCCAACGG-3 '(SEQ ID NO: 12) Reverse PCR primer 5'-CGCTCGATTCTCCATGTGCCTTCC-3' (SEQ ID NO: 13) Furthermore, synthetic oligonucleotide The hybridization probe was made from the DNA54187 sequence with the following nucleotide sequence: Hybridization probe 5'-GACGGAGTGTGTGGACCCTGTGTACGAGCCTGATCAGTGCTGTCC-3 '(SEQ ID NO: 14) RNA of the structure of the cDNA library was isolated from human retinal tissue (LIB94). By DNA sequencing of the clones isolated as described above, DNA6
The PRO1434 full length DNA sequence designated 8818-2536 (Fig7, SEQ ID NO: 10); and the derived protein sequence of PRO1434 were obtained. The complete nucleotide sequence of DNA68818-2536 is shown in Figure 7 (SEQ ID NO: 10). Clone DNA68818-2536 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 581-583 and ending at the apparent stop codon at nucleotide positions 1556-1558 (Fig7). The predicted polypeptide precursor is 325 amino acids long (Fig8). The full-length PRO1434 protein shown in FIG.
It has an estimated molecular weight of 296 daltons and a pI of approximately 5.37. Analysis of the full-length PRO1434 sequence shown in Figure 8 (SEQ ID NO: 11) demonstrated the presence of various important protein domains as shown in Figure 8. Clone DNA688
18-2536 was deposited with the ATCC on February 9, 1999 and was assigned ATCC deposit no. 203657. The Dayhoff database (version 35.4) using the WU-BLAST2 sequence alignment analysis method of the full-length sequence shown in FIG. 8 (SEQ ID NO: 11).
5 SwissProt 35) analysis of the PRO1434 amino acid sequence and the following Dayhoff
Array: NEL_MOUSE, APMU_PIG, P_W37501, NEL_RAT, TSP1_CHICK, P_W37500, NEL2
An important homology between _HUMAN, MMU010792_1, D86983_1 and 10MUCS_BOVIN was revealed.

Example 8: Isolation of a cDNA clone encoding human PRO1863 By use of the signal sequence algorithm described in Example 3 above, ES designated Incyte EST cluster SEQ ID NO: 82468 from the Incyte database.
It became possible to identify the T cluster sequence. This EST cluster sequence is then
Public EST database (eg GenBank) and independently developed EST DNA
Existing homologies were identified by comparison with various expressed sequence tag (EST) databases, including databases (Lifeseq ™, Incyte Pharmaceuticals, Palo Alto, CA). The homology search is performed by the computer program BLAST or BLAST2 (Altschul
Et al., Methods in Enzymology 266: 460-480 (1996)). Comparables that do not encode a known protein and have a BLAST score of 70 (sometimes 90) or higher are those of the program "phrap" (Phil Green, University of Washingto
n) were assembled to construct a consensus DNA sequence. The consensus sequence obtained therefrom is herein designated DNA56029. In view of the observed sequence homology between the DNA56029 sequence and the EST sequence contained in Incyte EST clone no. 2186536, Incyte EST clone no. 2186536 was purchased and the cDNA insert was obtained and sequenced. The sequence of this cDNA insert is shown in Figure 9 and is herein designated as DNA59847-2510. Clone DNA59847-2510 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 17-19, and ending at the stop codon at nucleotide positions 1328-1330 (Fig9). The predicted polypeptide precursor is 437 amino acids long (Figure 10). Fig1
The full-length PRO1863 protein shown in 0 has a predicted molecular weight of approximately 46,363 and a pI of approximately 6.22. Full length PRO1 shown in FIG. 10 (SEQ ID NO: 16)
Analysis of the 863 sequence revealed the presence of: a signal peptide of about amino acids 1 to about amino acids 15, a transmembrane domain of about amino acids 243 to about amino acids 260, about amino acids 46 to about amino acids 49, about amino acids 189. Potential N-glycosylation sites of about amino acids 192 and about amino acids 382 to about amino acids 385, about amino acids 51 to about amino acids 54 and about amino acids 359 to about amino acids 362 glycosaminoglycan attachment sites, about amino acids 54 to about amino acids. 5
9, about amino acid 75 to about amino acid 80, about amino acid 141 to about amino acid 14
6, about amino acids 154 to about amino acids 159, about amino acids 168 to about amino acids 173, about amino acids 169 to about amino acids 174, about amino acids 198 to about amino acids 203, about amino acids 254 to about amino acids 259, about amino acids 261 to about amino acids 266, About amino acid 269 to about amino acid 274, about amino acid 284 to about amino acid 289, about amino acid 333 to about amino acid 338, about amino acid 34
7 to about amino acid 352, about amino acid 360 to about amino acid 365, about amino acid 361 to about amino acid 366, about amino acid 388 to about amino acid 393, about amino acid 408 to about amino acid 413, and about amino acid 419 to about amino acid 424.
Is a potential N-myristoylation site. Clone DNA59847-251
0 was deposited with the ATCC on January 12, 1999, and has an ATCC deposit number of 20357.
6 is given. Dayhoff database (version 35.45 Swis) using WU-BLAST-2 sequence alignment analysis of the full length sequence shown in FIG. 10 (SEQ ID NO: 16).
sProt 35) analysis revealed that the PRO1863 amino acid sequence and the following Dayhoff sequence: A
F041083_1, P_W26579, HSA223603_1, MMU97068, RNMAGPIAN_1, CAHX_FLABR, S61
Homology was demonstrated between 882, AB007899_1, CAH1_FLALI and P_W13386.

Example 9: Isolation of a cDNA clone encoding human PRO1917 Using the signal sequence algorithm described in Example 3 above, LIFESEQ (
The identification of the EST cluster sequence of the registered trademark) was revealed and named as EST cluster number 85496. Then, this EST cluster sequence is
The presence of homology was identified by comparison with the ST database. Homology searches were performed using the computer programs BLAST and BLAST2 (Altshul et al.,
Methods in Enzymology 266: 460-480 (1996)). Does not encode a known protein,
Comparables with a BLAST score of 70 (sometimes 90) or higher are programs "phrap" (Phil Green, University of Washington, Seattle, Washington)
Were assembled into a consensus DNA sequence. The consensus sequence obtained is named DNA56415. Obtained DNA56415 and EST contained in EST No. 3255033
In view of sequence homology with the sequences, EST clones derived from ovarian tumor libraries were purchased, cDNA inserts were obtained and sequenced. The sequence of this cDNA insert is shown in Figure 11 and is herein designated as DNA76400-2528. The full-length clone shown in Fig11 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 6-9 and ends at the stop codon at nucleotide positions 1467-1469 (Fig11, SEQ ID NO: 17). . The predicted polypeptide precursor (Fig12, SEQ ID NO: 18) is 48
It is 7 amino acids long. PRO1917 has an estimated molecular weight of approximately 55,051 daltons and an estimated pI of approximately 8.14. Additional features include: a signal peptide at about amino acids 1-30; potential N-glycosylation sites at about amino acids 242-245 and 481-484; about amino acids 95-97, 182-184, and 4
Protein kinase C phosphorylation site at 27-429; about amino acids 107-112
, 113-118, 117-122, 118-123, and 128-133.
A myristoylation site; and an endoplasmic reticulum targeting sequence at about amino acids 484-487. The Dayhoff database (version 35.45 Swi) using the WU-BLAST2 sequence alignment analysis method of the full length sequence shown in FIG. 12 (SEQ ID NO: 18).
ssProt 35) analysis revealed that the amino acid sequence of PRO1917 and the Dayhoff sequence AF0127.
Significant homology with 14_1 was proved. It was also reported that significant homology between the PRO1917 amino acid sequence and the transition from the proliferation to the hypertrophy stage (International Patent Application Publication No. WO9801468-A1) in the chondrocyte sequence designated "P_W52286" in the Dayhoff database. Proved in. Also, the homology with the PRO1917 amino acid sequence and the following additional Dayhoff sequences: P_W52286, GGU59420_1, P_R25597, PPA3_YEAST, PPA.
Proven between 1_SCHPO, PPA2_SCHPO, A46783_1, DMC165H7_1, and AST8_DROME. Clone DNA76400-2528 has been deposited with the ATCC on January 12, 1999 and is assigned ATCC deposit no. 203573.

Example 10: Isolation of a cDNA clone encoding human PRO1868 As described in Example 1 above, conrap was used to assemble consensus DNA sequences against other EST sequences. This consensus sequence is now DNA49
It is named 803. Based on the homology observed between the DNA49803 consensus sequence and the EST sequence contained in Incyte EST clone no. 2994689, Incyte EST clone no. The sequence of the insert is shown in Figure 13, where DNA77624-
It was named 2515. The complete nucleotide sequence of DNA77624-2515 is shown in Figure 13 (SEQ ID NO: 19). Clone DNA77624-2515 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 51-53 and ending at the stop codon at nucleotide positions 981-983 (F
ig13). The predicted polypeptide precursor is 310 amino acids long (Fig.
14). The full-length PRO1868 protein shown in FIG.
It has a calculated molecular weight of 20 and a pI of about 7.90. Analysis of the full-length PRO539 sequence shown in Figure 14 (SEQ ID NO: 20) demonstrated the following: a signal peptide from about amino acid 1 to about amino acid 30, a transmembrane domain from about amino acid 243 to about amino acid 263, from about amino acid 104. N-glycosylation sites from about amino acid 107 and about amino acid 192 to about amino acid 195, cAMP- and cGMP-dependent protein kinase phosphorylation sites from about amino acid 107 to about amino acid 110, about amino acid 106 to about amino acid 109, about amino acid 296. To about 299 amino acid casein kinase II phosphorylation site, about amino acid 69 to about amino acid 77 tyrosine kinase phosphorylation site and about amino acid 26 to about amino acid 31
, About amino acid 215 to about amino acid 220, about amino acid 226 to about amino acid 2
31, potential amino acids 243 to about amino acids 248, about amino acids 244 to about amino acids 249, and about amino acids 262k to about amino acids 267 are potential N-myristoylation sites. Clone DNA77624-2515 was deposited with the ATCC on December 22, 1998 and was assigned ATCC deposit no. 203553. Dayhoff database (version 35.45 SwissPr) using the WU-BLAST2 sequence alignment analysis method of the full-length sequence shown in FIG. 14 (SEQ ID NO: 20).
ot 35), the PRO1868 amino acid sequence and the following Dayhoff sequence: HGS_
RC75, P_W61379, A33_HUMAN, P_W14146, P_W14158, AMAL_DROME, P_R77437, I38
Homology was demonstrated between 346, NCM2_HUMAN and PTPD_HUMAN.

Example 11: Isolation of a cDNA clone encoding human PRO3434 Using the signal sequence algorithm described in Example 3 below, the identification of EST cluster sequences in the Incyte database was revealed. This EST sequence can be found in public EST databases (eg GenBank) and / or corporate EST DNA.
Database (LIFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto
, CA) to identify the presence of homology.
Homology searches were performed using the computer programs BLAST and BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparables that do not encode a known protein and have a BLAST score of 70 (sometimes 90) or higher can be found in the program "phrap" (Phil Green, University of Washington, S.
eattle, Washington) to construct a consensus DNA sequence. The consensus sequence obtained is named DNA56009. In view of the sequence homology between the obtained DNA56009 and the EST sequence contained in Incyte EST clone No. 3327089, Incyte EST derived from ovarian tumor
Clone number 327089 was purchased and the cDNA insert was obtained and sequenced. The sequence of this cDNA insert is shown in Figure 15, where DNA77631-253
Name it 7. Clone DNA77631-2537 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 46-48 and ending at the stop codon at nucleotide positions 3133-3135 (FIg15).
The predicted polypeptide precursor is 1029 amino acids long (Figure 16). F
The full-length PRO3434 protein shown in ig16 has a predicted molecular weight of approximately 114,213 daltons and a pI of approximately 6.42. Analysis of the full-length PRO3434 sequence shown in Figure 16 (SEQ ID NO: 22) demonstrated the presence of various important polypeptide domains as shown in Figure 16. Clone DNA77631
-2537 was deposited with the ATCC on February 9, 1999 and has ATCC Registration No. 20.
3651 is assigned. The Dayhoff database (version 35.45 Swi) using the WU-BLAST2 sequence alignment analysis method of the full-length sequence shown in FIG. 16 (SEQ ID NO: 22).
ssProt 35) analysis, the amino acid sequence of PRO3434 and the following Dayhoff sequences: VATX_YEAST, P_R51171, POLS_IBDVP, IBDVORF_2, JC5043, IBDVPIV_1, VE7_HP
Homology with V11, GEN14220, MUTS_THETH and COAC_CHICK was proved.

Example 12: Isolation of a cDNA clone encoding human PRO1927 By using the signal sequence algorithm described in Example 3 above, LIFESEQ
It is now possible to identify EST cluster sequences from the
It was named ST cluster number 1913. This EST cluster sequence was then translated into additional company EST DNA databases (Genentech, South San Francisc
o, CA) was added to the various expressed sequence tag (EST) databases described above to identify existing homologies. Homology search is a computer program BL
It was performed using AST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparables that do not encode a known protein and have a BLAST score of 70 (sometimes 90) or higher can be found in the program “phrap” (Phil Green, U.
Niversity of Washington, Seattle, Washington)
The NA sequence was constructed. The consensus sequence obtained from it is
It is named 896. In view of the sequence homology between the DNA73896 sequence and the EST sequence contained in EST No. 3326981H1, EST clone 3326981H1 was purchased from a library consisting of RNA isolated from aortic tissue and the cDNA insert was obtained and sequenced. Were determined. The sequence of this cDNA insert is shown in Figure 17 and is herein designated as DNA82307-2531. The full-length clone shown in Figure 17 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 51-53 and ending at the stop codon at nucleotide positions 1695-197 (Fig17; SEQ ID NO: 23). . The predicted polypeptide precursor (Fig 18, SEQ ID NO: 24) is 5
It is 48 amino acids long. PRO1927 has an estimated molecular weight of about 63,198 and about 8
． It has an estimated pI of 10. Additional features include: about amino acids 1-2
3 a signal peptide; about amino acids 6-25 a putative transmembrane domain; about amino acids 5-8, 87-90, 103-106, and 465-469 potential N-
Glycosylation site; contains an N-myristoylation site at about amino acids 6-11, 136-141, 370-375, and 509-514. Dayhoff database (version 35.45 SwissProt 3) using WU-BLAST-2 sequence alignment analysis of the full length sequence shown in FIG. 18 (SEQ ID NO: 24).
Analysis of 5) revealed important sequence homology between the amino acid sequence of PRO1927 and the Dayhoff sequence AB000628_1. The PRO1927 amino acid sequence and the following Dayhoff sequences: HGS_A251, HGS_A197, CELC50H11_2, CPXM_BACSU, VF03_VACCC
, VF03_VACCV, DYHA_CHLRE, C69084 and A64315 were also identified. Clone DNA82307-2531 was cloned into ATCC on December 15, 1998.
And has been assigned ATCC deposit no. 203537.

Example 13: Inhibitory activity assay in mixed lymphocyte reaction (MLR) (Assay 6
7) This example demonstrates that one or more polypeptides of the present invention are active as growth inhibitors of stimulated T lymphocytes. Compounds that inhibit lymphocyte proliferation are
Suppression of the immune response is useful in preferred therapies. The basic protocol for this assay is Immunology, unit3.12; JE Coligan, A
M Kruisbeek, DH Marglies, EM Shevach, W Strober, National Institutes of Health, Jo
It is described in Current Protocols published by hn Wiley & Sons, Inc. More specifically, in one variation of this assay, peripheral blood mononuclear cells (PBMCs) are isolated by leukopheresis from mammalian individuals, eg, human volunteers.
(Supply PBMC to one donor and Responder PB to other donors)
Supply MC). If desired, after isolation, the cells can be treated with fetal bovine serum and DM.
Freeze in SO. Frozen cells were thawed overnight in assay medium (37 ° C., 5% CO ₂ ), then washed and 3 × 10 ⁶ cells / ml assay medium (RPMI; 10% fetal bovine serum, 1% penicillin / streptomycin, 1). % Glutamine, 1% HEPES, 1% non-essential amino acids, 1% pilbert). The stimulant PBMC is prepared by exposing the cells to light (approximately 3000 rads). This assay involves plating in triplicate wells a mixture: 100: 1 diluted 1% or 0.1% test sample, stimulator cells exposed to 50: 1 light, and 50: 1 responder PBMC cells. Is prepared by. 100 microtiter cell culture medium or 100 microtiter CD4-IgG are used as controls. The wells are then incubated for 4 days at 37 ° C., 5% CO ₂ . On day 5, apply tritiated thymidine (1.0 mC / well; Amersham) to each well. After 6 hours, cells are washed 3 times and then label uptake is assessed. In another variation of this assay, PBMCs are isolated from the spleens of Balb / c and C57B6 mice. Cells are for microscopic examination from spleens freshly collected in assay medium (RPMI; 10% fetal bovine serum, 1% penicillin / streptomycin, 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1% pilbert). PMBCs were isolated by cutting them into small pieces and overlaying these cells on Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and washing the mononuclear cell layer in assay medium. Resuspend cells in assay medium to 1 × 10 ⁷ cells / ml. The assay is then performed as described above. All reductions below the control are considered positive results for the inhibitory compound, with reductions of 80% or less being preferred. However, all values below the control show an inhibitory effect on the test protein. The following test polypeptides were positive in this assay: PRO191.
7 and PRO1868.

Example 14 Skin Vascular Permeability Assay (Assay 64) This assay demonstrates that certain polypeptides of the invention immunoreact by inducing mononuclear cell, eosinophil and PMN infiltration at the injection site in mammals. It is shown to promote and induce an inflammatory response. Compounds that enhance the immune response are available for treatments that require enhancement of the immune response. This skin vascular permeability assay is performed as follows. Hairless guinea pigs weighing 350 grams or more are ketamine (75-8
Anesthetized with 0 mg / Kg) and 5 mg / Kg xylazine intramuscular injection (IM). A purified sample of the polypeptide of the invention or a conditioned medium test sample is injected intradermally in the back of a test animal at 100 μl per injection site. About 10-30 per animal
, Preferably about 16-24 sites can be injected. 1 μl of Evans blue dye (1% buffered in saline) is injected intracardially. The spots at the injection site are then measured (diameter mm) 1 and 6 hours after injection. Animals are sacrificed 6 hours after injection. Each skin injection site is biopsied and fixed with formalin. The skin is then prepared for histopathological examination. Each site is examined for inflammatory cell infiltration into the skin. Sites with visible inflammatory cell inflammation are scored as positive. Inflammatory cells can be neutrophils, eosinophils, monocytes, lymphocytes. At least the minimal perivascular invasion at the injection site is scored as positive and negative if there was no invasion at the injection site. The following polypeptides tested positive in this assay: PRO1434

Example 15: Proliferation of Rat Vesicle Supporting Cells (Assay 54) This assay demonstrates that certain polypeptides of the present invention act as mitogens for inner ear supporting cells, which are auditory hair cell precursors, and are therefore mammalian. It is shown that it can be used for inducing regeneration of auditory hair cells and treatment of softness in animals. The assay is performed as follows. Rat UEC-4 vesicle epithelial cells are aliquoted into 96-well plates at 33 ° C. and 200 μl of serum-supplemented medium at a density of 3000 cells / well. The cells are cultured overnight and then transferred to serum-free medium at 37 ° C. Various dilutions of PRO polypeptide (or no control) are then added to the medium and the cells are then incubated for 24 hours. After 24 hours of incubation, ³ H-thymidine (
1 μCi / well) and then incubated for an additional 24 hours. The medium is then washed to remove unincorporated radiolabel and the cells removed and 1
Cpm per well is determined. An assay is considered positive if it has at least 30% or more Cpm in the PRO polypeptide-treated medium compared to control medium. The following polypeptides tested positive in this assay: PRO982

Example 16: Gene Amplification This example demonstrates that PRO1800-, PRO539-, PRO3434-and PR.
It is shown that the O1927-encoding gene is amplified in the genome of certain human lung, colon and / or breast cancer and / or cell lines. Amplification involves overexpression of the gene product,
It has been shown that polypeptides are useful targets for therapeutic treatment in certain cancers such as colon, lung, breast and other cancers. The therapeutic agents are PRO1800, PRO539,
An antagonistic form of the PRO3434 or PRO1927 polypeptide, eg, a mouse-human chimeric, humanized or human antibody against the PRO1800, PRO539, PRO3434 or PRO1927 polypeptide. These amplifications are useful as diagnostic markers for the presence of certain types of tumor types. The starting material for the screen is genomic DNA isolated from various cancers. DN
A is quantitatively accurate, eg fluorescently. 1 DNA as negative control
DNA was isolated from cells of 0 normal healthy individuals, pooled and used as an assay control for gene copy in healthy individuals (not shown). 5'nuclease assay (eg TaqMan ™) and real-time quantitative PCR (eg ABI Prizm 7700
Sequence Detection System (trade name) (Perkin Elmer, Applied Biosystems Divi
(Sion, Foster City, CA)) was used to discover genes that are potentially amplified in certain cancers. The results are DNA-encoded PRO1800, PRO539, PRO34.
34 or PRO1927 was used to determine whether primary lung or colon cancer or cancer cell lines or breast cancer cell lines screened were overexpressed. Primary lung cancer was obtained from individuals with tumors of the types and stages shown in Table 6. An explanation of the abbreviations used to represent the primary tumors listed in Table 6 and the primary tumors and cell lines referenced throughout this example is provided below.

Results for TaqMan ™ are reported in Delta (Δ) Ct units. One unit corresponds to one PCR cycle or about two-fold amplification relative to normal, two units to four-fold, three units to eight-fold amplification and so on. Quantification was performed using primers and PRO1800-, PRO539-,
It was obtained using TaqMan (trade name) fluorescent probe derived from the PRO3434- or PRO1927-encoding gene. PRO1800, P, which appears to contain the most unique nucleic acid sequences and at least does not have spliced introns
Regions of RO539, PRO3434 or PRO1927 are primer and probe derived, eg 3-untranslated regions. PRO1800, PRO539, PRO
The sequences of primers and probes (forward, reverse and probe) used for 3434 or PRO1927 gene amplification analysis are as follows: PRO1800 (DNA35672-2508) Positive 5'-ACTCGGGATTCCTGCTGTT-3 '(SEQ ID NO: 27) Probe 5' -AGGCCTTTACCCAAGGCCACAAC-3 '(SEQ ID NO: 28) Inverse 5'-GGCCTGTCCTGTGTTCTCA-3' (SEQ ID NO: 29) PRO539 (DNA47465-1561) positive 5'-TCCCACCACTTACTTCCATGAA-3 '(SEQ ID NO: 30) probe 5'-CTGTGGTACCCAATTGCCGCCTTGT -3 '(SEQ ID NO: 31) Inverse 5'-ATTGTCCTGAGATTCGAGCAAGA-3' (SEQ ID NO: 32) PRO3434 (DNA77631-2537) Positive 5'-GTCCAGCAAGCCCTCATT-3 '(SEQ ID NO: 33) Probe 5'-CTTCTGGGCCACAGCCCTGC-3 '(SEQ ID NO: 34) Inverse 5'-CAGTTCAGGTCGTTTCATTCA-3' (SEQ ID NO: 35) PRO1927 (DNA82307-2531) Positive 5'-CCAGTCAGGCCGTTTTAGA-3 '(SEQ ID NO: 36) Probe 5'-CGGGCGCCCAAGTAAAAGCTC-3 '(SEQ ID NO: 37) Reverse 5'-CATAAAGTAGTATATGCATTCCAGTGTT-3' (SEQ ID NO: 38) 5'Nuclease assay reaction is a fluorescent PCR-based technique for real-time amplification monitoring. The 5'exonuclease activity of the Taq DNA polymerase enzyme is used. Two oligonucleotide primers are used to generate the amplicon typical of PCR reactions. A third oligonucleotide, or probe, was designed to detect the nucleotide sequence located between the two PCR primers. The probe is non-extendable by the Taq DNA polymerase enzyme and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. When the two dyes are located close together on the probe, the laser-induced emission from the reporter dye is quenched by the quenching dye. During the amplification reaction, the probe is cleaved by the Taq DNA polymerase enzyme in a template-dependent manner. The resulting probe fragment dissociates in solution,
The signal from the released reporter dye does not undergo the quenching effect from the second fluorophore. One molecule of reporter dye is labeled for each newly synthesized molecule, and detection of unquenched reporter dye provides the basis for quantitative interpretation of the data. The 5'nuclease method is used for real-time quantitative PCR such as ABI Prism 7700TM sequence detection.
It is implemented in the device. The system consists of a temperature cycler, laser, charge-coupled device (CCD) camera and computer. The system amplifies samples in 96-well on a temperature cycler. During amplification, the laser-induced fluorescent signal is collected in real time in a 96-well fiber optic cable and detected by the CCD. The system includes software for instrument execution and data analysis. 5'nuclease assay data is initially expressed in Ct or border cycle. This is defined as the cycle in which the reporter signal accumulates above the fluorescent background. The ΔCt value was used as a quantitative measure of the relative starting copy number of a particular target sequence in a nucleic acid sample when comparing normal human DNA results with cancer DNA results. Table 6 shows PRO1800, PRO539, PRO3434 or PRO of the present invention.
Various primary tumor stages, T stages and N used to screen 1927 compounds
Describe the steps.

[0139]

DNA preparation: DNA was prepared from cultured cell lines, primary tumors, normal human blood. Isolations were all performed using the purification kit, buffer set and protease from Quiagen according to the manufacturer's instructions and the following. Cell culture lysis: cells are washed, trypsinized at a concentration of 7.5x10 8 per chip, 5 minutes at 4 ° C
Pelletized by centrifugation at 1000 rpm and then washed with 1/2 volume PBS re-centrifuge. The pellet was washed 3 times and the suspended cells were collected and washed with 2xPBS. The cells were then suspended in 10 mL PBS. Buffer C1 was equilibrated at 4 ° C. Quiagen
Protease # 19155 was diluted with 6.25 ml cold ddH ₂ O to a final concentration of 20 mg / ml and 4
Equilibrated at ° C. 10 mL of G2 buffer was added to the Quiagen RNAse A stock (1
00 mg / ml) was prepared by diluting to a final concentration of 200 μg / ml. Buffer C1 (10 ml, 4 ° C) and ddH ₂ O (40 ml, 4 ° C), then 10 ml
Cell suspension, mixed by inversion and incubated on ice for 10 minutes. Cell nuclei were pelleted by centrifugation in a Beckman swinging bucket rotor at 2500 rpm for 15 minutes at 4 ° C. Discard the supernatant and vortex the nuclei
It was suspended in ₂ ml of buffer C1 (4 ° C.) and 6 ml of ddH ₂ O, and after 1 second, centrifuged at 2500 rpm for 15 minutes at 4 ° C. The nuclei were then resuspended in the remaining buffer using 200 μl per chip. A gentle vortex was applied while adding G2 buffer (10 ml) to the suspended nuclei. When the buffer addition was complete, a strong vortex was applied for 30 seconds. Quiagen protease (200 μl prepared as above) was added and incubated at 50 ° C. for 60 minutes. The incubation and centrifugation was repeated until the lysate was clear (eg, incubated for an additional 30-60 minutes and pelleted at 3000 xg for 10 minutes at 4 ° C).

Preparation and lysis of solid human tumor samples: Tumor samples were weighed and placed in 50 ml conical tubes and kept on ice. Processing was limited to less than 250 mg tissue per preparation (1 tip / preparation). 6.25 ml of protease solution
Prepared freshly by diluting to a final concentration of 20 mg / ml in cold ddH ₂ O 4
Stored at ° C. G2 buffer (20 ml) was prepared (from 100 mg / ml stock) by diluting DNAse A to a final concentration of 200 mg / ml. Tumor tissue was homogenized in 19 ml of G2 buffer for 60 seconds and kept at room temperature using a polytron large tip in a laminar flow TC hood to avoid aerosol inhalation. The polytron was clarified between samples by spinning with ₂ L each of ddH ₂ O for 2 × 30 seconds and then with G2 buffer (50 ml). If tissue was on the generator chip, the device was disassembled and cleaned. Quiagen protease (prepared as above, 1.0 ml) was added, then vortexed and incubated at 50 ° C. for 3 hours. The incubation and centrifugation was repeated until the lysate was clear (eg, incubated for an additional 30-60 minutes and pelleted at 3000 xg for 10 minutes at 4 ° C). Human Blood Preparation and Lysis: Blood was drawn from healthy volunteers using standard infectious agent protocols and citrated to 10 ml samples per chip. Quiagen Protease was freshly prepared by diluting in 6.25 ml cold ddH ₂ O to a final concentration of 20 mg / ml at 4 ° C.
Stored in. G2 buffer (20 ml) was prepared by diluting RNAse A from a 100 mg / ml stock to a final concentration of 200 μg / ml. Blood (10 ml) was placed in a 50 ml conical tube, 10 ml of C1 buffer and 30 ml of ddH ₂ O (both equilibrated at 4 ° C.) were added, mixed by inversion and kept on ice for 10 minutes. Beckma
Nuclei were pelleted on a swinging bucket rotor at 2500 rpm for 15 minutes at 4 ° C and the supernatant discarded. While vortexing, add 2 ml of C1 buffer (4 ° C) to the nucleus.
And 6 ml of ddH ₂ O (4 ° C.). The vortex was repeated until the pellet turned white. The nuclei were then suspended in the remaining buffer using a 200 μl tip. G2 buffer (10 ml) was gently vortexed while adding to the suspension nuclei, then vortexed vigorously for 30 seconds. Add Quiagen protease (200 μl
) And incubated at 50 ° C. for 60 minutes. The incubation and centrifugation was repeated until the lysate was clear (eg, incubated for an additional 30-60 minutes and pelleted at 3000 xg for 10 minutes at 4 ° C).

Purification of clarified lysates: (1) Isolation of genomic DNA: Genomic DNA was equilibrated with 10 ml QBT buffer (1 sample per maximum chip preparation). The QF elution buffer was equilibrated at 50 ° C. The sample was vortexed for 30 seconds, then loaded onto an equilibrated tip and drained by gravity. The chip was washed with 2x15 ml QC buffer. 30 ml of DNA, 30 ml of silanized autoclave
The Cortex tube was eluted with 15 ml QF buffer (50 ° C). Isopropanol (10
0.5 ml) was added to each sample, the tubes were coated with paraffin and mixed by inversion repeatedly until the DNA was precipitated. Samples are run on an SS-34 rotor at 4 ° C for 10 at 15,000 rpm.
Centrifuge for minutes to pellet. Mark the position of the pellet, discard the supernatant, and
70% ethanol (4 ° C) was added. Specimen on SS-34 rotor at 4 ° C for 10,0
Centrifuge at 00 rpm for 10 minutes and pellet again. The pellet position was marked and the supernatant was discarded. The tubes were then placed on each side of the drying cabinet and dried for 10 minutes at 37 ° C, but care was taken to avoid overdrying. After drying, the pellet was dissolved in 1.0 ml TE (pH 8.5) and placed at 50 ° C. for 1-2 hours. The sample was kept at 4 ° C. overnight to continue lysis. The DNA solution was then transferred to a 1.5 ml tube equipped with a 26 gauge needle on a tuberculin syringe. The transfer was repeated 5x to shear the DNA. The sample was then placed at 50 ° C. for 1-2 hours.

(2) Quantification of genomic DNA and preparation for gene amplification assay: The DNA level in each tube was analyzed by standard A ₂₆₀ , A ₂₈₀ spectroscopic analysis of Beckman DU640 at a 1:20 dilution (5 μl DNA + 95 μl ddH ₂ O). It was quantified using a spectrophotometer 0.1 ml quartz cuvette. The A ₂₆₀ / A ₂₈₀ ratio was in the range 1.8-1.9. Each DNA sample was then diluted in TE (pH 8.5) to approximately 200 ng / ml. If the starting material was in high concentration (about 700 ng / μl), the material was left at 50 ° C. for several hours until resuspended. Fluorescent DNA quantification was then performed on the diluted material (20-600 ng / ml) with the manufacturer's instructions modified as follows. This was done by warming the Hoeffer DyNA Quant 200 Fluorometer for about 15 minutes. Hoechst dye working solution (# H33258, 10 μl, prepared within 12 hours of use) was diluted in 100 ml of 1 × TNE buffer. A 2 ml cuvette was filled with the fluorometer solution, placed on the machine and the machine was zeroed. pGEM3Zf (
+) (2 μl, lot # 360851026) was added to 2 ml of fluorometer solution and calibrated to 200 units. An additional 2 μl of pGEM3Zf (+) DNA was then tested to confirm the reading at 400 +/- 10 units. Each sample was then read at least 3 times. 3 samples are 10% of each other
When they were found to be within, they were averaged and this value was used as the quantified value. The concentration determined fluorometrically was then used to dilute each sample to 10 ng / μl in ddH ₂ O. This was done simultaneously for all template samples for one TaqMan plate assay, with enough material to perform the 500-1000 assay. The sample was tested with Taqman (trade name) primer and probe three times, and B-
Both actin and GAPDH are on a single plate with normal human DNA and no template control. The diluted sample was used, but the CT value of normal human DNA subtracted from the test DNA was +/- 1 Ct. The diluted, lot-qualified genomic DNA was stored at -80 ° C in 1.0 ml aliquots. Aliquots that were subsequently inherited and used for amplification assays were stored at 4 ° C. Each 1 ml aliquot is sufficient for 8-9 plates or 64 tests. Gene amplification assay: PRO1800, PRO539, PRO3434 or PRO1927 of the present invention.
The compounds were screened in the following primary tumors and the resulting ΔCt values are reported in Table 7.

[0144]

Example 17: Induction of Pancreatic β-Cell Precursor Proliferation (Assay 117) This assay serves to induce an increase in a large number of pancreatic β-cell progenitor cells in the presence of certain polypeptides of the invention, and thus diabetes. It is shown that it can be used for the treatment of various incomplete insulin stages in mammals including. The assay is performed as follows. The assay uses primary cultures of mouse fetal pancreatic cells, with the initial measurement being changes in expression of markers corresponding to either β-cell precursors or mature β-cells. Marker expression is measured by real-time quantitative PCR (RTQ-PCR);
The marker evaluated here is a transcription factor called Pdx1. The pancreas is removed from E14 embryo (CD1 mouse). Then the pancreas is 4 degrees at 37 degrees
Digested with collagenase / dispase in F12 / DMEM for 0 to 60 minutes (
Collagenase / dispase, 1.37 mg / ml, Boehringer Mannheim, # 1097113).
The digest is then neutralized with an equal volume of 5% BSA and the cells are washed once with RPMI1640. On day 1, cells are plated in 12-well tissue culture plates (20 μ / PBS)
Pre-coated with ml laminin, Boehringer Mannheim, # 124317). 1
-2 embryonic pancreas-derived cells are distributed per well. This primary culture medium is 1
It is 4F / 1640. On the second day, the medium was removed and the adhered cells were RPMI /
Washed at 1640. In addition to the protein to be tested, 2 ml minimal medium is added. On day 4, medium is removed, RNA is prepared from cells and marker expression is analyzed by real-time quantitative RT-PCR. A protein is considered to be active in an assay if it increases the expression of the relevant β-cell marker relative to an untreated control. 14F / 1640 is RPMI1640 (Gibco) with the addition of the following: Group A 1: 1000 Group B 1: 1000 Recombinant human insulin 10 μg / ml Aprotinin (50 μg / ml) 1: 2000 (Boehringer manhein # 981532) Bovine Pituitary Extract (BPE) 60 μg / ml Gentamicin 100 ng / ml Group A: (in 10 ml PBS) Transferrin, 100 mg (Sigma T2252) Epidermal growth factor, 100 μg (BRL100004) Triiodothyronine, 10 μl of 5x10 ⁻⁶ M (Sigma T5516) ) Ethanolamine, 100 μl of 10 ⁻¹ M (Sigma E0135) Phosphoethanolamine, 100 μl of 10 ⁻¹ M (Sigma P0503) Selenium, 4 μl of 10 ⁻¹ M (Aesar # 12574) Group C: (10 ml of 100% ethanol) medium) hydrocortisone, 2 [mu] l of 5x10 ^-3 M (sigma # H0135) progesterone, 1x10 ^-3 M in 100 [mu] l (sigma # P6149) forskolin, 20 mM of 500μl Calbiochem # 344270) Minimal medium: RPMI1640 transferrin (10μ / ml), insulin (1μg / ml), gentamicin (100ng / ml), is added aprotinin (50μg / ml) and BPE (15μg / ml).
Defined medium: Transferrin (10 μ / ml), insulin (1 μg / ml), gentamicin (100 ng / ml) and aprotinin (50 μg / ml) are added to RPMI1640. The following polypeptide was active in this assay: PRO1868.

Example 18: Induction of Pancreatic β-Cell Precursor Differentiation (Assay 89) This assay is derived from pancreatic β-cells matured with certain polypeptides of the invention to pancreatic β-cells.
We show that it acts to induce differentiation into progenitor cells and thus can be used to treat various insulin-deficient stages in mammals, including diabetes. The assay is performed as follows. The assay uses primary cultures of mouse fetal pancreatic cells, with the initial measurement being changes in expression of markers corresponding to either β-cell precursors or mature β-cells. Marker expression is performed by real-time quantitative PCR (RTQ-PC
R); the marker evaluated here is insulin. The pancreas is removed from E14 embryo (CD1 mouse). Then the pancreas is 4 degrees at 37 degrees
Digested with collagenase / dispase in F12 / DMEM for 0 to 60 minutes (collagenase / dispase, 1.37 mg / ml, Boehringer Mannheim, # 1097113)
. The digest is then neutralized with an equal volume of 5% BSA and the cells are washed once with RPMI1640. On day 1, cells are plated in 12-well tissue culture plates (2 in PBS).
Coated with 0 μ / ml laminin, Boehringer Mannheim, # 124317). Cells from pancreas of 1-2 embryos are distributed per well. This primary culture medium is 14F / 1
640. On the second day, the culture medium was removed and the adhered cells were RPMI / 1640.
To be washed. In addition to the protein to be tested, 2 ml minimal medium is added. On day 4, medium is removed, RNA is prepared from cells and marker expression is analyzed by real-time quantitative RT-PCR. Compared to untreated controls
A protein is considered active in the assay if it increases expression of the relevant β-cell marker. 14F / 1640 is RPMI1640 (Gibco) with the addition of the following: Group A 1: 1000 Group B 1: 1000 Recombinant human insulin 10 μg / ml Aprotinin (50 μg / ml) 1: 2000 (Boehringer manhein # 981532) Bovine Pituitary Extract (BPE) 60 μg / ml Gentamicin 100 ng / ml Group A: (in 10 ml PBS) Transferrin, 100 mg (Sigma T2252) Epidermal growth factor, 100 μg (BRL100004) Triiodothyronine, 10 μl of 5x10 ⁻⁶ M (Sigma T5516) ) Ethanolamine, 100 μl of 10 ⁻¹ M (Sigma E0135) Phosphoethanolamine, 100 μl of 10 ⁻¹ M (Sigma P0503) Selenium, 4 μl of 10 ⁻¹ M (Aesar # 12574) Group C: (10 ml of 100% ethanol) medium) hydrocortisone, 2 [mu] l of 5x10 ^-3 M (sigma # H0135) progesterone, 1x10 ^-3 M in 100 [mu] l (sigma # P6149) forskolin, 20 mM of 500μl Calbiochem # 344270) Minimal medium: RPMI1640 transferrin (10μ / ml), insulin (1μg / ml), gentamicin (100ng / ml), is added aprotinin (50μg / ml) and BPE (15μg / ml).
Defined medium: Transferrin (10 μ / ml), insulin (1 μg / ml), gentamicin (100 ng / ml) and aprotinin (50 μg / ml) are added to RPMI1640. The following polypeptide was active in this assay: PRO1863.

Example 19: Mouse Kidney Mesangial Cell Proliferation Assay (Assay 92) In this assay certain polypeptides of the invention serve to induce mammalian kidney mesangial cell proliferation, and, thus, such as, for example, Burger's disease. It has been shown to be useful in the treatment of renal disorders associated with decreased mesangial cell function, or other nephropathy associated with Shaneline-Henoho purpura, celiac disease, herpes dermatitis or Crohn's disease. The assay is performed as follows. On day 1, mouse kidney mesangial cells in 96-well plates in growth medium (Dulbecco's modified Eagle's medium and 95% fetal bovine serum, 5% in a 3: 1 mixture of heme F12 medium supplemented with 14 mM HEPES). And cultivated overnight. On day 2, PRO polypeptide is diluted to two concentrations in serum-free medium and added to cells. The control sample is serum medium only. Day 4, Cell Titer 96 Aqueous 1-liquid reagent (P
rogema) 20 μl is added to each well and the color reaction is allowed to proceed for 2 hours. The absorbance (OD) is then measured at 490 nm. A positive in the assay is one that gives an absorbance reading greater than at least 15% of the control reading. The following polypeptides tested positive in this assay: PRO1917.

Example 20: Proliferation of Fibroblasts (BHK-21) (Assay 98) This assay serves for any polypeptide of the invention to induce the proliferation of mammalian fibroblasts in culture, thus It has been shown to function as a growth factor in mammalian systems. The assay is performed as follows. BHK-21
Fibroblasts are plated in standard growth medium at 2500 cells / well in a total volume of 100 μl. Then, PRO polypeptide, β-FGF (positive control) or nothing (negative control) was added to give a final total volume of 200 μl.
Wells are added in the presence of μl / ml heparin. Then the cells are at 6 ° C for 6
Are incubated for 7 days. After incubation the medium is removed, the cells are washed with PBS and the acid phosphatase substrate reaction mixture (100 μl / well) is added. The cells are then incubated at 37 ° C for 2 hours. Then 10 μl of 1N NaOH is added per well to stop the acid phosphatase reaction. The plate is then read at OD405 nm. Positive in the assay is acid phosphatase activity that is at least 50% above the negative control. The following polypeptide tested positive in this assay: PRO982

Example 21: Chondrocyte Redifferentiation Assay (Assay 110) This assay allows certain polypeptides of the present invention to act on the induction of chondrocyte redifferentiation, and thus various assays such as sports disorders and arthritis. It has been shown to be expected to be useful in the treatment of bone and / or cartilage disorders. The assay is performed as follows. Porcine chondrocytes are isolated by overnight collagenase digestion of the articular cartilage of the metacarpophalangeal joints of 4-6 month old sows. The isolated cells were then supplemented with 25.0% heme F-12 containing 10% FBS and 4 μg / ml gentamicin.
It is plated at 00 cells / cm ² . The medium was changed every 3 days, then the cells were seeded in 96 wells in serum-free 100 μl of the same medium at 5,000 cells / well, 1
00 μl test PRO polypeptide 5 nM staurosporine (positive control)
Alternatively, medium alone (negative control) is added to give a final total volume of 200 μl / well. After 5 days of incubation at 37 ° C, each well is photographed to determine the chondrocyte differentiation status. A positive result in the assay is when chondrocyte redifferentiation was determined to be more similar to the positive control than the negative control. The following polypeptides tested positive in this assay: PRO1863.

Example 22: Use of PRO as a Hybridization Probe The following method describes the use of a nucleic acid sequence encoding PRO as a hybridization probe. The DNA containing the full-length or mature PRO coding sequence disclosed herein is homologous DNA (PR) in human tissue cDNA library or human tissue genomic library.
Used as a probe for screening of naturally occurring variants of O polypeptides, etc.). Hybridization and washing of filters containing either library DNA was performed under the following high stringency conditions. Hybridization of radiolabeled PRO-derivatized probe to the filter consisted 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%.
It was carried out at 42 ° C. for 20 hours in a solution of dextran sulfate. The filter was washed at 42 ° C. in an aqueous solution of 0.1 × SSC and 0.1% SDS. DNAs having the desired sequence identity with the DNA encoding the full length native sequence PRO can then be identified using standard methods known in the art.

Example 23: Expression of PRO in E. coli This example illustrates the preparation of the desired non-glycosylated form of PRO by recombinant expression in E. coli. The DNA sequence encoding PRO was first amplified using selected PCR primers. The primer must have a restriction enzyme site corresponding to the restriction enzyme site of the selected expression vector. Various expression vectors can be used. An example of a suitable vector is pBR322 (derived from E. coli; Boliva
r et al., Gene, 2:95 (1977)) and includes genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzymes and dephosphorylated. The PCR amplified sequence is then ligated into the vector. The vector preferably contains an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first 6 STII codons, polyhis sequences, and an enterokinase cleavage site), a PRO coding region, a lambda transcription terminator, and an argU gene. . The ligation mixture is then used to transform a selected E. coli strain using the method described by Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and then antibiotic resistant clones are selected. Plasmid DNA is isolated and confirmed by restriction analysis and DNA sequence analysis. Selected clones can be grown overnight in liquid media such as LB broth supplemented with antibiotics. The overnight culture is subsequently used to seed large scale cultures.
The cells are then grown to optimal optical density, during which the expression promoter activates. After culturing for a few more hours, the cells can be harvested and centrifuged. The cell pellet obtained by centrifugation is solubilized using various reagents known in the art and then the solubilized PRO protein is purified using metal chelation columns under conditions that allow for tight protein binding. be able to. The following procedure may be used to express PRO in E. coli in the poly-His tagged form. DNA encoding PRO is first amplified using selected PCR primers. Primers provide restriction enzyme sites corresponding to those of the selected expression vector, and efficient and reliable translation initiation, rapid purification on metal chelate columns, and proteolytic removal with enterokinase. Includes other useful sequences. The PCR-amplified poly-His tag sequence was then ligated into an expression vector which was used to strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (la.
cIq)) based transformation of E. coli host. Transformants are initially 50 mg / m
in LB containing 1 carbenicillin at 30 ° C. with shaking at an OD of 3-5. D. 600
Grow until it reaches. The culture was then cultivated in CRAP medium (3.57 g (NH4) 2S
O ₄ , 0.71 g sodium citrate 2H 2 O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Shefield hycase SF in 500 mL water, and 110 mM MPOS, pH 7.3.
, 0.55% (w / v) glucose and 7 mM MgSO ₄ ) in 100)
Diluted twice and grown at 30 ° C. with shaking for about 20-30 hours. Take out the sample and S
Expression was confirmed by DS-PAGE and bulk cultures were centrifuged to pellet cells. The cell pellet was frozen until purification and refolding.

E. coli paste from 0.5 to 1 L of fermentation (6-10 g pellets) was resuspended at 10 volumes (w / v) in 7M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfate and sodium tetrathionate were added to give final concentrations of 0.1M and 0.02, respectively.
M and the solution was stirred at 4 ° C. overnight. This step resulted in a denatured protein with all cysteine residues blocked by sulfite. Beckman solution
Centrifuge for 30 minutes at 40,000 rpm in an Ultracentrifuge. Dilute the supernatant with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and
Clarified by filtration through a 22 micron filter. The clarified extract was loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated with the metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4 ° C. The protein concentration was estimated by its absorption at 280 nm using the extinction coefficient calculated on the basis of its amino acid sequence. The protein was diluted by gradually diluting the sample into a freshly prepared refolding buffer consisting of 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Was refolded. Refolding volume is 50-100 micrograms / ml for final protein concentration
Selected to be. The refolding solution was gently stirred at 4 ° C for 12-36 hours. The refolding reaction was stopped by adding TFA at a final concentration of 0.4% (pH of about 3). Before further purifying the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to a final concentration of 2-10%. The refolded protein was loaded onto a Poros R1 / H reverse phase column with 0.1% TFA.
Chromatographed using 10% migration buffer and elution with a 10-80% acetonitrile gradient. Aliquots of fractions with A280 absorption were analyzed on SDS polyacrylamide gels and fractions containing homologous refolded proteins were pooled. In general, the correctly refolded species of most proteins elute at the lowest concentration of acetonitrile because these species are the most compact and their hydrophobic inner surface is shielded from interaction with reverse phase resin. To be done. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to removing the misfolded form of the protein from the desired form, the reverse phase step also removes endotoxin from the sample. Fractions containing the desired folded PRO polypeptide were pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins were subjected to dialysis or gel filtration on G25 Superfine (Pharmacia) resin equilibrated with preparation buffer and sterile filtration to 20 mM containing 0.14 M sodium chloride and 4% mannitol.
Hepes, pH 6.8. Many PRO polypeptides disclosed herein were successfully expressed as described above.

Example 24: Expression of PRO in Mammalian Cells This example illustrates preparation of a potentially glycosylated form of PRO by recombinant expression in mammalian cells. The vector pRK5 (see EP 307,247 published on Mar. 15, 1989) was used as an expression vector. In some cases, pRK5 with a restriction enzyme that selected PRODNA
And PRODNA is inserted using the ligation method as described in Sambrook et al., Supra. The resulting vector is called pRK5-PRO. In one embodiment, the host cell selected can be 293 cells. Human 293 cells (ATCC CCL 1573) were grown to confluence in tissue culture plates in media such as DMEM supplemented with fetal bovine serum and optionally nutrients and / or antibiotics. About 10 μg of pRK5-PRO polypeptide DNA was added to about 1
μg of VA RNA gene-encoding DNA [Thimmappaya et al., Cell, 31: 543 (1982
))] And mixed with 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227M CaCl ₂
Dissolved in. To this mixture, drop-wise, 500 μl of 50 mM HEPES (pH 7.35), 28
0 mM NaCl, 1.5 mM NaPO ₄ was added and a precipitate was formed at 25 ° C. for 10 minutes. The precipitate was suspended, added to 293 cells, and fixed at 37 ° C. for about 4 hours. The medium was aspirated and 2 ml of 20% glycerol in PBS was added for 30 seconds. The 293 cells were then washed with serum free medium, fresh medium was added and the cells were incubated for about 5 days. Approximately 24 hours after transfection, the medium was removed and the medium (only) or 200 μCi / ml ³⁵ S-
The medium was replaced with a medium containing cysteine and 200 μCi / ml ³⁵ S-methionine. After a 12 hour incubation, the conditioned medium is harvested, concentrated on a spin filter and 15%
Loaded on SDS gel. The treated gel was dried and exposed to film for a selected time that revealed the presence of PRO polypeptide. The medium containing the transformed cells was subjected to a further incubation (in serum-free medium) and the medium was tested in the selected bioassay. In an alternative technique, PRO is based on Somparyac et al., Proc. Natl. Acad. Sci.
., 12: 7575 (1981) and transiently introduced into 293 cells using the dextran sulfate method. 293 cells were grown to maximum density in spinner flasks,
Add 700 μg of pRK5-PRODNA. The cells were first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate was incubated on the cell pellet for 4 hours. Treat cells with 20% glycerol for 90 seconds, wash with tissue culture medium, tissue culture medium, 5 μg / ml bovine insulin and 0.1 μg / ml.
The spinner flask containing bovine transferrin was reintroduced. After about 4 days, the conditioned medium was centrifuged and filtered to remove cells and cell debris. Then expressed P
The RO-containing sample was concentrated and purified by a selected method such as dialysis and / or column chromatography. In another embodiment, PRO can be expressed in CHO cells. pRK5
-PRO can be transfected into CHO cells using known reagents such as CaPO ₄ or DEAE- dextran. As described above, the cell culture medium can be incubated and the medium replaced with culture medium (only) or medium containing a radiolabel such as ³⁵ S-methionine. After identifying the presence of PRO polypeptide, the medium may be replaced with serum free medium. Preferably, the medium is incubated for about 6 days and then the conditioned medium is collected. The medium containing the expressed PRO can then be concentrated and purified by any selected method. The epitope tag PRO may also be expressed in host CHO cells.
PRO may be subcloned from the pRK5 vector. The subclone insert can be subjected to PCR to fuse in frame with a selected epitope tag such as a poly-His tag in a baculovirus expression vector. The poly-his tagged PRO insert can then be subcloned into an SV40 driven vector containing a selectable marker such as DHFR for selection of stable clones. Finally, change the CHO cells to SV4
Transfected with 0-derived vector (as above). Labeling may be performed as described above to confirm expression. The medium containing the expressed poly-his tagged PRO can then be concentrated and purified by a selected method such as Ni2 + -chelate affinity chromatography. Alternatively, PRO may be expressed in CHO and / or COS cells by a transient expression method and in CHO cells by another stable expression method.

Stable expression in CHO cells was performed using the following method. Proteins are IgG constructs (immunoadhesins) in which the soluble sequences of the respective proteins are fused to a constant region sequence containing the hinge, CH2 and CH2 domains of the coding sequence (eg extracellular domain), or poly-. It was expressed as a His-tagged form. Following PCR amplification, the corresponding DNA was prepared using Ausubel et al., Current Protocols of
Subcloned into CHO expression vector using standard techniques as described in Molecular Biology, Unit 3.16, John Wiley and Sons (1997). The CHO expression vector has restriction sites compatible with the 5'and 3'of the DNA of interest, and has a cD
It is created so that NA can be conveniently shuttled. Vectors are Lucas et al., Nuc
cDNAs of interest and dihydrofolate reductase (DHFR) using expression in CHO cells as described in L. Acids res. 24: 9, 1774-1779 (1996).
The SV40 early promoter / enhancer is used to control the expression of (1). DHFR
Expression allows selection for stable maintenance of the plasmid following transfection. Twelve micrograms of the desired plasmid DNA is transferred to the commercial transfection reagent Superfec
t® (Quiagen), Dosper® and Fugene® (Boehringer M
annheim) introduced into about 10 million CHO cells. Cells were grown as described in Lucas et al., Supra. About 3x10 ^-7 cells are frozen in an ampule for further growth and production as described below. The ampoule containing the plasmid DNA was placed in a water bath, thawed, and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 mL of medium and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are mixed with 10 mL of selective medium (0.2 μm filtered PS20, 5%
0.2 μm diafiltered fetal bovine serum) was added). The cells are then split into 100 ml spinners containing 90 ml of selective medium. After 1-2 days, cells are transferred to a 250 mL spinner filled with 150 mL selective medium and incubated at 37 ° C. After another 2-3 days, 250 mL
, 500 mL and 2000 mL spinner were seeded at 3 × 10 ⁵ cells / mL. The cell medium was replaced with fresh medium by centrifugation and resuspended in production medium. Although any suitable CHO medium may be used, in practice the production medium described in US Pat. No. 5,122,469 issued Jun. 16, 1992 was used. A 3 L production spinner was seeded at 1.2 × 10 ⁶ cells / mL. On day 0, cell number and pH were measured. On day 1, spinners were sampled and sprayed with filtered air. On the second day, sample the spinner, change the temperature to 33 ° C and add 500 g / L glucose and 0.6 mL 10% antifoam (eg 35% polydimethylsiloxane emulsion, 30 mL of Dow Corning 365 Medical Grade Emulsion) with 30 mL. did. The pH was adjusted and maintained at around 7.2 throughout the production. After 10 days, or until viability was below 70%, cell culture medium was collected by centrifugation and filtered through a 0.22 μm filter.
The filtrate was either stored at 4 ° C or immediately loaded on a purification column.

For poly-His tagged constructs, proteins were purified using a Ni-NTA column (Qiagen). Prior to purification, imidazole was added to the conditioned medium to a concentration of 5 mM.
Conditioned medium containing 20 mM Hepes, pH 7 containing 0.3 M NaCl and 5 mM imidazole.
A 6 ml Ni-NTA column equilibrated with 0.4 buffer was pumped at 4 ° C at a flow rate of 4-5 ml / min. After packing, wash the column with additional equilibration buffer to remove protein.
Elution was done with equilibration buffer containing 0.25M imidazole. The highly purified protein was subsequently desalted with 25 ml G25 Superfine (Pharmacia) in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, and -80
Stored at ° C. The immunoadhesin (Fc containing) construct was purified from conditioned medium as follows. Conditioned medium was pumped to a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After packing, the column was washed extensively with equilibration buffer and then eluted with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions in a tube containing 275 μl of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted in storage buffer as described above for poly-His tagged proteins. Homogeneity was tested on SDS polyacrylamide gels and N-terminal amino acid sequenced by Edman degradation. Many of the PRO polypeptides disclosed herein have been successfully expressed as described above.

Example 25: Expression of PRO in Yeast The following method describes recombinant expression of PRO in yeast. First, create a yeast expression vector for intracellular production or secretion of PRO from the ADH2 / GAPDH promoter. DNA encoding PRO and a promoter are inserted into the appropriate restriction enzyme sites of the selected plasmid to direct intracellular expression of PRO. A DNA encoding the ADH2 / GAPDH promoter, a native PRO signal peptide or other mammalian signal peptide, or a yeast alpha factor or invertase secretion signal / leader, for example, is selected on a plasmid of choice for the DNA encoding PRO for secretion. Sequence and PR (if necessary)
It can be cloned with a linker sequence for the expression of O. Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatant can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gel with Coomassie blue stain. Recombinant PRO can then be isolated and purified by removing yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing PRO may be further purified using a selected column chromatography resin. Many of the PRO polypeptides disclosed herein have been successfully expressed as described above.

Example 26: Expression of PRO in Baculovirus-Infected Insect Cells The following method describes recombinant expression of PRO in Baculovirus-infected insect cells. The PRO coding sequence was fused upstream of an epitope tag contained in a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (such as the Fc region of IgG). pV _L 1393 (Nova
Various plasmids can be used, including plasmids derived from commercially available plasmids such as gen). Briefly, certain portions of PRO or PRO-encoding sequences, such as sequences encoding the extracellular domain of transmembrane proteins or sequences encoding mature proteins when the protein is extracellular, have 5'and 3'regions. Amplified by PCR with primers complementary to. The 5'primer may include flanking (selected) restriction enzyme sites. The product is then digested with the selected restriction enzymes and subcloned into the expression vector. Recombinant baculovirus was obtained by using Spodoptera frugiperda (“Sf9”) cells (ATCC) containing the above-mentioned plasmid and BaculoGold (trade name) viral DNA (Pharmingen).
CRL 1711) by co-transfection with lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28 ° C, released virus was collected and used for further amplification. Viral infection and protein expression are
illey et al., Baculovirus expression vectors: A laboratory Manual, Oxford: Ox
Performed as described in ford University Press (1994). The expressed poly-his tagged PRO is then purified as follows, eg by Ni ²⁺ -chelate affinity chromatography. Extraction is Rupert et al.,
Prepared from virus-infected recombinant Sf9 cells as described in Nature, 362: 175-179 (1993). Briefly, Sf9 cells were washed and sonicated buffer (25 mM Hepes, pH 7.9; 12.5 mM MgCl ₂ ; 0.1 mM EDTA; 10
% Glycerol; 0.1% NP-40; 0.4M KCl) and resuspended twice on ice 20
Sonicated for 2 seconds. The sonicate was clarified by centrifugation, the supernatant was diluted 50-fold with loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. Ni ²⁺ -NTA agarose column (Qiagen
Commercially available) was prepared in a total volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract was loaded onto the column at 0.5 mL / min. The column was washed with loading buffer to the baseline of A ₂₈₀ , which is the point where fraction collection begins. The column was then washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0) that elutes non-specifically bound proteins. After reaching the A ₂₈₀ baseline again, the column was placed in the secondary wash buffer.
The gradient was developed from 0 to 500 mM imidazole. Fractions of 1 mL were collected and SDS-PAG
E and Ni 2 ⁺ -NT complexed with silver stain or alkaline phosphatase (Qiagen)
Analyzed by Western blot at A. Eluted His 10 _- was dialyzed fractions containing the tagged PRO in loading buffer pool. Alternatively, purification of IgG-tagged (or Fc-tagged) PRO can be performed using known chromatographic techniques, including, for example, protein A or protein G column chromatography. Many of the PRO polypeptides disclosed herein have been successfully expressed as described above.

Example 27: Preparation of Antibodies that Bind PRO This example illustrates preparation of monoclonal antibodies that can specifically bind PRO. Techniques for the production of monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be used are purified PRO, PRO
And a fusion protein containing recombinant PRO on the cell surface. The choice of immunogen can be made by one of ordinary skill in the art without undue experimentation. Mice, such as Balb / c, are immunized with PRO immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally at 1-100 micrograms. Alternatively, the immunogen was replaced with MPL-TDM adjuvant (Ribi Immunochemical Researh, Hamil).
ton, MT) and injected into the hind footpad of the animal. The immunized mice are then boosted 10-12 days later with an additional immunogen emulsified in the selected adjuvant. The mice are then boosted with additional immunization injections for several weeks. Anti PR
Serum samples from retro-orbital hemorrhage may be taken periodically from mice for testing in an ELISA assay for detection of O antibodies. After a suitable antibody titer has been detected, the animals "positive" for the antibody are
The final infusion of RO intravenous injection can be given. After 3 to 4 days, the mice were sacrificed and spleen cells were removed. The spleen cells were then (using 35% polyethylene glycol) P3X63AgU.P3, available from the ATCC under the number CRL1597. Fused to selected mouse myeloma cell lines such as 1. Hybridoma cells were generated by the fusion and then plated on 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit the growth of unfused cells, myeloma hybrids, and spleen cell hybrids. Hybridoma cells are screened in an ELISA for reactivity against PRO. The determination of "positive" hybridoma cells secreting the desired monoclonal antibody against PRO is within the skill of the art. Positive hybridoma cells were injected intraperitoneally into syngeneic Balb / c mice for anti-PR
Ascites fluid containing O monoclonal antibody is generated. Alternatively, hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibody produced in the ascites can be performed using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the affinity of the antibody for protein A or protein G can be used.

Example 28: Purification of PRO Polypeptides Using Specific Antibodies Native or recombinant PRO polypeptides can be purified by a variety of standard protein purification methods in the art. For example, the pro-PRO polypeptide, mature polypeptide, or pre-PRO polypeptide is purified by immunoaffinity chromatography using an antibody specific for the PRO polypeptide of interest. In general, immunoaffinity columns are made by covalently attaching an anti-PRO polypeptide antibody to an activated chromatography resin. Polyclonal immunoglobulins are prepared from immune sera by either precipitation with ammonium sulfate or purification with immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). Similarly, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. The partially purified immunoglobulin is covalently bound to a chromatography resin such as CnBr-activated Sepharose (trade name) (Pharmacia LKB Biotechnology). The antibody is bound to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions. Such immunoaffinity columns are utilized in the purification of PRO polypeptide by preparing fractions from cells containing soluble form of PRO polypeptide. This preparation is derived by the addition of detergents or the solubilization of whole cell or subcellular fractions obtained via differential centrifugation by methods known in the art. Alternatively, soluble PRO polypeptide containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown. The solubilized PRO polypeptide-containing preparation is passed through an immunoaffinity column and the column is washed under conditions that allow for favorable adsorption of PRO polypeptide (eg, high ionic strength buffer in the presence of detergent). The column is then eluted under conditions that cleave the antibody / PRO polypeptide bond (eg, a low pH, such as about 2-3, a high concentration of chaotrope such as urea or thiocyanate ion) to recover PRO polypeptide. .

Example 29: Drug Screening The present invention is particularly useful for screening compounds by using PRO polypeptides or binding fragments thereof in various drug screening techniques. The PRO polypeptide or fragment used in such a test may be free in solution, immobilized on a solid support, carried on the cell surface or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells that are stably transfected with recombinant nucleic acids expressing PRO polypeptides or fragments. Agents are screened against such transfected cells in a competitive binding assay. Such cells, either in viable or immobilized form, can be used for standard binding assays. For example, the formation of complexes between the PRO polypeptide or fragment and the reagent being tested may be measured. Alternatively, the reduction in complex formation between the PRO polypeptide and its target cells caused by the agent being tested can be tested. Accordingly, the present invention provides agents or agents that can affect PRO polypeptide-related diseases or disorders. A method for screening any other reagent is provided. These methods involve contacting the reagent with a PRO polypeptide or fragment, (I) for the presence of a complex between the reagent and the PRO polypeptide or fragment, or (ii) between the PRO polypeptide or fragment and the cell. Including assaying for the presence of a complex between. In these competitive binding assays, the PRO polypeptide or fragment is typically labeled. After appropriate incubation, the free PRO polypeptide or fragment is separated from the bound form and the amount of free or unconjugated label is such that the particular reagent binds to the PRO polypeptide or PRO polypeptide / cell complex. Is a measure of the ability to inhibit. Other techniques for drug screening provide high throughput screening for compounds with suitable binding affinity for polypeptides, 1984 9
It is described in detail in WO 84/03564 published 13th March. Briefly, a number of different small peptide test compounds are synthesized on a solid support such as plastic pins or some other surface. When applied to a PRO polypeptide, the peptide test compound is reacted with the PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well known in the art. Purified PRO polypeptide can also be coated directly onto plates for use in the drug screening techniques described above. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. The present invention also contemplates competitive drug screening assays in which a neutralizing antibody capable of binding to a PRO polypeptide specifically competes with a test compound for a PRO polypeptide or fragment thereof. In this way, the antibody can be used to detect the presence of any peptide in the PRO polypeptide which carries one or more antigenic determinants.

Example 30: Rational Drug Design The purpose of rational drug design is to identify biologically active polypeptides of interest (eg, P
RO polypeptides) or small molecules with which they interact, eg, structural analogs of agonists, antagonists, or inhibitors. Any of these examples can be used to create more active and stable forms of the PRO polypeptide or to create agents that enhance or inhibit the function of the PRO polypeptide in vivo (reference,
Hodgson, Bio / Technology, 9: 19-21 (1991)). In one method, the three-dimensional structure of a PRO polypeptide, or PRO polypeptide-inhibitor complex, is determined by x-ray crystallography, computer modeling, and most typically by a combination of the two methods. Both the shape and charge of the PRO polypeptide must be confirmed in order to elucidate the structure of the molecule and determine the active site. Although small in number, useful information regarding the structure of PRO polypeptides may sometimes be obtained by modeling based on the structure of homologous proteins. In both cases, the relevant structural information is used to design similar PRO polypeptide-like molecules or to identify effective inhibitors. Useful examples of rational drug design include molecules with improved activity or stability as shown in Braxton and Wells, Biochemistry, 31: 7796-7801 (1992), or Athauda et al., J. Biochem. .,
113: 742-746 (1993), including molecules that act as inhibitors, agonists, or antagonists of natural peptides. It is also possible to isolate the target-specific antibody selected by the functional assay as described above and elucidate its crystal structure. This method in principle produces a pharmacore upon which subsequent drug design can be based. Protein crystallography can be bypassed by generating anti-idiotypic antibodies (anti-ids) to functional, pharmacologically active antibodies. As a mirror image of a mirror image, the binding site of anti-ids can be expected to be an analog of the first receptor. Anti-id
Can then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptide will function as the pharmacore. In accordance with the present invention, sufficient amounts of PRO polypeptides are available to carry out analytical experiments such as X-ray crystallography. Further, the knowledge of the PRO polypeptide amino acid sequence provided herein provides the knowledge used in computer modeling techniques in place of, or in addition to, x-ray crystallography.

Material Deposits The following materials have been deposited with the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland, USA (ATCC): Material ATCC Deposit Number Deposit Date DNA35672-2508 203538 1998 12 March 15 DNA47465-1561 203661 February 9, 1999 DNA57700-1408 203583 January 12, 1999 DNA68818-2536 203657 February 9, 1999 DNA59847-2510 203576 January 12, 1999 DNA76400-2528 203573 19991 December 12, DNA77624-2515 203553 December 22, 1998 DNA77631-2537 203651 February 9, 1999 DNA82307-2531 203537 December 15, 1998 These deposits relate to the international recognition of microbial deposits for the purpose of patent procedure. It was conducted in accordance with the provisions of the Budapest Treaty and its Regulations (Budapest Treaty). This guarantees that the viable culture of the deposit will be maintained for 30 years from the date of deposit. The deposit may be obtained from the ATCC in accordance with the terms of the Budapest Treaty and in accordance with the agreement between Genentech and ATCC, whichever is first, at the time of issuance of the relevant U.S. patent or at any time. At the time of publication of a U.S. or foreign patent application, we ensure that the progeny of the deposited culture will be permanently and unrestrictedly available, and will be subject to United States Patent Law Section 122 and the Patent Office Commissioner's Regulations accordingly (especially reference number 886OG638
(Including 37 CFR §1.14) of this document, guaranteeing that descendants will be available to those who have been determined by the US Patent Office Commissioner to have rights. The assignee of the present application agrees that if the deposited culture dies or is lost or destroyed if cultured under the proper conditions, the material will be replaced immediately with the same at the time of notification. To do. The availability of deposited material is not to be construed as a license to practice the invention in breach of any rights recognized under any governmental authority under patent law. The written specification given above is considered sufficient to enable one skilled in the art to practice the invention. The deposited embodiment is intended as an illustration of one of the aspects of the invention and any functionally equivalent construct is within the scope of the invention, so that the deposited deposits may It is not limited. No deposit of material herein is admitted that the written description contained therein is sufficient to enable the practice of any aspect of the invention, including its best mode. It is not to be construed as limiting the scope of the claims to the particular illustrations presented. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[Brief description of drawings]

Figure 1 shows the nucleotide sequence of the native sequence PRO1800 cDNA (SEQ ID NO: 1), SEQ ID NO: 1 is the clone designated herein "DNA35672-2508".

[Figure 2] This shows the amino acid sequence (SEQ ID NO: 2) derived from the coding sequence of SEQ ID NO: 1 shown in Figure 1.

[Figure 3] This shows the nucleotide sequence of the native sequence PRO539 cDNA (SEQ ID NO: 6), and SEQ ID NO: 6 is a clone herein designated "DNA47465-1561".

[Figure 4] This shows the amino acid sequence (SEQ ID NO: 7) derived from the coding sequence of SEQ ID NO: 6 shown in Figure 3.

[Figure 5] Shows the nucleotide sequence of the native sequence PRO982 cDNA (SEQ ID NO: 8), SEQ ID NO: 8 is the clone designated herein as "DNA57700-1408".

[Figure 6] This shows the amino acid sequence (SEQ ID NO: 9) derived from the coding sequence of SEQ ID NO: 8 shown in Figure 5.

[Figure 7] This shows the nucleotide sequence of the native sequence PRO1434 cDNA (SEQ ID NO: 12), wherein SEQ ID NO: 12 is herein referred to as "DNA68818-2536".
It is a clone named.

[Figure 8] This shows the amino acid sequence (SEQ ID NO: 13) derived from the coding sequence of SEQ ID NO: 12 shown in Figure 7.

[Figure 9] This shows the nucleotide sequence of the native sequence PRO1863 cDNA (SEQ ID NO: 17), wherein SEQ ID NO: 17 is referred to herein as "DNA59847-2510."
It is a clone named.

[Figure 10] This shows the amino acid sequence (SEQ ID NO: 18) derived from the coding sequence of SEQ ID NO: 17 shown in Figure 9.

[Figure 11] shows the nucleotide sequence of the native sequence PRO1917 cDNA (SEQ ID NO: 19), wherein SEQ ID NO: 19 is referred to herein as "DNA76400-252".
8 ”is a clone.

[Figure 12] This shows the amino acid sequence (SEQ ID NO: 20) derived from the coding sequence of SEQ ID NO: 19 shown in Figure 11.

[Figure 13] This shows the nucleotide sequence of the native sequence PRO1868 cDNA (SEQ ID NO: 21), wherein SEQ ID NO: 21 is herein referred to as "DNA77624-251."
5 ”is a clone.

[Figure 14] This shows the amino acid sequence (SEQ ID NO: 22) derived from the coding sequence of SEQ ID NO: 21 shown in Figure 13.

[Figure 15] This shows the nucleotide sequence of the native sequence PRO3434 cDNA (SEQ ID NO: 23), wherein SEQ ID NO: 23 is herein referred to as "DNA77631-253.
It is a clone named "7".

[Figure 16] This shows the amino acid sequence (SEQ ID NO: 24) derived from the coding sequence of SEQ ID NO: 23 shown in Figure 15.

[Figure 17] This shows the nucleotide sequence of the native sequence PRO1927 cDNA (SEQ ID NO: 25), wherein SEQ ID NO: 25 is referred to as "DNA82307-253".
It is a clone named "1".

[Figure 18] This shows the amino acid sequence (SEQ ID NO: 26) derived from the coding sequence of SEQ ID NO: 25 shown in Figure 17.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12P 21/02 C 5/10 21/08 15/02 C12R 1:91 C12P 21/02 1 : 645 // C12P 21/08 1:19 (C12P 21/02 C12N 15/00 ZNAA C12R 1:91) 5/00 B (C12P 21/02 15/00 C C12R 1: 645) (C12P 21/02 C12R 1:19) (31) Priority claim number 60 / 113,511 (32) Priority date December 22, 1998 (December 22, 1998) (33) Country of priority claim United States (US) (31) Priority Right claim number 60 / 115,565 (32) Priority date January 12, 1999 (January 12, 1999) (33) Priority claim country United States (US) (31) Priority claim number 60 / 115,558 (32) Priority date January 12, 1999 (January 12, 1999) (33) Country of priority claim United States (US) (31) Priority claim number 60 / 115,733 (32) Priority date January 12, 1999 (January 12, 1999) (33) Priority claim country United States (US) (31) Priority claim No. 60 / 119,341 (32) Priority date February 9, 1999 (1999.2.9) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 119,537 (32) ) Priority date February 10, 1999 (February 10, 1999) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 119,965 (32) Priority date February 1999 12th (1999.12.12) (33) Priority claiming country United States (US) (31) Priority claiming number PCT / US99 / 12252 (32) Priority date June 2, 1999 (1999.6.2) ) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR , K Z, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN YU, ZA, ZW (72) Inventor Ferrara, Napoleon United States California 94109, San Francisco, Pacific Avenue 2090 Sharp 704 (72) Inventor Von, Sherman United States California 94502, Alameda, Basinside Way 19 (72) Inventor Gao, Way-Can United States California 94404, Foster City, Pilgrim Drive 641 (72) Inventor Goddard, Audrey United States California 94131, San Francisco, Congo Street 110 (72) Inventor Gurney, Austin, El. United States California 94002, Belmont, Debbie Lane 1 (72) Inventor Pan, James United States California 94002, Belmont, Coronet Boulevard 2705 (72) Inventor Roy Margaret, Ann United States California 94123, San Francisco, Webster Street 2960 Sharp 4 (72) Inventor Stewart Timothy A. United States California 94114, San Francisco, Douglas Street 465 (72) Inventor Tamas, Daniel United States California 94563, Olinda, Lae Avenue 3 (72) Inventor Watanabe, Colin, Kay. United States California 94556, Moraga, Corris Drive 128 (72) Inventor Wood, William, Ai. United States California 94010, Hillsborough, Southdown Court 35F Term (Reference) 4B024 AA01 BA44 BA80 CA04 CA06 CA07 DA02 DA06 DA12 EA04 FA18 GA11 4B064 AG01 AG27 CA02 CA06 CA10 CA19 CA20 CC24 DA01 4B065 AA26X AA72X AA90X AA90 CA24 AC24 CAA02 CA24 CAA02 CA01 CA24 4H045 AA10 AA11 AA20 BA10 BA41 CA40 DA76 EA22 EA28 FA72 FA74

Claims (21)

[Claims] 1. Fig2 (SEQ ID NO: 2), FIG4 (SEQ ID NO: 7), F
ig6 (SEQ ID NO: 9), Fig8 (SEQ ID NO: 11), Fig10 (SEQ ID NO: 16), Fig12 (SEQ ID NO: 18), Fig14 (SEQ ID NO: 20), F
ig16 (SEQ ID NO: 22) and FIG18 (SEQ ID NO: 24) isolated with at least 80% nucleic acid sequence identity to the nucleotide sequence encoding the amino acid sequence selected from the group consisting of Nucleic acid. 2. Fig1 (SEQ ID NO: 1), Fig3 (SEQ ID NO: 6), F
ig5 (SEQ ID NO: 8), FIG7 (SEQ ID NO: 10), FIG9 (SEQ ID NO :)
15), Fig11 (SEQ ID NO: 17), Fig13 (SEQ ID NO: 19), Fi
An isolated nucleic acid having at least 80% nucleic acid sequence identity with a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in g15 (SEQ ID NO: 21) and Fig17 (SEQ ID NO: 23). 3. Fig1 (SEQ ID NO: 1), FIG3 (SEQ ID NO: 6), F
ig5 (SEQ ID NO: 8), FIG7 (SEQ ID NO: 10), FIG9 (SEQ ID NO :)
15), Fig11 (SEQ ID NO: 17), Fig13 (SEQ ID NO: 19), Fi
An isolated nucleic acid having at least 80% nucleic acid sequence identity with a nucleotide sequence selected from the group consisting of the full length coding sequences of the nucleotide sequences set forth in g15 (SEQ ID NO: 21) and FIG17 (SEQ ID NO: 23). 4. ATCC registration numbers 203538, 203661, 20358
3, 203657, 203576, 203573, 203553, 203651
And an isolated nucleic acid having at least 80% nucleic acid sequence identity with the full length coding sequence of the DNA deposited at 203537. 5. A vector comprising the nucleic acid according to any one of claims 1 to 4. 6. The vector of claim 5, operably linked to a control sequence recognized by a host cell transformed with the vector. 7. A host cell comprising the vector of claim 5. 8. The host cell according to claim 7, wherein the cell is a CHO cell. 9. The host cell according to claim 7, wherein the cell is Escherichia coli. 10. The host cell according to claim 7, wherein the cell is a yeast cell. 11. Production of a PRO polypeptide comprising culturing the host cell of claim 7 under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture medium. Method. 12. Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO: 7),
Fig6 (SEQ ID NO: 9), Fig8 (SEQ ID NO: 11), Fig10 (SEQ ID NO: 16), Fig12 (SEQ ID NO: 18), Fig14 (SEQ ID NO: 20),
An isolated polypeptide having at least 80% amino acid sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequences shown in Figure 16 (SEQ ID NO: 22) and Figure 18 (SEQ ID NO: 24). 13. Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO: 7), F
ig6 (SEQ ID NO: 9), Fig8 (SEQ ID NO: 11), Fig10 (SEQ ID NO: 16), Fig12 (SEQ ID NO: 18), Fig14 (SEQ ID NO: 20), F
at least 80% when compared to an amino acid sequence selected from the group consisting of the amino acid sequences shown in ig16 (SEQ ID NO: 22) and Fig18 (SEQ ID NO: 24)
An isolated polypeptide exhibiting a positive score for. 14. ATCC registration numbers 203538, 203661, 2035
83, 203657, 203576, 203573, 203553, 20365
An isolated polypeptide having at least 80% amino acid sequence identity to the amino acid sequence encoded by the full length coding sequence of the DNA deposited at 1 and 203537. 15. A chimeric molecule comprising the polypeptide of any of claims 12-14 fused to a heterologous amino acid sequence. 16. The chimeric molecule according to claim 15, wherein the heterologous amino acid sequence is an epitope tag sequence. 17. The chimeric molecule according to claim 15, wherein the heterologous amino acid sequence is an immunoglobulin Fc region. 18. An antibody that specifically binds to the polypeptide according to any one of claims 12 to 14. 19. The antibody according to claim 18, wherein the antibody is a monoclonal antibody, a humanized antibody or a single chain antibody. 20. (a) FIG2 (SEQ ID NO: 2) and FIG4 (SEQ ID NO :)
7), Fig6 (SEQ ID NO: 9), Fig8 (SEQ ID NO: 11), Fig10 (
SEQ ID NO: 16), FIG12 (SEQ ID NO: 18), FIG14 (SEQ ID NO: 2)
0), Fig16 (SEQ ID NO: 22) or the polypeptide shown in Fig18 (SEQ ID NO: 24) and lacking its related signal peptide; (b) Fig2 (SEQ ID NO: 2), Fig4 (sequence) No .: 7), FIG6 (SEQ ID NO: 9), FIG8 (SEQ ID NO: 11), FIG10 (SEQ ID NO: 16), F
ig12 (SEQ ID NO: 18), Fig14 (SEQ ID NO: 20), Fig16 (SEQ ID NO: 22) or the extracellular domain of the polypeptide shown in Fig18 (SEQ ID NO: 24), with its associated signal peptide Nucleotide sequence; or (c) Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO: 7), Fig6 (SEQ ID NO: 9), Fig8 (SEQ ID NO: 11), Fig10 (SEQ ID NO: 16), F
ig12 (SEQ ID NO: 18), Fig14 (SEQ ID NO: 20), Fig16 (SEQ ID NO: 22) or the extracellular domain of the polypeptide shown in Fig18 (SEQ ID NO: 24), lacking its associated signal peptide An isolated nucleic acid having at least 80% nucleic acid sequence identity with the nucleotide sequence. 21. (a) FIG2 (SEQ ID NO: 2) and FIG4 (SEQ ID NO :)
7), Fig6 (SEQ ID NO: 9), Fig8 (SEQ ID NO: 11), Fig10 (
SEQ ID NO: 16), FIG12 (SEQ ID NO: 18), FIG14 (SEQ ID NO: 2)
0), Fig16 (SEQ ID NO: 22) or Fig18 (SEQ ID NO: 24) and lacking its related signal peptide; (b) Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO: 7), Fig6 (SEQ ID NO: 9), Fig8 (SEQ ID NO: 11), Fig10 (SEQ ID NO: 16), F
ig12 (SEQ ID NO: 18), Fig14 (SEQ ID NO: 20), Fig16 (SEQ ID NO: 22) or Fig18 (SEQ ID NO: 24), the extracellular domain of the polypeptide with its associated signal peptide; or ( c) Figure2 (SEQ ID NO: 2), Figure4 (SEQ ID NO: 7), Figure6 (SEQ ID NO: 9), Figure8 (SEQ ID NO: 11), Figure10 (SEQ ID NO: 16), F
ig12 (SEQ ID NO: 18), Fig14 (SEQ ID NO: 20), Fig16 (SEQ ID NO: 22) or Fig18 (SEQ ID NO: 24), and the extracellular domain of the polypeptide lacking its associated signal peptide and at least 80 An isolated polypeptide having% amino acid sequence identity.