AU772759B2 - Secreted and transmembrane polypeptides and nucleic acids encoding the same - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): GENENTECH, INC.

Invention Title: SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS ENCODING THE SAME The following statement is a full description of this invention, including the best method of performing it known to me/us: la SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS ENCODING THE SAME FIELD OF THE INVENTION The present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides encoded by that DNA.

BACKGROUND OF THE INVENTION All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the Sreferences states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Extracellular and membrane-bound proteins play important roles in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, proliferation, migration, 25 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 (for instance, mitogenic factors, survival Sfactors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment, usually at a membrane-bound receptor protein.

Secreted proteins have various industrial applications, including use as pharmaceuticals, diagnostics, biosensors and bioreactors. In fact, most protein drugs available at present, such as thrombolytic agents, interferons, Ib interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secretory proteins. Their receptors, which are membrane-bound proteins, also have potential as therapeutic or diagnostic agents. Receptor immunoadhesins, for instance, can be employed as therapeutic agents to block receptor-ligand interaction. Membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. Such membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. Transduction of signals that regulate. cell growth and differentiation is regulated in party by phosphorylation of 15 various cellular proteins. Protein tyrosine kinases, enzymes that catalyze that process, can also act as growth factor receptors. Examples include fibroblast growth factor receptor and nerve growth factor receptor.

Efforts are being undertaken by both industry and academia to identify new, native secreted and membrane-bound receptor proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound receptor proteins. Examples of screening methods and techniques are 25 described in the literature [see, for example, Klein et al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S. Patent No.

5,536,637].

We herein describe the identification and characterization of novel secreted and transmembrane polypeptides and novel nucleic acids encoding those polypeptides.

The entire disclosure in the complete specification of our Australian Patent Application No.

93178/98 is by this cross-reference incorporated into the present specification.

1. PR0211I and PRO217 Epiderna growth factor (I3GF) is a conventional nitogenic factor that stimuhies the proliferation of various types of cells inchidia epithelial cells and fibroblasts. EGF binds to and activates dhe EGF receptor (EGFR), which initiates intracellular signaling and subsequnt effects. The EGFR is expressed in neurons of the cerebral cortex.

cerebellum, Sand hippocampus in addition to other regions of tie central nervous system (CNS). In addition, EGF is also expessd in various regions of the CNS. Therefore, EGF acts not only on initotic cells. but also on postiiotic neurons. In fact. many studies have indicated that EGF has nrurotrophic or neazromodulatory effects on various types of neurons in die CNS. For example. EGF acts directly on cultured cerebral cortical and cerebellar neurons, enhancing neurite outgrowth and survival. On the other handl, EUF also acts on other cell types, including septal cholinrgic and mesencephalic dopaininergic neurons, indirectly through glial cells. Evidence of the effects of EGF on neurons in die CNS is accumulating, but the mechanisms of action remnain essentially unknmown. EGF-rnduced sigaling in snitotic cells is betterunderstood dhan in postnitotic neurons. Studies of coned pheochromocytoma PCI2 oell and cultured cerebral cortical neurons have suggested that the EGF-induccd neurotrophic actions are mediated by sustained activation of the EGFR and mnitogen-activated protein kinase (MAPK) in response to EGF. The sustained inraicellular signaling correlates with the decreased rate of EGFR down-regulation, which miight determine the response of neuronai cells to EGF. It is likely that EGF is a multi-potent growth factor that acts upon various types of cells including mitotic cells anid posimitotic neurons.

is produced by the salivary and Brnner's glands of the gastrointestinal system, kidney, pancreas, thyroid gland, pituitary gland, and the nervous system, and is found in body fluids such as saliva, blood, ccrebrospinal fluid (CS urine, amnniotic fluid, prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman, 20 Peptides .12: 653-663 (1991).

EUF is mediated by its miembrane specific receptor, which containis an intrinsic tyrosine kinase. Stoscbeck d al., J. Cell Bioclzem. 31: 135-152 (1986). EGF is believed to function by binding to the extraceliular portion of its receptor which induces a traosmenibrane signal that activates the intrinsic tyrosine kidae.

Purification and sequence analysis of the EGF-like domain has revealed dhe presence of six conserved a 25 cysteine residues which cross-bimi to create three peptide loops, Savage et al.. J. Biol Chem. W4: 7669-7672 (1979).

It is now generally known that several other peptides; can react with the EGF receptor which share the same generalized motif X.CX XCX 5 0

CXCX

5

GX

2 CX. where X represents any non-cysteine amin acid, and n is a variable repeat nimbr. Non isolated peptides having this motif include TGF-t, anmphiregulin, schwannoma-derived growth factor (SDGI-). heparin-binding EGF-Iike growth factors and certain virally encoded peptidcs Vaccinia virus, Reisner, Nature 2n3: 801-803 (1985), Shope fibromna virus, Chang et al., Mol Cell Biol. 7: 535-540 (1987), Molluscum ontagiosurn, Porter and Ardiard, J. Gen. Virol. 673-68 (1987). and Myxoma virus, Upton el al..

J. Virol. Al: 1271-1275 (1987), Prigent and Lemoine. Prog. Growh Factor Res. 1: 1-24 (1992).

EGFlikc domains arc not confihed to growth factors but have been observed in a variety of cell-surface and extracellilar proteins which have interesting properties in coil adhesion, protein-protein interaction and development, Luia=c and Gusterson, 71wnor Biol. 11: 229-261 (1990). These proteins include blood coagulation factors (factors VI. Ex. X. XII. protein C, protein S. protein Z. tissue plasminogen activator, urokinase), extracelluar matrix components (laminin, cytotactin, entactin), cell surface receptors (LDL receptor, thrombomodulin receptor) and iiumity-rclated proteins (comaplemient Cir. urornodulna).

Even more interesting. the genera structure pattern of EGp-like precursors is Preserved through lower Organisms as well as in mammalian cells. A number of genes with developmental significance have been identified in invertebrates with EGP-like repeats. For example. the notch gene of Drosophila encodes 36 tandemly arranged amino acid repeats which show homology to EGF, Wharton ei aL. Cell 41: 557. '581 (1985). Hydropathy-plots indicate a putative membrane spanning domain. with the EGF-related sequences being located on the extracellular side of the membrane. Other homeotic genes with EGF-like repeats include Delta, 95F and SZD which were identified using probes based on Notch, and the nemabode gene Lin42 which encodes a putative receptor for a developmental signal transmitted between two specified cells.

Specifically. EGF has been shown to have potential in the preservation and maintenance of gastrointestinal mucosa and the repair of acte and chronic nmucosal lesions, Konturek et at., Eur.). Gasrroenterol Heparol. 2(10), 933-37 (1995), including dhe treatment of necrotizing enterocolidis, Zollinger-Ellison syndrome, gastrointestinal ulceration gastrointestinal ulcerations and congenital microvillus atrophy, Guglietta and Sullivan, Eur. Gsontemrvi, liepaol, 2(10), 945-50 (1995). Additionally. EGF has been implicated in hair follicle differentiation; dur Cros, Invest. Dernatol. 1I1 (I Suppl.). 106S-1 13S (1993), Hillier, in. Endocrnol. U3(4), 427-28 (1990); kidney function, Hanme a l, Semin. Nephrol. 13 109-15 (1993), Hamfs. Am. J. Kidney Dis. 11(6):,627-30 (1991); tear fluid, van Settene a aL, Int. Ophthainwi 359-62 (1991); vitamin K mediated blood coagulation, Stcnflo a aL. Blood 28(T: 1637-51 (1991). EGF is also implicated various skin disease characterized by abnormal keratinocyte differentiation, psoriasis, epithelial cancers such as squamous cell carcinomas of the lung, epidermold carcinoma of the vulva and gliomas. King et at.. Am. Med. Sci. 226: 154-158 (1988).

~*20 Of great interest is mounting evidence that genetic alterations in growth factors signaling pathways are closely linked to developmental abnormalities and to chronic diseases including cancer. Aaronson, Science 1146-1153 (1991). For example, c-erb-2 (also known as HER-2), a proto-oncogene with close structural similarity to EGF receptor protein. is overxpressed in human breast cancer. King at Science 22: 974-976 (1985); Gullick, Honmones and their actions, Cooke et at., cds, Amsterdam, Elsevier. pp 349-360 (1986).

We herein describe the identification and characterization of novel polypeptides having homology to EGF.

wherein those polypeptides are herein designated PRO211I and PRO2 17.

2. EBRO230 Nephritis is a condition characterized by inflammation of the kidney affecting the structure and normal function of the idney. This condition can be chronic or acute and is generally caused by infection, degenerative process or vascular disease. In all cases, early detection is desirable so that the patient with nepbxitls can begin treatment of the condition.

An approach to detecting ncphritis is to determine the antigens associated with nephritis and antibodies thereto. In rabbit, a tubulointerstitial nephritis antigen (TIN-ag) has been reported in Nelson, T. et al., 1. iol.

Q=m, 270C27):16265-70 (July 1995) (GENBANK/U24270). This study reports that the rabbit TIN- ag is a basement membrane glycoprotein having a predicted armmo acid seqiznce which has a carboxyl-terminal region exhibiting 30 homology with human preprocathepsin B, a member of the cystein proteinase family of proteins. It is also reported that the rabbit TIN-ag has a domain in the amino-terminal region containing an epidermal growth factor-like motif that shares homology with laminin A and S chains, alpha 1 chain of type I collagen, von Willebrand's factor and mucin, indicating structural and functional similarities. Studies have also been conducted in mice. However, it is desirable to identify tubulointerstitial nephritis antigens in humans to aid in the development of early detection methods and treatment of nephritis.

Proteins which have homology to tubulointerstiial nephritis antigens are of particular interest to the medical and industrial communities. Often, proteins having homology to each other have similar function. It is also of interest when proteins having homology do not have similar functions, indicating that certain structural motifs identify information other than function, such as locality of function. We herein describe the identification and characterization of a novel polypeptide, designated hgerein as PR0230, which has homology to tubulointerstitial nephritis antigens.

3. PRO23 Stem cells are undifferentiated cells capable of proliferation, self maintenance, the production of a large number of differentiated functional progeny. regeneration of tissue after injury and/or a flexibility in ""15 the use of these options. Stem cells often express cell surface antigens which are capable of serving as cell specific markers that can be exploited to identify stem cells, thereby providing a means for identifying and isolating specific stem cell populations.

Having possession of different stem cell populations will allow for a number of important applications. For example, possessing a specific stem cell population will allow for the identification of growth factors and other S, 20 proteins which are involved in their proliferation and differentiation. In addition, there may be as yet undiscovered o. *proteins which are associated with the early steps of dedication of the stem cell to a particular lineage, (2) prevention of such dedication, and negative control of stem cell proliferation, all of which may be identified if one has possession of the stem cell population. Moreover, stem cells are important and ideal targets for gene therapy where the inserted genes promote the health of the individual into whdm the stem cells are transplanted. Finally, stem cells may play important roles in transplantation of organs or tissues, for example liver regeneration and skin grafting.

Given the importance of stem cells in various different applications, efforts are currently being undertaken by both industry and academia to identify new, native stem cell antigen proteins so as to provide specific cell surface markers for identifying stem cell populations as well as for providing insight into the functional roles played by stem cell antigens in cell proliferation and differentiation. We herein describe the identification and characterization of novel polypeptides having homology to a stem cell antigen, wherein those polypcptides are herein designated as PR0232 polypeptides..

4. PRQ187 Growth factors are molecular signals or mediators that enhance cell growth or proliferation, alone or in concert, by binding to specific cell surface receptors. However, there are other cellular reactions than only growth upon expression to growth factors. As a result, growth factors are better characterized as multifunctional and potent 4 ceMUla regulsors. Their biological effect include prolifration, chemotaxis and stinnalaion of extracellular matrix production. Growth factors can have both stimulatory and inhibitory effects. For example. transforming growth factor (rGF-A) is highl pleiotropc and can stirmulate proliferation in some cells, especially connective tissue, while being a potent inhibitor of proliferation in others. such as lymphocytes and epithelial cells.

711e physiological effect of growth stimulation or inhibition by growth factors depends upon the s tate of developinnt and differentiation of the target tissue. The mechan;im of local cellular regulation by classical endocrine molecules involves comprehends autocnine (sanme cell), juxtacrine (neighbor cell), and paracrine (adjacent cells) pathways. Peptde growth factors ame clnnrir of a complex biological language, providing the basis for intercellular comunication. They permit cells to convey information between each other, mediate interaction between cells and change gem expresion. The effect of these mnultifuinctional and pluripotent factors is dependent on the presence or absence of other peptdes.

FGP-S is a member of the fibroblast growth factors (FO~s) which are a family of beparin-binding, potent mtogens for both normal diploid fibroblasts and established cell lines, Gospodarowicz ea a. (1984). Proc. Nail.

Acod Sd. LISA 1:96. The FGF family comprises acidic FGF (PGF-l), basic FGF (FGF-2), INT-2 (PGF-3), K- FGF/HST (FGF-4), FGP-S. FGF-6, KGF (FGF-7). AIGF (FGF-8) among others. All FGFs have two conserved cysreine residues and share 30-50% sequence homology at the amino acid level. These factors are niitogenic for a wide variety of normal diploid mesoderm-derived and neual crest-derived cells, including granulosa cells, adrenal cortical ells, chondrocytes, myoblasts. cornea] and vascular endothelial cells (bovine or human), vascular smooth muscle cells, lens, retina and prostatic epithelial cells, oligodendrocytes, astrocytes, chrondocytes. myoblasts; and osteoblasts.

Fibroblast growth factors can also stimulate a large number of cell types in a non-mitogenic manner. These activities include promotion of cell migration into wound area (chemtotaxis), initiation of new blood vessel formulation (anigiogenesis), modulation of nerve regeneration and survival (rieurotrophism), modulation of endocrine functions, and stimulation or suppression of specific cellular protein expression, extracellular matrix production and cell survival. Baird Bohlen, Handbook of Exp. Pharmacol. 95(1): 369-418. Springer, (199). These properties provide :*25 a basis for using fibroblast growth factors in therapeutic approaches to a clrate wound healing, nerve repair, collateral blood vessel formation, and the like. For example, fibroblast growth factors have been suggested to miiiemyocardium damage in heart disease and surgery 4.378.347).

F-S. also known as androgen-induced growth factor (AIGF), is a 215 amino acid protein which shares 30-40% sequence homology with the other members of the FGF family. FGF-8 has been proposed to be under androgenic regulation and induction in the mouse mammary carcinoma cell line SCI. Tanaka ar aL, Proc. Nat!.

Aci. Sd. USA 12: 892848932 (199); Sato r al., Steroid Biochem. Molec. Biol. 47: 91-98 (1993). As a result, FGP-S may have a local role in the prostate, which is known to be an androgen-responsive organ. FGF-8 can also be oncogenic, as it displays transforming activity when trantsfected into NIH-3T3 fibroblasts. Kouhara et aL, OMwogene 2 455-462 (1994). While FGF-8 has been detected in heart, brain, lung, kdmy, testis. prostate and ovary, expression was also detected in the absence of exogenous androgens. Schmitt er al., J. Steroid Blochemn. Mot. Biol.

.U 173-78 (1996).

FGF-8 shares the property with several other FO~s of being expressed at a variety of stages of murine embryogenests. which supports the theory that the various FGFs have: multiple and perhaps coordinated roles in differentiation and embryogenesis. Moreover, FGF-8 has also been identified as a protooncogenc that cooperates with Wnt-l in the process of mammary tumongetesis (Shackleford et at.. Proc. Nat!. 4 d. Sci. USA 2Q. 740-744 (1993); Heikinheimo et al.. Mechz. Dev. 129-138 (1994)).

In contrast to the other FOFs, FGF-8 exists as three protein isoforms, as a result of alternative splicing of the primary transcript. Tanaka et al.. supra. Normal adult expression of FGF-8 is weak and confined to gonadal tissue, however northern blot analysis has indicated that FOF8 xnRNA is present from day 10 through day 12 or marine gestation, which suggests that FGF-8 is important to normal development. Heildnheinio e al.. Mech Dev.

41(2): 129-38 (1994). Further in sit hybridization assays between day 8 and 16 of gestation indicated initial expression in the surface ectodernm of the first bronchial arches, the frontonasal process, the forebrain and the midbrain-hindbrain junction.. At days 10-12, FGF-8 was expressed in the surface ectoderm of the forelimb and hindlimb buds, the nasal its and nasophazynx, the infundibujum and in the telencephaloni. diencephalon and metencephalon. Expression continues in the developing hindilirobs through day 13 of gestation, but is undetectble thereafter. The results suggest that FGF-8 has a unique temporal and spatial pattern in cmbryogenesis and suggests a role for this growth factor in multiple regions of ectodermal differentiation in the post-gastrulation embryo.

We herein describe the identification of novel poypeptides having homology to FGF-8, wherein those polypeptides are heein designated PRO 187 polypeptides.

S. PRO265 Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the structural and futnctional mechanisms underlying protein-protein interactions, protein-protein interactions can be more easily manipulated to regulate the particular result of the protein-protein interaction. Thus.

the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community.

All proteins containing ieucine-rich repeats are thought to be involved in protein-protein interactions.

Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. The crystal structure of ribotuelease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha. structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual. nonglubular shape. These two features have been indicated as responsible for the protein-binding functions of proteins containing leucine-rich repeats. See, Kobe and Deisenliofer, Trends iociem- Sd, 19(10):415-421 (Oct. 1994).

A study has been reported on leucine-rich proteoglycans which serve as tissue organizers, orienting and ordering collagen fibrils during ontogeny and are involved in pathological processes such as wound healing, tissue repair, and tumor stroma formation. lozzo. R. Crit. Rev. ioiem. Mat. iol., 32(2):141-174 (1997). Others studies implicating leucine rich proteins in wound healing and tissue repair are De IA Salle. et al., XVlL...Rll.

Fr- Heratg1. (Germany), 37(4):215-2 (1995), reporting mutations in the leucine rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome and Chlemetson, K. Thrmb. Haernost. (Germany), 74(1): 111-116 (July 1995), reporting that platelets have leucine rich repeats. Another protein of particular interest which has been reported to have leucine-rich repeats is the SLIT protein which has been reported to be useful in treating noiro-egeneadve diseases such as Alzheirnr's disease. nerve: damage such as in Parkinson's disease, and for diagnosis of cancer, see, Artavanistsakonas, S. and Rotberg. J. W09210518-Al by Yale University. Other studies reporting on the biological functions of proteins having Icucine-rich repeats include: Tayar. et al.. MQL QUIEnadg~~o., (Ireland), 125(1-2):65-70 (Dec. 1996) (ganadotropin receptor inlvolvenment); Miura. et al., VjiDL.RIZIIgl (Japan), 54(7):1784-1789 (July 1996) (apoptosis involvement); Harris, P. et al., L-m Sc ftm. 6(4):1125-1133 (Oct. 1995) (kidney diseae involvement); and Ruoslalit, E. L. et al.. W09110727-A by LA Jolla Cancer Research Foundation (dccorin binding to tranforming growth factor-p involvement for trcarrcnt for cancer. wound healing and scarring). Also of particular interest is fibromodulin and its use to prevent or reduce dermal scarring. A study of fibromodulin is found in. U.S. Patent No. 5,654,270 to Ruoslalir, et al.

Effoarts are therefore being undertaheni by both industry ard academia to identify new proteins having icucine rich repeats to better understand protein-protein interactions. Of particular interest are those proteins having leucine rich repeats and! homology to known proteins having leucine rich repeats such as fibrorodulin, the SLIprotein and *..*platelet glycoprotein V. Many efforts are focused on the screening of miammnalian recombinant DNA libraries to **.identify the coding seqluences for novel secreted and membrane-bound proteins having leucine rich repeats. We herecin describe the identification and characterization of novel polypeptides having homology to fibrornodulin, herein designated as PR0265 polypeptides.

6. PROa29 Human matrilin-2 polypeptide is a member of the von Wiliebrand factor type A-like module superfamily.

von Willebrand fa=o is a protein which plays an imnportant role in the maimsenence of hemostasis. More specifically, von Wilecbrand factor is a protein which is known to participate in platelet-vessel. wall interactions at the site of vaclrhMvaisaiiyt neatadfr ope ihFco VIII. Ihe absence of von Wilebrand factor in the blood causes an abnormality with the blood platelets that prevents platelet adhesion to the vascular wall at the site of the vascular injury. The: result is the propensity for bnzsing, nose bleeds, intestinal bleeding, and the like comprising von Willebrand's disease.

Given tie physiological importance of the blood clotting factors, efforts are currently being undertaken by both industry and! academia to identify new, native proteins which may be involved in the coagulation process. We herein describe the identification of a novel full-length polypeptide which possesses homology to the human matrilin-2 precursor polypeptide.

7. PROQZ4 The cell surface protein HCAR is a membrane-bound protein that acts as a receptor for subgroup C of the adenoviruses and subgroup B of the coxsackievirues. Thus, HCAR mnay provide a means for mediating viral infection of cells in that the presetc= of the HCAR receptor on the cellular surface provides a binding site for viral particles, thereby facilitating viral infection.

In light of the physiological importance of membrane-bound proteins and specficially those which serve a cell surface receptor for viruses, efforts are currently being undertaken by both industry and academia to identify new, nattive membrane-bourd receptor proteins. Many of these efforts are focused on the screening of mammalian recombinant DNA libraries to ideify die coding sequence for novel receptor proteins. We herein describe a novel membrane-bournd polypeptide (designated heren as PR0246) having homology to the cell surface protein HCAR and to various ano= antigens inchuding A33 and carcinoe~mh~c antigen, wherein this polypeptide may be a novel cell surface virus receptor or tumor antigen.

S. PR02ZU There am a number of known seven traniembraiie protis anid within this family is a group which includes CD97 and EMR1. CD97 is a seven-span transmemhrane receptor which has a cellular ligand, CDSS, DAF.

Hamann, et al., J.E ,Md 184(3):1199 (1996). Additionally, CD97 has been reported as being a deifrntain marker in htnsn thyroid carcinomas and as associated with inflammation. Aust, et al.. CacrRs 57(9):1798 (1997); Gray, et al., J. I~numol. IS7(M2:5438 (1996). CD97 has also been reported as being related to the secrcma receptor superfamily, but unlike known members of that family, CD97 and EMRI have extended extracellular regions that possess several EGF domains at the N-tminus. Hamann, -et al.. GenoMjcs.

32(l): 144 (1996); Harmann, et al., J.JImmuzml., 155(4):1942 (1995). EMRI is further described in Lin, et al., Geois 41(3).301 (1997) and Baud. et al., Genomics. 26(2):334 (1995). While CD97 and EMRI appear to be related to the secretin receptors, a known member of the secredin family of G protein-coupled receptors includes the alpha-latroxin receptor, latrophilin, which has been described as calcium independent and abundant among neuronal tissues. Uliantova. et al.. J. Biolhmi. 272C34). 2150.4 (1997); Davletov, et al., L il h 271(38):23239 (1996). Both members of the sccretin receptor superfamily and non-members which are related to the secretin receptor superfamily, or CRF and ealcitonin receptors are of interest. In particular, new members of these families, identified by their homology to knorwn proteins, are of interest.

Efforts are being undertaken by both industry and academia to identify new membrane-boirod receptor proteins, particularly transtembrane proteins with EGF repeats and large N-terminuses which may belong to the family of scven-transmembranc proteins of which CD97 and EMRI are members. We herein describe the identification and charactization of novel polypeptides having homology to CD97 and EMR1. designated herein as PRO22 polypeptides.

PR 3 Growth factors are molecular signals or mediators that enhance cell growth or proliferation, alone or in concert. by binding to specific cell surface receptors. however, there are other cellular reactions than only growth upon expression to growth factors. As a result, growth factors are better characterized as multifunoctional and potent celluar regulators. Their biological effects include proliferation, chemnotaxis and stimulation of extracelkilar matrix production. Growth factors can have both stimulatory and inhibitory effects. For example, transforming growth factors CTGF-P) is higbly pleictropic and can stimnulate prolifeation in some cells, especially connective tissues, while being a potent inhibitor of proliferation in others, such as lymphocytes and epithelial cel The physiological effect of growth stimulation or inhibition by growth factors depends upon the state of developmnt and differentiation of the target tisse. The mechanism of local cellular regulation by classical endocrine WO 99/14329 WO 9914328PCTIUS9SlI933O molecules comprehends autoajace (same cell). jw acrine (neighbor cell). and paracrine (adjacent cell) pathways.

PePtide growth factors are elcments of a complex biological language, providing the basis for intercellular communication. They permit cells to convey information between eiach other. mediate interaction between cells and change gee expression. the effect of these multifuanctional and phunpotent factors is dependent on the presence or absence of 9ther peptidcs.

Fibroblast growth factors (FGFs) are a family of heparin-binding, potent mitogens for both normal diploid fibroblasts and established cell lines, Godpodarowicz. D. et al. (1984), Proc. Natl. AM&d Sci. USA 8 1: 6983. the FOF family comprises acidic FOF (FGF-l1), basic FGF (FGF-2). INT-2 (FGF-3), K-FGF/HT (FGF-4), FGF-S, FGF-6. KGF (FGF-7). AIOF (FGF-8) among others. All FGFs have two conserved cysteine residues and share 30-S/oh sequence homology at the amino acid level. These factors are mitogenic for a wide variety of normal diploid mesoderm-desived and neural crest-derived cells inducing granulosa cells, adrenal cortical cells, chrondocytes myoblasts, cortical and vascular eadothelial cells (bovine or human), vascular smooth uiscle cells, lens, retina and prostatic epithelial cells, :9 oligodaidrocytes, astrocytes, chrondocytes, Myoblasts and osteoblASs.

Fibroblast growth factors can also stimulate a large number of cell types in a non-initogenic maniner. These* 9 9activities include promotion of cell migration into a wound area (chemotaxis), initiation of new blood vessel formulation (arigiogaiesis). modulation of nerve regeneration and survival (neurotrophism). modulation of endocrine functions, and stimulation or suppression of specific cellular protein expression, extracellular matrix production and cell survival.

A. Bohlen, Handbook of Ep. Phrmoal. 25( 369-418 (1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healing, nerve repair. collateral blood vessel formation, and~thc like. For example, fibroblast growth factors, have beeni suggested to minimize myocardium damage in heart disease and surgery (IJ.S.P. 4,378,437).

herein describe the identification and characterization of novel polypeptides having homology to FGF, herein designated PR0533 polypeptides.

PRQ24 Somte of the most important proteins involved in the above described regulation and modulation of cellular processes are the enzymes which regulate levels of protein phosphorylation in the cell. For example, it is known that dhe tanduction of signals that regulate cell growth and differentiation is regulated at least in part by phosphorylation and dephosphorylaton of various cellular proteins. The enzymes that catalyze these processes include the protein kinases, which function to phosphorylate various cellular proteins, and the protein phosphatases, which function to remove phosphate residues from various cellular proteins. The balance of the level of protein phosphorylaeicm in the cell is thus mediated by the relative activities of these two types of enzymes.

Althouh manny protein kinas enzymies have been identified, the physiological role played by many of these catalytic proteins has yet to be elucidated. It is well known, however, that a numnber of the known protein kinases functioni Sophosphorylate tyrosine residues in proteins, thereby leading to a variety of different effects. Perhaps most importantly, there has been a great deal of interest in the protein tyrosine kinases since the discovery that many orrcogene products and growth factors possess intrinsic protein tyrosine kinase activity. There is, therefore, a desire to identify new members of the protein tyrosine kinase family.

Given the physiological importune of the promein kinases, efforts are being undertaken by both industry and academia to dentif new, native Itinase proteins. Many of these efforts are focused on the screening of mammralian recombinant DNA libraries to identify the coding sequences for novel kinase proteins. We herein describe the identification and characterization of novel polypeptides having homology to tyrosine kinase proteins, designated herein as PR0245 polypeptides.

11. PRO220. PR0221and PRO227 Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the structural and fiuncionmal mechani ms underlying protein-protein interactions, protein-protein interactions can be niore easily manipulated to regulate die particular result of the protein-prortein interaction. Thus, the underlying mechanisms of protein-protein interactions are of interet to the scientific and medical community.

All proteins containing leucine-ricm repeats are thoughit to be involved in protein-protein intractions.

Leucine-rich repeats are short sequence motifs present in a numiber of proteins with diverse functions and ellular locations. 1Te crystal structure of ribomiuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglubular shape. These two features have been indicated as responsible for the protein-binding functions of proteins containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends iochem- Sci.. 19(10):.415-421 (Oct. 1994).

A study has been reported on leucine-ridh proteoglycans which serve as tissue organizers. orienting and ordering collagen fibrils during ontogeny and are involved in pathological processes such as wound healing, tissue :20 repair, and rmnor stroina formation. Iozzo, Rt. Crnt.-Rev. iocheni. Mol. Biol., 32(2):141-174 (1997). Others studies implicating leucine rich proteins in wound healing and tissue repair are De La Salle, et aL. Vouv. Rev- 000 e matolzg. (Germany), 37(4).215-222 (1995). reporting mutations in the leucine rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome and Cblenietson. K. Throm. tiHamio. (Germany), *74(l):111-116 (July 1995), reporting that platelets have leucine rich repeats. Another protein of particular interest *0 ~25 which has been reported to have leucine-rich repeats is the SJT protein which has been reported to be useful in treating neuro-degenerative diseases such as Alzheiier's disease, nerve damage such as in Parkinson's disease, and for diagnosis of cancer. see, Artavanistsakoaas, S. and Rothberg. J. W09210518-AI by Yale University. Other studies reporting on the biological functions of proteins having leucine-rich repeats include: Tayar. e t al.. M-L.

CelEndorimmi., (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor involvement); Miura, et al..

ftn Rinsh (Japan), 54C7):1784-1799 (July 1996) (apoptosis involvement); Harris. P. et al., J.Am..Socftkpl.. 6(4):1125-1 133 (Oct. 1995) (kidney disease involvement); and Ruoslahti. E. L. er al.. W09110727-A by La Jolla Cancer Research Foundation (decorin binding to trantsformning growth factor3 involvement for treatment for cancer, wound healing and scarring).

Efforts are therefore being undertaken by both industry and academia to identify new proteins having leucine rich repeats to better understandl protein-protein interactions. Of particular interest are those proteins having leucine rich repeats and homology to kniown proteins having leucine rich repeats such as the SLIT protein and platelet glycoprotein V.

12. PROUS Immunoglobulins are antibody molecules, the proteins that function both as receptors for antigen on the Bcell membrane and as the secreted products of the plasma cell. Like all antibody molecules, immunoglobulins perform two major functions: they bind specifically to an antigen and they participate in a limited number of biological effector unctions. Therefore, new members of the Ig superfamily are always of interest. Molecules which act as receptors by various viruses and those which act to regulate immune function are of particular interest. Also of particular interest are those molecules which have homology to known Ig family members which act as virus receptors or regulate immune function. Thus, molecules having homology to poliovirus receptors, CRTAM and CD166 (a ligand for lymphocyte antigen CD6) are of particular interest.

Extracellular and membrane-bound proteins play important roles in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, 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 (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or 15 signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extraccllular environment, usually at a membrane-bound receptor protein.

We herein describe the identification and characterization of novel polypeptides having homology to CRTAM, designated herein as PRO258 polypeptides.

13. PROQ66 Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be more easily manipulated to regulate the particular result of the protein-protein interaction. Thus, .the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community.

25 All proteins containing leucine-rich repeats are thought to be involved in protein-protein interactions.

Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. The crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface S exposed to solvent, so that the protein acquires an unusual nonglobular shape. These two features have been indicated as responsible for the protein-binding functions of proteins containing leucine*rich repeats. See. Kobe and Deisenhofer, Trends Biochem Sci. 19(10):415-421 (Oct. 1994).

A study has been reported on leucine-rich proteoglycans which serve as tissue organizers, orienting and ordering collagen fibrils during ontogeny and are involved in pathological processes such as wound healing, tissue repair, and tumor stroma formation. lozzo, R. Crit. Rev. Biochem. Mol Bil.. 32(2):141-174 (1997). Others studies implicating leucine rich proteins in wound healing and tissue repair are De La Salle, ct al., Youv. Rev.

r. I maol. (Germany), 37(4):215-222 (1995). reporting mutations in the leucine rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome and Chlcmenson. K. Thromb. Haemost. (Germany), 7401):111-116 (July 1995). reporting that platelets have leucin rich riepeats. Another protein of particular interest which has been reported to have leucine-rich repeats is the SWT protein which has been reported to be useful in treating narm-degenerative diseases such as Alzheimer's disecase, nerve damage such as in Parkinson's disease, and for diagnosis of cancer sec. Antavanistsaoas, S. and Rodhherg, J.1M.. W09210518-Al by Yale University. Other studies reporting on the biological functions of proteins having leucine-rich repeats include: Tayar. et al., M2L Cell Enorio. (Ireland). 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor involvement); Miura. et al., N~ipponRinmho (Japan). 54M7:1784-1789 (July 1996) (apoptosis involvement); Harris, P. et al., L-An...Soc.

Nqt. 6(4):1125-1 133 (Oct. 1995) (kidney disease involvement); and Ruoslahui, E. et al., W09110727-A by La Jolla Cancer Research Foundation (decorin binding to transforming growth factorP involvement for treatment for cancer, wound healing and scarring).

Efforts are therefore being undlertaken by both iudustry and academia to idea*f~ new proteins having leucine rich repeats to better understand protein-protein interactions. neuronal development and adhesin molecules. Of particular imercst are those proteins having leucine rich repeats and homology to knawn proteins having leucine rich repcats such as the SLI protein. We herein describe novel polypeptkdes having homology to SLIT, designated herein as PR0266 polypeptides.

14. PRO26 7Tnrombomodulin binds to and regulates the activity of thrombin. Iz is important in the control of blood .coagulation. Thrombomodulin functions as a naftura anticoagulant by accelerating the activation of protein C by thronmbin. Soluble thrombornodulin may have theapeutic use as an witithrombotic agent with reduced risk for 20 hemnorrhage as compared with heparin. Thrombomodujin is a cell surface um~ns-muembrAne glycoprotein, present on crudothelial cells and platelets. A smaller, functionally active: form of thrornbomodulin circulates in the plasma and is also found in urine. (in Haeberli, Human Protein Data, VCH Dub., 1I92). Peptides having homtology to throinbornodulin are particularly desirable.

S S**We herein describe the identification and characterization of novel polypeptides having homology to 25 thrombomodulin, designated herein as PR0269 polypeptides.

515. PRQ287 Procollagen C-protein aseehanccr protein binds to and enhances the activity of bone morphogenic protein "BMPI lprocollagen C-proteinasc (PCP). It plays a role in extracellular matrix deposition. BMP I proteins may be used to induce bone and/or cartilage formation and in wound healing and tissue repair. Therefore, procollagen Cproteinase enhaner~ protein, BMP I and proteins having homology thereto, are of interest to the scientific and medical communities.

We herein describe the identification and characterization of novel polypeptides having homology to procollagen C-proteinas Cenmn protein peursor and procollagen C-proteinasc enhiancr protein, designated herein as PR0287 polypeptides.

16. PRZ214 Growth factors are molecular signals or mediators that enhances cell growth or proliferation, alone or in concert, by binding to specific cell surface receptors. However, there are other cellular reactions than only growth upon expression to growth factors. As a result, growth factors are better characterized as multifunctional and potent cellular regulators. Their biological effects include proliferation, chemotaxis and stimulndation of extraccllular matrix production. Growth factors can have both stimulatory and inhibitory effects. For example, transforming growth factor P (IGF-P) is highly pleiotropic and can stimndate proliferation in some cells, especially connective tissue, while being a potent inhibitor of proliferation in others, such as lymphocytes and epithelial cells.

The physiological effect of growth stimulation or inhibition by growth factors depends upon the state of development and differentation of the target tissue. The mechanism of local cellular regulation by classical endocrine molecules involves comprehends autocrine (same cell), juxtacrine (neighbor cell), and paracrine (adjacent cells) pathways. Peptide growth factors are clements of a complex biological language, providing the basis for intercellular communication. They permit cells to convey information between each other, mediate interaction between cells and O change gene expression. The effect of these multifunctional and pluripotent factors is dependent on the presence or absence of other pcptidcs.

15 Epidermal growth factor (EGF) is a conventional mitogcnic factor that stimulates the proliferation of various types ofcells including epithelial cells and fibroblasts. EGF binds to and activates the EGF receptor (EGFR), which ,°initiates intracellular signaling and subsequent effects. The EGFR is expressed in neurons of the cerebral cortex, cerebelmn, and hippocampus in addition to other regions of the central nervous system (CNS). In addition, EGF is also expressed in various regions of the CNS. Therefore, EGF acts not only on mitotic cells, but also on postmitotic 20 neurons. In fact. many studies have indicated t ht EGF has neuroophic or neuromodulatory effects on various types of neurons in the CNS. For example, EGF acts directly on cultured cerebral cortical and cerebellar neurons, enhancing neurite outgrowth and survival. On the other hand, EGF also acts on other cell types, including septal cholinergic and mesencephalic dopaminergic neurons, indirectly through glial cells. Evidence of the effects of EGF on neurons in the CNS is accumulating, but the mechanisms of action remain essentially unknown. EGF-induced signaling in mitotic cells is better understood than in postnitoic neurons. Studies of cloned phcochromocytoma PC 12 cells and cultured cerebral cortical neurons have suggested that the EGF-induced neurotrophic actions arc mediated by sustained activation of the EGFR and mitogen-activated protein kinase (MAPK) in response to EGF. The sustained intracellular signaling correlates with the decreased rate of EGFR down-regulation, which might determine the response of neuronal cells to EGF. It is likely that EGF is a multi-potent growth factor that acts upon various types of cells including mitotic cells and postmitoic neurons.

EGF is produced by the salivary and Brnner's glands of the gastrointestinal system, kidney, pancreas.

thyroid gland, pituitary gland, and the nervous system, and is found in body fluids such as saliva, blood, cerebrospinal fluid (CSF). urine, amniotic fluid, prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman, CR Peptides 12: 653-663 (1991).

EGF is mediated by its membrane specific receptor, which contains an intrinsic tyrosine kinase. Stoscheck CM et al.. J. Cell Biochem. 31: 135-152 (1986). EGF is believed to function by binding to the extracellular portion of its receptor which induces a transmcmbrane signal that activates the intrinsic tyrosine kinase.

Purification and sequence analysis of the EUF-like domain has revealed the presence of six conserved cysteine residues which cross-bind to create three peptide loops. Savage CR et at., L. Biol. Ch~em. 2A3: 7669-7672 (1979). Itis now generally knvown that several other peptides can react with the EGF receptor which share the same generalized motif XCXCX,:CX,,CXCX,GXC)C.. where X represents any non-cysteine amio acid, mand a is a variable repeat nmber. Non isolated peptides having this motif include TGF-a. amphiregulin, schwannonia-derived growth ftctor (SDGF), heparin-bidn EGF-lik growth factors and certain vitally encoded peptides Vaccinia virus. Reisxtr All. Nature 113: 801-803 (198), Shope fibroma virus, Chang et al., Mal Cell Biol. 7: 53-SAC) (1987), Molluscum conagiosum. Porter CID Archard LC, L. Gen. Virol. 673-682 (1987), and Myxoma virus, Upton C et al.. J. Virol. Qj: 1271-1275 (1987). Prigent SA Lemoine Prog. Growh Factor Res. 4: 1-24 (1992).

EGF-lk domains are not confined to growth factors but have been observed in a variety of cell-surface and extracellular proteins which have interesting properties incell adhesion, protein-protein interaction and development, Laurence DiR Gusterson BLA, Tumor Biol. 11: 229-261 (1990). Thnese proteins include blood coagulation factors (factors VI, IX, X. XIM. prtein C, protein S. protein Z. tissue plasminogen activator, urokinase), extracellular matrix es.. components (laminin, cytotactin, entactin). cell surface receptors (LDL receptor. thrombomodulin receptor) and immunity-related proteins (complemnt Cir, uromodulin).

Even more interesting, the general structure pattern of EGE-like precursors is preserved through lower organisms as well as in mammalian cells. A number of genes with developmental significance have been identified in invertebrates with EGF-like repeats. For example, the notch gene of Drosophila encodes 36 tandemly arranged amino acid repeats which show homology to EUF, Wharton W et al., Cell 11- 557-581 (19W5. Hydropathy plots .:20 indicate a putative membrane spanning domain, with the ECIF-related sequences being located on the extracellular side of the membrane. Other homeotic, genes with EGF-like repeats include Delta. 9SF and 5ZD which were identified using probes based on Notch. and the nematode gene Lin-)2 which encodes a putative receptor for a developmental signal tamnsmitted between two specified cells.

Specifically, EGF has been shown to have potential in the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic tmucosai lesions, Konrurek, PC et al., Eur. J. Gastroenterol liepatol.

2 933-37 (1995). including the treatment of necrotizing enterocolitis, Zollinger-Ellison syndrome.

gastroaesimal ulceration gastrointestinal ulcerations and congenital microvillus atrophy, A. Guglietta PB Sullivan.

Eur. J. Gasiroenterol Heparol. j(10). 945-50 (1995). Additionally, ECIF has been implicated in hair follicle Gos diestiation; C.L do Cros, J. Invest. Dermarol. im (I Suppl.), 106S-1 13S (1993), SG Hillier, Clin. Endocino!.

33(4), 427-28 (1990); idney function, L.L. Hamm at al.. Semi,:. A'ephrol. 12 109-15 (1993), RC Harris, Am.

J. Kidney Dis. 627-30 (1991); tear fluid, GB van Setten et al., lInt. Ophthainol&Q-6);359-62 (1991): vitamin K mediated blood coagulation, J. Stenflo et al., Blood 71(7): 1637-Si (1991). ECIF is also implicated various sin disease characterized by abnormal keratinocyte differentiation. psoriasis. epithelial cancers such as squamous cell carcinomas of the lung, epidermoid carcnoma of the 'vulva and glionas. King. LE et al.. Am. J. Med. Sd. 226: 154-158 (1988).

Of great interest is mounting evidence that genetic alterations in growth factors signaling pathways are closely linked to devclopmenWa abnormalities and to chronic diseases including cancer. Aaronson SA, Science 1146-1153 (1991). For example, c-crb-2 (also known as HIER-2). a proto-oncogene with dose strutural similarity to EGF receptor protein, is overexpressed in human breast cancr. King ef al-. Science 22.9: 974-976 (1985); Gullick.

WJ. Hormones and their actions. Cooke BA er al.. eds. Amsterdam, Elsevier, pp 349-360 (1986).

17. PO1 ThE TG"-j sUPerga family. Or simply TGF-p superfamily, a group of secreted proteins, includes a large inimber of related growth and differentiation factors expressed in virtually all phyla. Superfamily members bind to specific cell surface receptors that activate signal tranisduction mechanisms to elicit their zritifunctionai cytokine effects. Kolodiejczyk and Hall, Biochemt Cell Biol., 7A: 299-314 (1996); Atusano and Wrana, Cvtk rgMb Factor Rev., 2: 327-339 (1996); and Hill. Cellular.ZignAljng 1: 533-544 (1996).

Members of this family inchade five distinct fonnis of TGF-f3 (Sporn and Roberts. in Peptide Growth Factors and Their Recentor, Spore and Roberts, eds. (Springer-Verlag: Berlin, 1990) pp. 419.472). as well -as the differentiation factors vgI (Weeks and Melton, Q&I 51: 861-867 (1987)) and DPP-C polypeptide (Padgett et al..

tulre, W2.: 81-84 (1987)). the hormones activin and ilibin (Mason et al., Naam. 311: 659-663 (1985); Mason et al.. Grwt Fatr.1: 77-88 (1987)), the Mullerian-inhibiting substance (]MIS) (Cate et al., Cell. 45: 685-698 (1986)). the bone morphogenetic, proteins (EMPs) (Wonaey et al., Sgec 2JZ: 1528-1534 (1988); PCT WO 8/00205 published January 14, 1988; U.S. 4.8M.864 issued October 31, 1989), the developmentally regulated proteins Vgr-1 (Lyons et al.. Proc. Nadl Acad. Sci. USA. U: 4554-4558 (1989)) and Vgr-2 (jones et al., MoDkc- ':Endocrinol. 6: 1961-1968 the mouse growth differentiation factor (GDI), such as GDF-3 and GDF-9 (Kin~gsley, Gee De- 1: 133-146 (1994); McPherron and Lee. L Bi~L. Chemg., 20: 3444-3449 (1993)). the mouse lefty/Stral (Meno et al.. Nature. 3M: 15 1-155 (1996); Boullt el al., D il.. M7: 420-433 (1995)). glial cell :line-derived neurotrophic factor (GDNF) (Lin as al., Science, 2fQ: 1130-1132 (1993), neurturin (Kotzbauer er al., N1aiw. BI- 467-470 (1996)), and endonietria bleeding-associated factor (EBAP) (Kothapalli et al., I- CIin. nvest-, 2:2342-235D0(1997)). The subset BMP-2A and BMP-2B is approximately 75% homologous in sequence to DPP-C and may represent dhe mnammalian equivalent of that protein.

The proteins of the TGF-P superfamily are disulfide-linked homo- or heterodin-ers encoded by larger 9 9 precursor polypeptide chains containing a hydrophobic signal sequence, a long and! relatively poorly conserved Nterminal pro region of several baindied amino acids, a cleavage site (usually polybasic). and a shorter and more highly conserved C-terminal region. Thbis C-terminal region corresponds to the processed mature protein and contains approximately 100 ano acids with a chazacteristic cystein motif, i the conservation of seven of the nine cysteine residues of TGF-P amonig all known fimily members. Although the position of the cleavage site between the mature and pro regions varies among the family members, the C-terminus of all of the proteins is in the identical position, ending in the sequence Cys-X-Cys-X. but differing in every case from the TGF-P consensus C-terminus of Cys-Lys- Cys-Ser. Sporn and Roberts, 1990, sup ra.

There art at least five forms of TUF-fi currally identified, TGF-P I. TGF-P2. TGF4I3, TGF-M4, and TGF- P5. The activated form of TGF-01 is a bitnodimer formed by dimerization of the carboxy4erminal 112 amino acids of a 3 90amn*acdprecursor. Recombinant TOF- Il has been cloned (Dcrynck et al.. Naiur, 3M~:701-705 (1985)) and! expressed in Chinese hamster ovary cells (Gentry elat..! MAL~sUoL..m 2: 3418-3427 (1987)). Additionally, recombinant human TGF-P2 (deMartin a at.. EMBID.L, fi: 3673 (1987)), as well as human and porcine TG-P3 (eryeck aL. EM1HL. 2: 3737-3743 (1988); ten Dijkcer at., Proc. Nan Acad. Sci. USA, L9: 4715 (1988)) have been cloned. TGF-.P2 has a precursor form of' 414 amino acids and is also processed to a homoodimer from the carboxy-terminal 112 amino acids that shares approximately 70% homology with the active farm of TGF431 (Marquard etal.,J Biol. be, 2: 12127 (1987)). See also EP 200,341; 169,016;,268,561; and! 267,463; U.S.

Pat. No. 4,774,322; Cheifetz ar aL. Ccfl. 41: 409-415 (1987); Jakowlew ea aL, Molecular Endocrin., 2: 747-755 .(1988); Derynck ai al.. J. Biol. Chem., 2U1: 4377-4379 (1986). Sharples et al.. fl1i 239-244 (1987) Derynck eal, Nul Ais Rs.11: 3188-3189 (1987); Derynek at al., Nucd Acids -Res,. 15: 3187 (1987). Seycdin at J.Biol. Chem.. 5693-5695 (1986); Madisen a al.. P-B, 2: 1-8 (1988); and Hanks el aL, Pro, Nd-Acad.

)SiLL(US.A., IS: 79-82 (1988).

TGF-P4 and TG-P5 were cloned from a chicken chondrocyte cDNA library (Jakowlew r Malec.

EndociDl 2: 1186-1195 (1988)) and from a frog oocyte cDNA library, respectively.

The pro region of TGF-P associates non-covalently with die mature TGF-P dimer (Wakefield ar al., J. iol.

Czgn. 20: 7646-7654 (1988); Wakefield a al., Growth Factr. 203-218 (1989)). and the pro regions are found to be necessary for proper folding and secretion of' the active mature dimers of both TGF-3 and activin (Gray and mason, sc 2E2: 1328-1330 (1990)). The association between the mature and pro regions of TGrLP masks the biological activity of' the mature dimer. resulting in formation of an inactive latent farm. Latency is not a constant of dhe TGF-P superthmily, since the presence of the pro region has no effect on activin or inhidbin biologicalI activity.

A unifying feature of the biology of the proteins from the TGF-P superfamily is their ability to regulate developmental processes. TGF-P has been shown to have numerous regulatory actions on a wide variety of' both normal and neoplastic cells. TGF-P is nmaltiftnctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell functon (Spom and Roberts, supra).

One member of the TG"-f superfamil, EBAF, is exptessed in-endometriun only in the late secretory phase and during abnormal endometrial bleeding. Kothapalli ar al., J.Clin, nvest., 22: 2342-2350 (1997). Human ernlonetrum is unique in that it is the only tissue in the body that bleeds at regular intervals. In addition, abnormal endometrial bleeding is one of the most common manifestations of gynecological diseases, and is a prime indication for hysterectomy. In situ hyrdization showed that the suRNA of EBAF was expressed in the stroma without any significant mRNA expression in the endometrial glands or endothelial cells.

The predicted protein sequence of EBAF showed a strong homology to the protein encoded by mouse lefty/rA=3 of the TGFLP superfamily. A motif search revealed that the predicted EBAF protein contain most of the cysteine residues which are conserved among the TGF-p-related proteins and which are necessary for the formation of the cysteirie knot strucr. The EBAF sequence contains an additional cysteine residue, 12 amino acids upstream from the first conserved cysteine residue. The only other family members kanown to contain an additional cysteine residue are TGF-Ps, inhibins, and GDF-3. EBAF, similar to LEFM, GDF-3i'Vgr2, and GDF-9, lacks the cysteine residue that is known to form the intermolecular disulfide bond. Therefore, EBAF appears to be an additional member of the TGF-P superfinnily with an unpaired cysteinc residue that may not exist as a dimer. However, hydrophobic contacts between the two monomer subunits may promote dimecr formation. Fluorescence in situ hybridization showed that the ebaf gene is located on human chromosome I at band q42. 1.

Additional members of the TGF-P superfamily, such as those related to EBAF, are being searched for by industry and academics. We herein describe the identification and characterization of novel polypeptides having homology to EBAF. designated herein as PR0317 polypcptides.

18. PR0301 The widspread occuence of cancer has prompted the devotion of considerable resources and discovering new treatments of treatment. One particular method involves the creation of tumor or cancer specific monoclonal antibodies (mAbs) which are specific to tumor antigens. Such mAbs, which can distinguish between normal and cancerous cells are useful in the diagnosis, prognosis and treatment of the disease. Particular antigens are known S to be associated with neoplastic diseases, such as colorectal cancer.

One particular antigen, the A33 antigen is expressed in more than 90% of primary or metastatic colon cancers as well as normal colon epithelium. Since colon cancer is a widespread disease, early diagnosis and treatment is an important medical goal. Diagnosis and treatment of colon cancer can be implemented using monoclonal antibodies (mAbs) specific therefore having fluorescent, nuclear magnetic or radioactive tags.

Radioactive gene, toxins and/or drug tagged mAbs can be used for treatment in sri with minimal patient description.

15 mAbs can also be used to diagnose during the diagnosis and treatment of colon cancers. For example, when the serum levels of the A33 antigen are elevated in a patient, a drop of the levels after surgery would indicate the tumor resection was successful. On the other hand, a subsequent rise in scrunnn A33 antigen levels after surgery would indicate that meastases of the original tumor may have formed or that new primary tumors may have appeared. Such monoclonal antibodies can be used in lieu of, or in conjunction with surgery and/or other chemotherapies. For example, U.S.P. 4,579.827 and U.S.S.N. 424.991 199.141) are directed to therapeutic administration of S* monoclonal antibodies, the latter of which relates to the application of anti-A33 mAb.

SMany cancers of pithelial origin have adenovirus receptors. In fact, adenovirus-derived vectors have been proposed as a means of inserting antisense nucleic acids into tumors 5,518,885). Thus, the association of viral receptors with neoplastic tumors is not unexpected.

25 We herein describe the identification and characterization of novel polypeptides having homology to certain cancer-associated antigens, designated herein as PRO301 polypeptides.

19. PRO224 Cholesterol uptake can have serious implications on one's health. Cholesterol uptake provides cells with most of the cholesterol they require for membrane synthesis. If this uptake is blocked, cholesterol accumulates in the blood and can contribute to the formation of atherosclerotic plaques in blood vessel walls. Most cholesterol is transported in the blood bound to protein in the form of complexes known as low-density lipoproteins (LDLs). LDLs are endocytosed into cells via LDL receptor proteins. hlerefore. LDL receptor proteins, and proteins having homology thereto, are of interest to the scientific and medical communities. Membrane-bound proteins and receptors can play an important role in the formation, differentiation and maintenance of multicellular organisms.

The LDL receptors are an example of membrane-bound proteins which are involved in the synthesis and formation of cell membranes, wherein the health of an individual is affected directly and indirectly by its function. Many membrane-bound proteins act as receptors such as the LDL receptor. These receptors can function to endocytose substrates or they can function as a receptor for a channel. Other membrane-bound proteins function as signals or antigens.

Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceucal and diagnostic agents. The membrane-bound proteins can also be employed for screening of potential peptide or small molecule regulators of the relevant receptor/ligand interaction. In the case of the LDL receptor, it is desirable to find molecules which enhance endocytosis so as to lower blood cholesterol levels and plaque formation.

It is also desirable to identify molecules which inhibit cndocyosis so that these molecules can be avoided or regulated by individuals having high blood cholesterol. Polypcptides which are homologous to lipoprotein receptors but which do not function as lipoprorcn receptors are also of interest in the determination of the function of the fragments which show homology.

The following studies report on previously known low density lipoprotein receptors and related proteins including apolipoproteins: Sawannmra, et al., Nippon Chemiphar Co, Japan patent application 109098787: Novak, S..

c t al., JL. i o L, 271:(20)11732-6 (1996); Blaas, Virol.. 69(11)7244-7 (Nov. 1995); Scott, Jnherit.

M.:tab. Di.. 9/Supp. 1 (3-16) (1986); Yamamoto, et al., cll,. 39:27-38 (1984); Rebece, et al., Nurobiol.

15 Aging 15:5117 (1994); Novak, et al., Biol. Chemistry 271:11732-11736 (1996); and Sestavel and Fruchart.

Mo. 40(4):461-81 (June 1994). These publications and others published prior to the filing of this application provide further background to peptides already known in the art.

Efforts are being undertaken by both industry and academia to identify new, native membrane-bound receptor proteins, particularly those having homology to lipoprotein receptors. We herein describe the identification and characterization of novel polypeptides having homology to lipoprotein receptors, designated herein as PRO224 polypeptides.

PR0222 Complement is a group of proteins found in the blood that are important in humoral immunity and inflammation. Complement proteins are sequentially activated by antigen-antibody complexes or by proteolytic enzymes. When activated, complement proteins kill bacteria and other microorganisms, affect vascular permeability, release histamine and attact white blood cells. Complement also enhances phagocytosis when bound to target cells.

In order to prevent harm to autologous cells, the complement activation pathway is tightly regulated.

Deficiencies in the regulation of complement activation or in the complement proteins themselves may lead to immune-complex diseases, such as systemic lupus crythematosus, and may result in increased susceptibility to bacterial infection. In all cases, early detection of complement deficiency is desirable so that the patient can begin treatment. Thus. research efforts are currently directed toward identification of soluble and membrane proteins that regulate complement activation.

Proteins known to be important in regulating complement activation in humans include Factor H and Complement receptor type 1 (CRI). Factor H is a 150 kD soluble serum protein that interacts with complement protein C3b to accelerate the decay of C3 convertase and acts as a cofactor for Factor I-mediated cleavage of complement protein C4b. Complement receptor type I is a 190-280 kD membrane bound protein found in mast cells and most blood cells. CR1 interacts with complement protein C3b. COb. and iC3b to accelerate dissociation of C3 convertases, acts as a cofactor for Factor I-mediated cleavagc of C3b and COb. and binds immune complexes and promotes their dissolution and pbagocytosis.

Proteins which have homnology to comnplemntx proteins am of particular interest to the medical and industral communities. Often. proteins having homology to each other have similar function. It is also Of interest when proteins having homology do not have similar functions, indicating that certain structural motifs identify information other than fuinction, such as locality of function.

Efforts arc being undlertakten by both industry and academia to identify new, native secreted and membrane-bound proteins, particularly those having homology to known proteins involved in .the complement pathway. Proteins involved in the complement pathway were reviewed in Birminghami DJ (1995), Critical Revis ialmwmtnolgg1, 15(2):133-154 and in Abbas AK. et al. (1994) Cellular and Molecular Immunology, 2nd Ed. W.B.

Saunders Company, Philadelphia, pp 295-315.

We herein describe the identfication and characterization of novel polypeptides having homology to complement receptors, designated herein as PR0222 polypeptides.

21. PRO234 The successful function of many systems within nilticeilular organisms is dependent on cell-cell interactions. Such interactions are affected by the alignment of particular ligands with particular receptors in a manner which allows for ligand-receptor binding and thus a cecellU adhesion. While protein-protein interactions in cell recognition have been recognized for some time, only recently has the role of carbohydrates in physiologically relevant recognition been widely considered (see B.K. Brandley e J1. Leidc. Biol. AQ: 97 (1986) and N. Sharon c at, Sdence 2A6: 227 (1989). Oligosaccharides are well positioned to act as recognition novel lectins due to their cell surface location and structural diversity. Many oligosaccharide structure can be created through the differential activities of a smaller number of glycosyltransferases. The diverse structures of oligosaccharides; can be generated by transcription of relatively few gene products, which suggests that the oliosaccharides are a plausible mechanism by which is directed a wide range of cell-cell interactions. Examples of differential expression of cell surface carbohydrates and putative carbohydrate binding proteins (lectins) on interacting cells have been described Dodd T.M. Jessel, J. Neurara. 1: 3278 (1985): U. Regan a al., Proc. Nail. Aced. Sci. USA 12: 2248 (1986); M.

Constantine-Patn et Nature 3Z~: 459 (1986); and M. Tiemeyer et al.. J. Biol. Chtem. W: 1671 (1989). One intereting member of the lectin family are seletins.

The migration of leukocytes to sites of acute or chronic inflammation involves adhesive interactions between these cells and the endothelim. This specific adhesion is the initial event in the cascade that is initiated by inflammatory insults. and it is, therefore, of paramount importance to the regulated defense of the organism.

The types of cell adhesion molecules that arc involved in the interaction between leukocytes and the endathelium during an inflammatory response currently stands at four. selectins; (carbohydrate and glycoproicin) ligands for selectns; integrins'. and integrin ligands, which are members of the immunoglobulin gene superfamily.

The selectins are cell adhesion molecules that are unified both structualy and functionally. Structurally, selectins are charcterizecd by the inclusion of a domain with homology to a calcium-dependent lcan (C-lectins). an epidermal growth factor (egf)-likc domain and several complement binding-Like domains, Bevihacqua. M.P. et al., Sdemce2&3: 1160-1165 (1989); Johnston et al., Cell 5& 1033-1044 (1989); Lasky ef al, Cell.%6: 1045-1055 (1989); Siegalmian, M. et al., Science W: 1165-1172 (1989); Stoolman, LM.. Cell M: 907-910 (1989). Functionally, selectins share the common property of their ability to mediate cell binding through interactions between their lectin domains and cell surface carbohydrate ligarids; (Braudley. B. ar al., Cell 861-463 (1990); Springer, T. and Lasky, Nature 3_2, 19-197 (1991); Bevilacqua, M.P. and Nelson, Cliii. Invest. 21 379-387 (1993) and Tedder eral, J. Erp. Mod. In~: 123-133 (1989).

There art tduee mnanbers identified so far in the selecin family of cell adhesion molecules: L-selectin (also Called peripheral lymph node homing receptor (pnHR), LEC-CAMl. LAM-I. gpg0NEL. gpL4EL. SpI 10"I- MEL- 14 antigen Lcu-S antigen, TQ-1 antigen, DREG antigen), E-selectin (LEC-CAM-2, LECAM-2, ELAM- 1) and Pselectin (LEC-CAM-3. LECAM-3, GMP-140, PADGEM).

The identification of the C-lectin domain has led to an intense effort to define carbohydrate binding ligands for proteins containing such domains. E-selectin is believed to recognize the carbohydrate sequence NeuNAca2- 3GalPl-4(FucaI-3)GlcNAc (sialyl-Lewis x. or sLe) and related oligosaccharides, Berg eal. J. Biol. Omem. Z: 14869-14872 (1991); Lowe d CeR fld: 475-484 (1990); Phillps et al.. Science 25Q: 1130-1132 (1990); Tiemeyer eal., Proc. Nati. Acad. Sd. USA 11: 1138-1142 (1991).

L-selectin, which comprises a lectin domain, performs its adhesive function by recognizing carbohydratecontaining liganda on endotlia cells. L-scectin is expressed on the surface of leukocytes, such as lymphocytes.

neurophils, monocytes and eosinophils, and is involved with the trafficking of lymphocytes to peripheral lympboid tissues (GalLatin et al.. Nature MD: 30-34 (1983)) and with acute neutrophil-medicated inflammatory responses (Watson, Nature 312-.164-167 The amino acid sequenice of L-selectin and the encoding nucleic acid sequence are, for example, disclosed in U.S. patent No. 5,098,833 issued 24 March 1992.

L-selectin (LECAM-1) is particularly interesting because at its ability to block neutrophil influx (Watson et al., Nature MA: 164-167 (1991). It is expressed in chronic lymphocytic leukemnia cells which bind to R1EV (Spertini aol., Nature M2: 691-69(1991). It is also believed that 11EV structures at sites of chronic inflammation arc associated with the symptoins of diseases such as rheumatoid arthritis, psoriasis and multiple sclerosis.

E-seiectin (EL.AM-l). is particularly interesting because of its transient expression on endothelial Cells in response to IL-1 or TNF. Bevilacqua er al., Science 2E~: 1160 (1989), The time course of this induced expression (2-8 b) suggests a role for this receptor in initial neutrophil induced extravasation in response to infection and injury.

It has further been reported that ana-ELAM-l antibody blocks die influx of neutrophils in a primate asthma model and thus is beneficial for preventing airway obstruction resulting from the Inflammatory response. Gundel el al., J.

Clin. Irnvest. L8-:1407 (1991).

The adhesion of circulating neutrophils to atirmaated vascular endothelium is a primary event of the inflammatory response. P-selectin has been reported to recognize die Lewis x structure (Galf 3 1-4(Fucal-3) GlcNAC), Larsen a al., Cell fi2: 467-474(1990). Others report that art additional terminal linked sialic acid is required for high affinity binding, Moore al.. 1. Cell. Not. 112: 491-499 (1991). P-selectin. has been shown to be significant in acute lung injury. Anti-P-sclctinanibody has been shown to have strong protective effects in a rodent lung injury model.

M.S. Mulligan et al. J. Clin. Invest. 29: 1600 (1991).

We herein describe the identification and characterization of novel polypeptides having homology to lectin protcins, herein designated as PR0234 polypeptides.

22. PR0231 Some of the most important proteins involved in the above described regulation and modulation of cellular processes are the enzymes which regulate levels of protein phosphorylation in the cell. For example, it is known that the transduction of signals that regulate cell growth and differentiation is regulated at least in part by phosphorylation and dephosphorylation of various cellular proteins. The enzymes that catalyze these processes include the protein Idnases, which function to phosphorylate various cellular proteins, and the protein phosphatases, which function to remove phosphate residues from various cellular proteins. The balance of the level of protein phosphorylation in the cell is thus mediated by the relative activities of these two types of enzymes.

Protein phosphatases represent a growing family of enzymes that are found in many diverse forms, including both membrane-bound and soluble forms. While many protein phosphatases have been described, the functions of 15 only a very few are beginning to be understood (Tonks, Semin. Cell Biol. 4:373-453 (1993) and Dixon, Recent Prog.

Horn. Res. 51:405-414 (1996)). However, in general, it appears that many of the protein phosphatases function to modulate the positive or negative signals induced by various protein Idnases. Therefore, it is likely that protein phosphatascs play critical roles in numerous and diverse cellular processes.

Given the physiological importance of the protein phosphatases, efforts are being undertaken by both industry and academia to identify new, native phosphatase proteins. Many of these efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel phosphatase proteins.

SExamples of screening methods and techniques are described in the literature see, for example, Klein et al., m Natl. Acad. Sci., 92:7108-7113 (1996); U.S. Patent No. 5,536,637)].

We herein describe the identification and characterization of novel polypeptides having homology to acid 25 phosphatases, designated herein as PRO231 polypptides.

23. PR0229 Scavenger receptors are known to protect IgG molecules from catabolic degradation. Riechmann and Hollinger, Nature Biotechnologv, 15:617 (1997). In particular, studies of the CH2 and CH3 domains have shown that specific sequences of these domains are important in determining the half-lives of antibodies: Ellerson ct al., LJ.mmnol., 116: 510 (1976); Yasmeen, et al.. LImmunol. 116: 518 (1976; Pollock, et al., Eur. J. Immunol., 2021 (1990). Scavenger receptor proteins and antibodies thereto are further reported in U.S. Patent No. 5,510,466 to Krieger, et al. Due to the ability of scavenger receptors to increase the half-life of polypeptides and their involvement in immune function, molecules having homology to scavenger receptors are of importance to the scientific and medical community.

Efforts are being undertaken by both industry and academia to identify new, native secreted and membranebound receptor proteins, particularly those having homology to scavenger receptors. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identifyr the coding sequences for novel secreted and membrane-bound receptor proteins. Examples of screening methods and techniques are described in the literature [sec. for example, Klein et aL. Proc. Natl_ Acad. Sci., 21:7108-7113 (1996); U.S. Patent No. 5,536.637)].

We herein describe the identification and characterization of novel polypeptides having homology to scavenger receptors, designated herein as PR0229 polypeptides.

24. P~RO23 Oxygen free radicals and antioxidants appear toplay an important rote in the central nervous system after cerebral imbanmia and reperfusion. Moreover, cardiac injury, related to ischaemia anid reperfusion has been reported to be caused by the action of free radicals. Additionally. studies have reported that the redox state of the cell is a pivotal determinant of the fatecof the cls. Funhermore, reactive oxygen spedies have been reported to be cytotoxic, causing inflammatory disease, including tissue necrosis, organ failure, atherosclerosis, infertility, birth defects, premature aging, mutations and malignancy. Thus, the control of oxidation and reduction is important for a number of measons including for control and prevention of strokes, heart affacin, oxidative stress and hypertension. In this :::.regard, reductases, and particularly, oxidoreductases, are of interest. Publications fiurher describing this subject mnatrincludecKelsey. et al., Br. J 76(7):952-4 (1997); Friedrich and Weiss, J. Theor. Biol., 187(4):529-40 (1997) and Piculle, ct al.. J. BaterioL. 179(18):56W4.92 (1997).

Efforts are being undertak en by both induistry and academia to identify new, native secreted and membranebound receptor proteins, particularly secreted proteins which have homology to reductasc. Many efforts arc focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound receptor proteins. Examples of screening methods and techiniques ame described in the literature [see, for example, Klein et al., Proc. Nati. Acad. Sci., 22:7108-7113 (1996); U.S. Patent No. 5,536,637)1.

We herein describe the identification and characterization of novel polypeptides having homology to reductase, designated herein as PR0238 polypeptdecs.

25. PR0233 Studies have reported that the redox state of die celi is an important determinant of the fate of the cell.

Furthermore, reactive oxygen species have been reported to be cytotoxic. causing inflammatory disease. including tisse necrosis, organ failure, atherosclerosis, infertilty, birth defects, premature aging, rrmtations and malignancy.' Thus, the control of oxidation and reduction is imoportant for a number of reasons, including the control and prevention of strokes, hear attacks, oxidative stress and hypertension. Oxygen frec radicals and antioxidants appear to play an important role in the central nervous system after cerebral ischerma and reperfusion. Moreover, cardiac injury, related to ischaeinia *and rcperfusion has been reported to be caused by the action of free radicals. In this regard, reductases. and partictularly, oxidoreductases, ame of interest. In addition, the transcription factors, KF-kappa B and AP-l, arc known to be regulated by redox tate and to affect the expression of a large variety of genes thought to be involved in the pathogenesis of AIDS, cancer, atherosclerosis arnd diabetic complications. Publications fulrther describing this subject matter include Kelsey, et al.. Br.J.Cancc 76(7):852-4 (1997); Friedrich and Weiss, _L.

Tber. io 19 7 (4):529-.40 (1997) arnd Piculle, et al., J. acteiol, 179(18):5684-92 (1997). Given the physiological inmportance of redox reactions in vivo, efforts are currently being under taken to identify new. native proteins which are involved in redox. reactions. We &escribe herein the identification of novel polypeptides which have homology to reductase, designated herein as PR0233 polypeptides.

26, PRO223 The carboxypeptidase famiily of exopeptidases constitutes a diverse group of enzymnis that hydrolyze carboxyl-terminal amide bonds in polypeptides, wherein a large number of mammalian tissues produce these enzymnes-. Many of the carboxypeptidase enzymes that have been identified to date exhibit rather strong cleavage specificities for certain amino acids in polypeptides. For example, carboxypepidase enzymes have been identified which prefer lysine. arginine, seine or amino acids with either aromatic or branched aliphatic side chains as substrates at the carboxyl terminus of the polypeptide.

Wiith regard to the serinc carboxypeptidases, such amino acid specific enzymes have been identified from a variety of different mammalian and non-mammualiant organisms. 'The mammalian scmie carboxypeptidase enzymes play important roles in many different biological processes including, for example. protein digestion. activation.

9**inactivation, or modulation of peptide hormone activity. and alteration of the. physical properties of proteins and enzymes.

In fight of the physiological importance of the seririe carboxypeptidases, efforts are being undertaken by both industry and academia to identif new, native secreted and membrane-bound receptor proteins and specifically novel carboxypeptidases. Many ofthese efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound receptor proteins. We describe herein novel polypeptides having homology to one or more serine carboxypeptidase polypeptides, designated herein as PR0223 polypeptides.

27. R235 Plexin was first identified in Xenopus tadpole nervous system as a membrane glycoprotein which was shown to mediiate cell adhesion via a homophilic binding mechanism in the presence of calcium ions. Strong evolutionary conservation between Xenopus, mouse and human homologs of plexin has been observed. [Kaneyama et al., Biochem. And Biophys. Res. Comm. 226: 524-529 (1996)). Given the physiological importanc of cell adhesion mechanisms in vivo. efforts are currently being under taken to identify new, native proteins which are involved in a...,cell adhesion. We describe herein the identification of a novel polypeptide which has homology to plexin, designated herein as PR0235.

28. PR10236 and PR2612 P-galactosidase is a well known enzymatic protein which functions to hydrolyze P-galactoside molecules.

ji-galactosidase has been employed for a variety of different applications, both in vitro and in vivo and has proven to be an extremely useful research tool. As such, there is an interest in obtaining novel polypeptides which exhibit homology to the 1-galactosidasc polypeptide.

Given the strong interest in obtaining novel polypeptides having homology to P-galactosidase. eforts are curently being undeetaken by both industry and academia to identify new, native J -galactosidase homnolog proteins.

Many of these efforts are focused on die screening of mnammnalian recombinant DNA libraries to identity the coding sequences for novel P-galactosidase-like proteins. Examples of acrening methods and techniques are described in the literature [see, for example. Klein et al.. Proc. Nadl Acad. Sci, 21:710-7113 (1996); U.S. Patent No.

5.536.637)]. We herein describe novel poylpeptides having siginificant homology to the P-galabtosidase enzyme, designated herein as PR0236 and PR0262 polypeptddes.

29. EBRg29 Densin is a glycoprotein which has beent isolated havm the brain which has all the hallmarks of an adhesion moleculc. It is highly concentrated at synaptic sites in the brain and is expressd prominently in denduitic processes in developing neurons. Densin has been characterized as a member of the 0-linked sialoglycoproteins. Densin has relevance to medically important processes such as regeneration. Given the physiological importance of synaptic processes and cell adhesion mechanisms~ in vivo, efforts are crrenhly being under taken to identify new, native proteins which are involved in synaptic machinery and cell adhesion. We describe herein the identification of novel polypeptides which have homology to densin, designated herein as PR0239 polypetides.

DR22 *Ebnerin Is a cell surface protein assoc:iated with von Elme glands in mammals. Efforts are being undertaken by both industry and academia to identify new, native cell surface receptor proteins and specifically those which possess sequence homology to cell surface proteins such as cbnerin. Many of these effort& are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel receptor proteins.

We herein describe the identification of novel potypeptides having significant homology to the von Ebner's glandassociated protein ebnerin, designated herein as PR0257 polypeptides.

31. PRO260Q Fucosidases are enzymes that remove fiacose residues from facose containing proteoglycans. In some Pathological conditions, such as cancer, rheumtoid arthritis, and diabetes, there is an abnormal fuicosylation of serum proteins. Therefore, fucosidases. and proteins having homology. to flacosidase, arc of importance to the study and abrogation of these conditions. In particular, proteins having homology to the alpha-l-fucosidase precursor are of interest. Fucosidases and fucosidase inhibitors are further described in U.S. Patent Nos. 5,637,490, 5.382.709.

5,240.707, 5,153,325, 5.100,797, 5.096,909 and 5,017,704. Studies arc also reported in Valk, et al., L.YirlI., 71(9):6796 (1997), Aktogu, et al.. Monai. Arh Cest Dis. (Italy). 52(2):1 18 (1997) and Focarelli, et al., Bjgojhjfl, Biophvs. Res. CommN. 234(l):54 (1997).

Efforts are being undertaken by both indlustiy and academia to identify new, native secreted and membranebound receptor proteins. Of particular interest are proteins having homology to the alpha-I-fucos idase precursor.

Many efforts are focused on t screening of mammalian recombinant DNA libraries to identity the coding sequences for novel secreted and membrane-bound receptor proteins. Examples of screening methods and techniques are described in the literature (sec. for example. Klein et al., Proc. Nat!. Acad. Sci-, 21:7108-7113 (1996); U.S. Patent No. 5.536,637)].

We herein describe the identification and characterization of novel polypeptides having homology to fucosidases, designated herein as PR0'260 polypeptides.

32. PRO263 CD44 is a cell surface adhes ion molecule involved in cell-cell and ceUl-matrix interactions. Hyaluronic acid, a component of the extracelilar matrix is a major ligand. Othe= Lgands include collagen, fibronectin, Laminin, dirondruitin sulfate. ucosal addressjn, serglycin and osteoponin. CD44 is also important in regulating cell traffic, lymph node homning, transission of growth signals, and presentation of chemokines and growth factors to traveling cells. CD44 surface proteins are associated with metastatic timors and CD44 has been used as a marker for IVY infection. Certain splice variants are associated with metastasis and poor prognosis of cancer patients. 1Therefore, molecules having homology with CD44 are of particular interest, as their homology indicates that they may have functions related to those functions of CD44. CD44 is further described in U.S. Patent Nos. S,506,119, 5,504.194 and 5,108,904; Gerberick, et al., Toxicol. Agnt. Phamacl., 146(l):l (1997); Wittig, et al., munol,. Uters (Netherlands), 57(1-3):217 (1997); and Oliveira and Odell, OrlLOzngI. (Englad, 33(4).260 (1997).

Efforts are being undertaken by both inustry and academia to identify' new, native secreted and membranebound receptor proteins, particularly transmemnbrAne proteins with homology to CD44 antigen. Many efforts are focued n te sreeingof mammalian recombinant DNA lib.raries to ienif the coding seq.uences fornoe secreted and membrane-bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Nal. Acad. Sci, 9:7108-7113 (1996); U.S. Patent No.

.5,536,637)].

.*We herein describe the identification and characterization of novel polypeptides having homnology to CD44 antigen, designated herein as PR0263 polypeptddes.

33. PRQ2701 Thioredoxins effecduction-caiatian (redox) stale. Many diseases are potentially related to redox state and reactive oxygen species may play a role in many important biological processes. The transcription factors, NE-kappa B and AP- 1. are regulated by redox state and are known to affect the expression of a large variety of genes thought to be involved in the pathogenesis of AIDS. cancer, atherosclerosis and diabetic complications. Such proteins mhay also play a role in cellular antioxidant defense, and in pathological conditions involving oxidative stress such as stroke and inflammation in addition to having a role in apoptosis. Therefore, thioredoxins, and proteins having homology thereto, are of interest to the scientific and medical communities.

We herein describe the identification and characterization of novel polypeptides having homology to thioredoxin, designated herein as PR0270 polypeptides.

34. ERQ271 7he procoglycan fink protein is a protein which is intimately associated with various extracellular matrix proteins and more specifically with proteins such as collagen. For example. one primazry component of collagen is a large proteoglycan called aggrecan. This molecule is retained by binding to the glycwsaxinoglycan hyaluronan through the amiuno terminal G1 globular domain of the core protein. This binding is stabilized by the proteoglycan link protein which is a protein that is also associated with other tissues contaiing hyaluronan binding proteoglycass such as versican.

LI nk protein has been identified as a potential target for alitoiniiune antibodies in individuals who suffer from juvenile rheumatoid arthritis (see Guerassimov et al., J. Rheumatology 24(5):959-964 (1997)). As such, there is strong interest in identifying novel proteins having homology to link protein. We herein describe the identification and characterization of novel polypeptides having such homology, designated herein as PR0271 polypeptides.

PRQ=7 Reiculocalbin is an endoplasmic reticular protein which my be involved in protein transport and lurninal :::.protein processing. Reticulocalbin resides in the lumen of the endopladsmic rerticulum, is known to bind calcium.

15 and may be involved in a lusninal retention mechanism of the endoplasmic reticulum. It contains six domnains of the EF-hand motif associated with high affinity calcium binding. We describe herein the identification and characterization of a novel polypeptdec which has homology to the reticulocalbin protein, designated herein as PROM7.

36. PRO294 *~:Collagen. a naturally occurring protein, finds wide application in industry. Chemically hydrolyzed natural collagen can be denatured and reentred by heating and cooling to produce gelatin, which is used in photographic and medical, among other applications. Collagen has important properties such as the ability to form interchain :aggregates having a conformation designated as a triple. helix. We herein describe the identification and 25 characteization of a novel polypeptide which has homology to portions of the collagen molecule, designated herein as PROM9.

*37. PRQ29 V7Th integ'rins comprise a supergene family of cell-surface glycoprotein receptors that prombote cellular adhesion. Each cell has numnerous receptors; that define its cell adhesive capabilities. Integrins are involved in a wide variety of interaction between cells and other cells or matrix components. The integrins are of particular importance in regulating movement and ftnction of immune system cells The platelet HII/IA integrin complex is of particular imnportanc in regulating platelet aggregation. A member of the integrin family, integrin P-6, is expressed on epithelial cells and modulates epithelial inflammnauion. Another integrin. leucocyte-associated antigen-l (LFA-1) is important in t adhesion of lymphocytes during an immune response. The integuins are expressed as heterodimers of noncovalently associated alpha and beta subunits. Given the physiological importance of cell adhesion mnechanisms in tio, efforts are currently being under taken to identify new. native proteins which are involved in cell adhesion. We describe herein the identification and characterization of a novel polypeptide which has homology to integrin.

designated herein as PR0295.

38. PRO293 Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the structural and functional miechanismn underlying protein-protein interactions, protein-protein interactions can be more easily manipulated to regulate the particular result of the protein-protein interaction. Thus, the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community.

All proteins containing leucine-rich repeats are thought to be involved in protein-protein interactions.

Lcucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. The crystal structure of ribonucleas inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. Thkese units are arranged so that they form a parallel beta-sheet wih one surface exposed to solvent, so that the protein acquires an unusual, nonglubular shape. These two features have been idctdas responsible for the protein-binding functions of proteins containing leucine-uich repeats. See, Kobe and :::.Deisenhofer, Trends Biochem.- Sci., 19(10):415-421 (Oct. 1994).

A study has been reported on leucine-rich proteoglycans which serve as tissue organizers, orienting and ordering collagen fibrils during ontogeny and are involved in pathological processes such as wound healing, tissue repair, and tumor stroma formation. lozzo, R. C-it, Rev, iocien. Mot- Bilt., 32(2):141-174 (1997). Others studies irripicating leucine rich proteins in wound healing and tissue repair are De La Salle, et al., Vouv. Rev.

Er- Hcatol. (Germany), 37(4):215-222 (1995), reporting miutations in the leucine rich motif in a complex associated with the bleeding disorder Bernard-Soqilier syndrome and Chlercson, K. Thromb.-Haemo. (Germany), 74(l):Ill-116 (July 1995), reporting that platelets have leucine rich repeats. Another protein of particular interest which has been reported to have leucine-rich repeats is the SLIT protein which has been reported to be useful in treating neuro-degenerative diseases such as Alzheirner's disease, nerve damage such as in Parkinson's disease, and for diagnosis of cancer, see, Artavanistskoa, S. and Rothberg. J. W09210518-Al by Yale University. Other studies reporting on the biological functions of proteins having leucine-rich repeats include: -Tayar. et al.. frigi Cell Enocrinoj., (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotrojin receptor involvement); Miura, et al., Wpjg..RigdIa (Japan). 54(7):1784-1789 (July 1996) (apopt osis involvement), Harris, P. et alI., J.Aml. Soc.

tbml.. 6(4):1 125-1 133 (Oct. 1995) (kidney disease involvemnent); and Ruoslahti, E, et al.. W091 10727-A by La Jolla Cancer Research Foundation (decorin binding to transforming growth factorp involvement for treatment for cancer, wound healing and scarring).

Efforts are therefore being undertaken by both industry and academia to identify new proteins having leucine rich repeats to better understand protein-protein interactions. Of particular interest are those proteins having Icucine rich repeats and homology to known neuronal leucior rich repeat proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and nlembrane-bound proteins having leucine rich repeats. Examples of screening mnethods and technique s are described in the literature (see, for example. Klein et al., Proc. NaI AcadSci, 91:7108-7113 (1996); U.S. Patent No. 5,536,637)].

We describe herein the identification and characterization of a novel polypeptide which has homology to leucine rich repeat proteins. designated herein as PR0293.

39. PROZ47 Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the stuctural and functional mechanisms underlying protein-protein interactiozis, protein-protein interations can be momt easily manipulated to regulate the particular result of the protein-protein interaction. Thus, the underlying mechanisms of protein-protein interactions arc of interest to dhe scientific and medical community.

Alt proteins containing leucine-rich repeats ame thought to be involved in protein-protein interactions.

Lcucine-rich repeats are short sequence motifs present in a mber of proteins with diverse functions and cellular locations. The crystal structure of ribonruclease inhibitor protein has revealed that leucine-ich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surfaceexposed to solvent, so that the protein acquires an unusual, nonglubular shape. These two features have been inilicat-d as responsible for the protein-binding functions of proteins containing leucine-rich repeats. See, Kobe and **Deisenhofer, Trends iochem. Sci.. 19(10):415-421 (Oct. 1994).

A study has been reported on leucine-rich proteoglycans which serve as tissue organizers, orienting and ordering collagen fibrils during ontogeny and are involved in pathological processes such as wound healing, tisse repair, and tumor stronia formation. lozzo, R. Crit. Rev. iochern Mal, Biol., 32(2):141-174 (1997). Other studies implicating leucine ich proteins in wound healing and tissue repair are De La Salle, et al., Ymix..Rey, a Jkmatoi. (Germany). 37(4):215-222 (1995). reporting mutations In the leucine rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome and Chlemetson. K. Tlirntb- Haernost. (Germany), :74(l):111-1 16 (July 1995), reporting that platelets have leucine rich repeats. Another protein of particular interest which has been reported to have leucine-rich repeats is the SLIT protein which has been reported to be useful in treating neuro-degeneative diseases such as Alzheimer's disease, nerve damage such as in Parkinson's disease, and .x :fordiagnosis of cancer, see, Aiiavanistsakonas, S. and Rothberg, 1. W09210518-Al by Yale University. Other studies reporting on the biological functions of protein s having leucine-rich repeats include: Tayar, et al., M2L CS1..Edgdinj.. (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor involvement); Mura, et al., HipjRwLEljnsb (Japan), 54(7):1784-1789 (July 1996) (apoptosis involvement); Harris, P. et al.. J. Amn...Soc ligpbwl., 6(4):1125-1133 (Oct. 1995) (ktidney disease involvement); and Ruoslahti, E. et al., W09110727-A by 909* La Jolla Cancer Research Foundation (decorin binding to transforming growth factorf involvement for treatmoent for cancer, wound healing and scarring).

Densin is a glycoprotein which has been isolated from the brain which has all the hallmarks of an adhesion molecule. It is highly concentrated at synaptic sites in the brain and is expressed prominently in dendzitic processes in developing neurons. Densin has been characterized as a member of the 0-linked sialoglycoproteins. Densin has relevance to medically important processes such as regeneration. Given the physiological importance of synaptic processes and cell adhesion mechanisms in vivo, efforts are currently being under taken to identify new, native proteins which are involved in synaptic machinery and cell adhesion. Densin is further described in Kennedy, M.B.

TredsNeroci (England), 20(6)-264 (1997) and Apperson, et al.. J1. Neurosci, 16(21):6839 (1996).

Efforts are therefore bein undertaken by both industry and academia to identify new proteins baving leucine rich repeats to better uderstand protein-protein interactions. Of particular interest arc those proteins having leucine rich repeats and homology to known proteins having leucine rich repeats such as KLAA0231 and densin. Many efforts arc focused on dhe scning of mamnmalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound proteins having leucine rich repeats. Examuples of screening methods and techniques are dscribed indie literature [see, for example, Kleinct al.. Proc. Nail. Acad. Sc- 9:7108-7113 (1996); U.S. Patent No. 5,536,637)].

We describe herein the identification and characterization of a novel polypeptide which has homology to leucine rich repeat proteins, designated herein as PR0247.

40. PR0302. PR0303. PR0304. PRO307 and PR0343 Proteases are enzymatic proteins which are involved in a large number of very important biological processes in mammalian and non-mammalian organisms. Numerous different protease enzymes from a variety of different mammalian and non-mammalian organisms have been both identified and characterized. The mammalian, protease enzymes play important roles in many different biological processes including, for example, protein 0eO15 digestion, activation. inactivation, or modulation of peptide hormone activity, and alteration of the physical properties of proteins and enzymes.

In light of the important physiological roles played by protease enzymes, efforts are currently being undertaken by both industry and academia to identify new, native protease homologs. Many of these efforts are focused on the screening of mamnmalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bounid receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Nat]. Acad. Sci :7108-7113 (1996); U.S. Patent No.

5,536,637)]. We herein describe the identification of novel polypeptides having homology to various proteasc enzymes, designated herein as PRO3(02, PR0303, PR0304. PR0307 and PR0343 polypeptides.

41. PRO328 The GUIP protein family has been characterized as comprising zinc-finger proteins which play important roles in enibmyogenesis. T7hese proteins may function as transcriptional regulatory proteins and are known to be S0SSSamplified in a subset of human tumors. Glioma pathogenesis protein is stucurally related to a group of plan **se*pathogenesis-reated proteins. It is higjhly expressed in glioblastomna. See US Pat. Nos. 5,582,981 (issued Dec. 1996) and 5.322.801 (issued June 21, 1996), Ellington, A.D. et al., kNtur, 3:818 (1990). Grindley, J.C. el al., Dev..Iiol-. IMfL2:337 (1997). Marine, J.C. et al., Moh De21 O:211 (1997). The CRISP or cysteine rich secretory protein family are a group at proteins which are also structurally related to a group of plant pathogenesis proteins. [Schwidetzky, Biochem.J.. M2:325 (1997), Pfisterer. hsgL cIL.B~jQL, 1=(f:6160 (1996), 1Cj,-.cmar. Eur Bo 827 (1996)]. We describe herein the identification at a novel polypeptide which has homology to CUIP and CRISP. designated herein as PR0328 polypeptides.

42. PR033S. PRO331 and PR0326 Protein-Protein interactions include receptor And antigen complexes and signaling mechanisms. As more is known about dhe structural and functional mechanisms underlying protein-protein interactions, protein-protein itefractions can be morm easily manipulated to regulate die particular result of the protein-protein interaction. Thus, the underlying mechanisms of protein-protein interactions are of interest to dhe scientific and medical community.

AllI proteins containing leucine-rich repeats are thought to be involved in protein-protein interactions.

Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations.- The crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual. nonglubular shape. These two features have been indlicated as responsible for the protein-binding functions of proteins containing leucine-rich repeats. See, Kobie and Deisenhofer. Trend-- iochem. Sci. 19(10):415-421 (Oct. 1994).

A study has been reported on leucine-rich pmoteoglycans which serve as tissue organizers, orienting and :4....ordering collagen fibrils during ontogeny and are involved in pathological processes such as wound healing, tissue .4 repair, and tumor stronia formation. lozzo, R. Crit. Rev. Biochem. Mol. Bliol., 32(2):141-174 (1997). Others sue mlctn ecn ihpoen nwudhaigadtm earaeD aSle taVu.Rv Fr. Hmatol. (Germany), 37(4):215-m (1995). reporting mutatin in the Icucine rich motif in a complex associated with dhe bleeding disorder Bernard-Soulier syndrome, Chlemetson, K. J1., ]]irnin. Haemos. (Germany). 74(1):I 11- 116 (July 1995), reporting that platelets have leucine rich repeats and Ruoslahti. E. L. et al.. W091 10727-A by La Jolla Cancer Research Foundation reporting that decorin binding to transforming growth factorp has involvement in a treatment for cancer. wound healing and scarring. Related by function to this group of proteins is the insulin like growth factor (IGF). in that it is useful in wound-healing and associated therapies concerned with re-growth of tissue, such as connective tissue, skin and bone; in promoting body growth in humans and animalls; and in stimulating other growth-related processes. The acid labile subunit of IGF (ALS) is also of interest int that it increases the half-life of IGF and is part of the lOP complex ix~ .25 Another protein which has been reported to have Jeucine-rich repeats is the SLIT protein which has been reported to be useful in treatin neuro-degenerative diseases such as Alzhemer's disease, nerve damage such as in Parkinson's disease, and for diagnosis of cancer, see, Artavanistsakonas. S. and Rothbcrg, J. W09210518-Al by Yale University. Of particular interest is UG-1 a membrane glycoprotein that is expressed specifically in glial cells in the mouse brain, and has leucine rich repeats and inunuoglobulin-likc domains. Suzuki, et al., 1. fBi.

Chem. 271(37):22522 (1996). Othier studies reporting on the biological functions of proteins having leucine rich repeats include: Tayar, ct al.. MolCell..Ezsocrinoj.. (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor involvement); Miura, et al., N1IVnR~ircsla (Japan), 54(7):1784-1789 (July 1996) (apoptosis involvement); Harris. P. et al., L Am. Soc. Nebrol., 1125-1133 (Oct. 1995) (kidney disease involvement).

Efforts are therefor being undertaken by both industry and academia to identify new proteins having leucine rich repeats to better understand protein-protein interactions. Of particular interest are those proteins having leucine rich repeats and homology to known proteins having leucine rich repeats such as LIG-1, ALS and decorin. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound proteins having leucine rich repeats. Examples of screening methods and tchiques am described in th iterature [see, for exaniple, Klein et at., Proc. Nati. Acad. Sci-, 9:7108-7113 (1996); U.S. Patent No. 5,536,637)].

We describe herein the iden tification and characterization of novel polypeptdecs which have homology to proteins of t leucinec rich repeat superfamnily, designated herein as PROM3, PR0331 and PR0326 polypeptides.

43. PH=~ Secreted proteins comprising a repeat characterized by an arrangement of conserved leucine residues (leucine-rich repeat motif) have diverse biological roles. Certain proteoglycans, such as biglycan, fibromodulin and decorin, ame, for example, characterized by the presence of a leucine-rich repeat of about 24 amino acids (Ruoslahti, Ann. Rev. Cell. iot- A 229-255 (1988): Oldberg et aL, -EMOO J. 1, 2601-2604 (1989)]. In general, proteoglycans ame believed to play a role in regulatn exracellular matrix, cartilage or bone fuanction. The proteoglycan decorin binds to colla2gen type I and H and affects the rate of fibril formation. lFibromodulin also binds collagen and delays fibril formati. Both fibromodulin and decorin inhibit the activity of transforming growth factor beta (TGF-P) (U.S.

Patent No. 5,583,103 issued December 10. 1996). TGF-P is known to play a key role in the induction of extracellular matrix and has been implicated in the development of fibrotic diseases, such as cancer and gloniculomphritis. Accordingly, proteoglycans have been proposed for the treatment of fibrotic cancer, based upon their ability to inhibit TGFP s growth stimulating activity on the cance r cell. Proteoglycans have also been described as potentially useful in the treatmenit of other proliferative pathologies, including rheumatoid arthritis, arteriosclerosis, adult respiratoty distress syndrome, cirrhosis of the liver, fibrosis of the lungs, post-myocardial infarction, cardiac fibrosis. post-angioplasty restenosis, renal interstitial fibrosis and certain dermal fibrotic conditions, such as keloids and scarring, which might result from burn injuries, other invasive skin injuies, or cosmetic or reconstructive surgery Patent No. 5,654,270, issued August 5, 1997).

We describe herein the identification and characterization of novel polypeptides which have homology to :proteins of the leucine rich repcat supcrfamily, designated herein as PR0332 polypeptides.

44. P~RO334 Microfibril bundles and proteins found in association with these bundles, particularly attachment molecules, **are of interest in the field of dermatology, particularly in the study of sin which has been damaged from aging, injuries or the sun. Fibrillin microfibrils define the continuous elastic network of skin, and are present in dermis as microfibril bundles devoid of measurable elastin extending from the dermal-epiuielial junction arnd as components of t thick elastic fibres present in the deep reticular dermis. Moreover, Marfan syndrome has been linked to mutations which interfere with multimerization of fibrillin monomers or other connective tissue elements.

Fsbulin-l is a modular glycoprotein with amino-terminal anaphlatoxin-like modules foMowed by nine epidermral growth factor (EGF)-like modules and, depending on alternative splicing, four possible carboxyl termini.

Fibulin-2 is a novel extritcellular matrix protein frequently found in close association with microfibrils containing either fibronectin or fibrijlin. Thus, fibrillin, fibulin. and molecules related thereto are of interest, particularly for die use of preventing sin from being damaged from aging, injuries or the sun, or for restoring skin damaged from same. Moreover, thes molecules are generally of interest in the study of connective tissue and attachment molecules a! 'related rmcanisins. Fibrillin filiulin and related moleculs are further described in Adams, et al.. LXgL-jMi., 272(2):226-36 (1997); Kielty and Shuttlcworth, Microsc.kzRes bc.. 38(4):413-27 (1997); and Chilt J Crd 591L.. 12C2Supp.):131.S (1997).

Currently. effrts are being undertaken by both industry and academia to identify new, native secreted and membrne-bmund receptor proteins. particularly secreted protein which have homology to fibulin and fibrillin. Many efforts are focused on the screening of mammalian, recombinant DNA libraries to identify the coding sequences for novel secreted and membranc-bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example. Klein et al., Proc, Nat!- Acad. Sd 22:7108-7113 (1996); U.S. Patent No.

5.536.637)].

We herein describe the identiication and charcterizaton of novel polypeptides having homology to fibulin and fibrillin, designated herein as PR0334 polypeptides.

45. PRO346 71= widespread occurrence of cancer has prompted the devotion of considerable resources and discovering new treatments of treatment. One particular method involves the creation of tumor or cancer specific monoclonal antibodies (niAbs) which are specific to tumor antigens. Such snAbs. which can distinguish between normal and cancerous cells are useful in the diagnosis, prognosis and treatment of the disease. Particular antigens are known to be associated with neoplastic diseases. such as coloreccal and breast cancer. Since colon cancer is a widespread disease, early diagnosis and treatment is an important medical goal. Diagnosis and treatment of cancer can be implemented using monoclonal antibodies (mAbs) specific therefore having fluorescent. nuclear magnetic or *:radioactive tags. Radioactive genes. toxins and/or drug tagged m.Abs can be used for treatment in situ with minimal patient description.

Carcinoemrbryonic antigen (CEA) is a glycoprotein found in human colon cancer and the digestive organs :of a 2-6 month human embryos. CEA is a known human tumor marker and is widely used in the diagnosis of neoplastic diseases, such as colon cancer. For example, when the scrum levels of CEA are elevated in a patient, a drop of CEA levels after surgery would indicate the tumor resection was successful. On the other hand, a subsequent rise in serum CEA levels after surgery would indicate that metastases of the original tumor may have formed or that new primary tumors may have appeared. CEA may also be a target for mAb. antisense nucleotides P6 RQZ268 Protein disulfide isomerase is an enzymatic protein which is involved in the promotion of correct refolding of pruteins through the establishment of correct disulfide bond formation. Protein disullide isomerase was initially identified based upon its ability to catalyze the renaturation of reduced denatured RNAse (Goldberger ea al.. J. Biol.

Ozem 239:1406-1410 (1964) and Epstein et al.. Cold .Spring Hlarbor S"m. Quant. Bid 28:439-449 (1963)). Protein disulfide isomerase has been shown to be a resident enzyme of the endoplasmic reticuluim which is retained in the endoplasmic reticulum, via a -KDEL or -HDEL amino acid sequence at its C-terminus.

Given the importance of disulfide bond-forming enzymes and their potential uses in a number of different applications, for example in increasing the yield of correct refolding of recombinantly produced proteins, effortsare currently being undertaken by both industry and academia to identify new, native proteins having homology to protein disulfide isomerase. Many of these efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel protein disulfide isomerase homologs. We herein describe a novel polypeptide having homology to protein disulfide isomerase, designated herein as PR0268.

47. Prolyl 4-hydroxylase is an enzyme which functions to post-translationally hydroxylate proline residues at S the Y position of the amino acid sequence Gly-X-Y, which is a repeating three amino acid sequence found in both collagen and procollagen. Hydroxylation of proline residues at the Y position of the Gly-X-Y amino acid triplet to form 4-hydroxyproline residues at those positions is required before newly synthesized collagen polypeptide chains may fold into their proper threedimensioal triple-helical conformation. If hydroxylation does not occur, synthesized collagen polypeptdes remain non-helical, are poorly secreted by cells and cannot assemble into stable functional collagen fibrils. Vuorio et al.. Proc. Nat. Acad. Sci. USA 89:7467-7470 (1992). Prolyl 4-bydroxylase is comprised of at least two different polypeptide subunits, alpha and beta.

S: Efforts are being undertaken by both industry and academia to identify new, native secreted and membranebound receptor proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Natl. Acad. Sci., 2:7108-7113 (1996); U.S. Patent No. 5,536.637)]. Based upon these efforts. Applicants have herein identified and describe a novel polypeptide having homology to the alpha subunit of prolyl 4-hydroxylase. designated herein as PR0330.

48. PRO339 and PRO310 25 Fringe is a protein which specifically blocks serrate-mediated activation of notch in the dorsal compartment ofthe Drosophila wing imaginal disc. Fleming, et al., Development. 124(15):2973-81 (1997). Therefore, fringe is of interest for both its role in development as well as its ability to regulate serrate, particularly serrate's signaling abilities. Also of interest are novel polypeptides which may have a role in development and/or the regulation of serrate-like molecules. Of particular interest are novel polypeptides having homology to fringe as identified and described herein, designated herein as PR0339 and PRO310 polypeptides.

49. R0244 Lcctim are a class of proteins comprising a region that binds carbohydrates specifically and non-covalently.

Numerous lectins have been identified in higher animals, both membrane-bound and soluble, and have been implicated in a variety of cell-recognition phenomena and tumor metastasis.

Most lectins can be classified as either C-type (calcium-dependent) or S-type (thiol-dependent).

34 Lectins are thought to play a role in regulating cellular events that are initiated at the level of the plasma membrane. For example, plasma membrane associated molecules are involved in the activation of various subsets of lymphoid cells, e.g. T-lymphocytes, and it is known that cell surface molecules are responsible for activation of these cells and consequently their response during an immune reaction.

A particular group of cell adhesion molecules, selectins, belong in the superfamily of C-type lectins.

This group includes L-selectin (peripheral lymph node homing receptor (pnHR), LEC-CAM-1, LAM-1, gp 9 0 MEL, gpl00MEL GPllO0EL, MEL-14 antigen, Leu-8 antigen, TQ-1 antigen, DREG antigen), E-selectin (LEC-CAM-2, LECCAM-2, ELAM-1), and P- 15 selectin (LEC-CAM-3, LECAM-3, GMP-140, PADGEM). The structure of selectins consists of a C-type lectin ((carbohydrate binding) domain, an epidermal growth factorlike (EGF-like) motif, and variable numbers of complement regulatory (CR) motifs. Selectins, are associated with S. 20 leukocyte adhesion, e.g. the attachment of neutrophils to venular endothelial cells adjacent to inflammation (Eselectin),. or with the trafficking of lymphocytes from .blood to secondary lymphoid organs, e.g. lymph nodes and Peyer's patches (L-selectin).

Another exemplary .lectin is the cell-associated macrophage antigen, Mac-2 that is believed to be involved in cell adhesion and immune responses. Macrophages also express a lectin that recognizes Tn Ag, a human carcinomaassociated epitope.

Another C-type lectin is CD95 (Fas antigen/APO-l) that is an important mediator of immunologically relevant regulated or programmed cell death (apoptosis).

"Apoptosis" is a non-necrotic cell death that takes place in metazoan animal cells following activation of an intrinsic cell suicide program. The cloning of Fas antigen is described in PCT publication WO 91/10448, and European patent application EP510691. The mature Fas molecule 34a consists of 319 amino acids of which 157 are extracellular, 17 constitute the transmembrane domain, and 145 are intracellular. Increased levels of Fas expression at T cell surface have been associated with tumor cells and HIVinfected cells. Ligation of CD95 triggers apoptosis in the presence of interleukin-1 (IL-2).

C-type lectins also include receptors for oxidized low-density lipoprotein (LDL). This suggests a possible role in the pathogenesis of atherosclerosis.

We herein describe the identification and characterization of novel polypeptides having homology to C-type lectins, designated herein as PR0244 polypeptides.

Throughout the description and claims of this .:specification, the word "comprise" and variations of the 15 word, such as "comprising" and "comprises", means "including but not limited to", and is not intended to exclude other additives, components, integers or steps".

SUMMARY OF THE INVENTION 1. PRO211 and PR0217 20 Applicants have identified cDNA clones that encode novel polypeptides having homology to EGF, designated in the present application as "PRO211" and "PR0217" polypeptides.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO211 or PR0217 polypeptide. In one aspect, the isolated -nucleic acid comprises DNA encoding EGF-like homologue PRO211 and PRO217 polypeptides of Fig. 2 (SEQ ID NO:2) and/or 4 (SEQ ID NO:4) indicated in Fig. 1 (SEQ ID NO:1) and/or Fig. 3 (SEQ ID NO:3), respectively, or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO211 and PRO217 EGF-like homologue PRO211 and PR0217 polypeptides. In particular, the invention provides isolated native sequence PR0211 and PR0217 EGFlike homologue polypeptides, which in one embodiment, includes an amino acid sequence comprising residues: I to 353 of Fig. 2 (SEQ ID NO:2) or 1 to 379 of Fig. 4 (SEQ ID NO: 4).

2. Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0230".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0230 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0230 polypeptide having amino acid residues 1 through 467 of Figure 6 (SEQ ID NO: 12). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0230 polypeptide. In particular, the invention provides isolated native sequence PRO230 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 through 467 of Figure 6 (SEQ ID NO:12).

In another embodiment, the invention provides an expressed sequence tag (EST) comprising the nuclcotide 15 sequence ofSEQ ID NO:13 (Figure 7) which is herein designated as DNA20088.

3. PR023 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PRO232'.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0232 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0232 polypeptide having amino acid residues 1 to 114 of Figure 9 (SEQ ID NO:18). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0232 polypeptide. In particular, the invention 25 provides isolated native sequence PRO232 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 114 of Figure 9 (SEQ ID NO:18).

4. PRO187 Applicants have identified a cDNA clone that encodes a novel polypeptide, designated in the present application as "PRO187".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0187 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO187 polypeptide of Figure 11 (SEQ ID NO:23), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the invention provides a nucleic acid comprising the coding sequence of Figure 10 (SEQ ID NO:22) or its complement. In another aspect, the invention provides a nucleic acid of the full length protein of clone DNA27864-1155, deposited with the ATCC under accession number ATCC 209375, alternatively the coding sequence of clone DNA27864-1155, deposited under accession number ATCC 209375.

In yet another embodiment, the invention provides isolated PRO187 polypeptide. In particular, the invention provides isolated native sequence PRO187 polypcptide. which in one embodiment, includes an amino acid sequence comprising residues 1 to 205 of Figure 11 (SEQ ID NO:23). Alternatively, the invention provides a polypeptide encoded by the nucleic acid deposited under accession number ATCC 209375.

PRO26S Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as 'PR0265".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0265 polypcptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0265 polypeptide having amino acid residues I to 660 of Figure 13 (SEQ ID NO:28). or is complementary to such encoding nucleic acid sequence, and remain stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0265 polypeptide. In particular, the invention 15 provides isolated native sequence PR0265 polypeptide. which in one embodiment, includes an amino acid sequence comprising residues 1 to 660 of Figure 13 (SEQ ID N0O28). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0265 polypeptide.

6. PR0219 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0219".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0219 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO219 polypeptide having amino acid residues 1 to 915 of Figure 15 (SEQ ID NO:34), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0219 polypeptide. In particular, the invention provides isolated native sequence PR0219 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 915 of Figure 15 (SEQ ID NO:34).

7. PRZ46 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0246".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0246 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0246 polypeptide having amino acid residues 1 to 390 of Figure 17 (SEQ ID NO:39), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides usolated PR0246 palYPePtide. In particular, t invention provides isolated native sequence PR0246 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 390 of Figure 17 (SEQ ID NO:39). An additional embodiment of the present invention is directed to an isolated cxtracellular domain of a PR0246 polypptide.

8. IPRO228 Applicants have identified a cDNA clone that encodes a novel polypepUde having homology to CD97, EMRl and latrophilin, wherein the polypeptide is designated in the present application as 'PRO228".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0228 polypeptidc. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO228 polypeptide having amino acid residues I to 690 of Figure 19 (SEQ ID NO:49). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

another embodiment, the invention provides isolated PR0228 polypepdec. In particular, the invention provides isolated native sequence PRO228 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 690 of Figure 19 (SEQ ID NO:49). An additional embodiment of the present invention is directed to an isolated cxtracellular domain of a PR0229 polypeptide.

In another embodiment, the invention provides an expressed sequence tag (EST) comprising the nucleotide sequence of SEQ ID NO:50, designated herein as DNA219SI.

PR0533 Applicants have identified a cDNA clone (DNA49435-1219) that encodes a novel polypeptide, designated in the present application as PROM3.

In one embodiment, the invention provides -an isolated ncleic acid molecule having at least about sequence identity to a DNA molecule encoding a PR0533 polypeptide comprising the sequence of amino acids 23 to 216 of Figure 22 (SEQ ID NO59), or the comiplement ofdie DNA mnolecule of The sequence identity preferably is about 85%, more preferably about 90%, most preferably about 95%. In one aspect, the isolated nucleic acid has at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95 sequence identity with a polypeptide having amino acid residues 23 to 216 of Figure 22 (SEQ ID NO:59). Preferably, the highest degree of sequence identity occurs within the secreted portion (ammno, acids 23 to 216 of Figure 22, SEQ ID NO:59). In a further embodiment, tie isolated nucleic acid molecule comprises DNA encoding a PR0533 potypeptide having amino acid residues 1 to 216 of Figure 22 (SEQ ID NO:59), or is complentary to such encoding nucleic acid sequence, and remains stably bounid to it under at least moderate, and optinally, uner. high stingency conditions. In another aspect, the invention provides a nucleic acid of the full length protein of clone DNA49435-1219, deposited with the ATCC under accession number ATCC 209480.

In yet anoither embodin~t. the invention provides isolated PR0533 polypeptide. In particular, the invention provides isolated native sequence PR0533 polypeptide, which in one embodiment, includes an amino acid sequence comprising reidties 23 to 216 of Figure 22 (SEQ MD N0:59). Native PR0533 polypeptides with or without the native signal sequence (amino acids I to 22 in Figure 22 (SEQ MI 140:19)). and with or witu the initiating methionine art specifically included. Alternatively, the invention provides a PR0533 polypcptde encoded by the nucleic acid deposited wnder accession number ATCC 209480.

10. PRO245 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as *PRO245'.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0245 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0245 polypeptide having amno aid residties I to 312of Fig. 24 (SEQ ID NO:64), or is complementary to such encoding nucleic acd sequence, and remains stably bound to it under at least moderate, and optionally, nder high stringency conditions.

In another embodiment, the invention provides isolated PR0245 polypeptde. In particular, the invention provides isolated native sequence PR0245 polypeptide. which in one embodiment, includes an amino acid sequenc *..*comprising residues I to 312 of Figure 24 (SEQ ID NO:64).

It. PR0220. PRO221 and PR0227 Applicants have identified cDNA clones that cad encode rovel polypeptides, all having leucine rich repeats.

These polypeptides are designated in the present application as PR0220. PR0221 and PR0227.

In one embodimnent, tie invention provides isolated nucleic acid molecules comprising DNA respectively encoding PR0220. PR0221 and PR0227. respectively. In one aspect, provyided herein is an isolated nucleic acid comprses DNA encoding the PR0220 polypeptide having amino acid residues I throgh 708 of Figure 26 (SEQ ID NO:69). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate. and optionally, under high stringency conditions- Also provided herein is an isolated nucleic acid cmrssDNA encoding the PR0221 polypeptide having amino acid residues 1 through 259 of Figure 28 (SEQ ID NO:71), or is complementary to such encoding nucleic acid sequecen and remains stably bound to-it under at least moderate, and optionally, under high stringency conditions. Moreover, also provided herein is an isolated nucleic; acid comnprises DNA encoding the PROM polypeptde having amino acid residues I through 620 of Figure 30 (SEQ ED NO:.73). or is complementary to sixh encoding nucleic acid sequnace, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another emibodiment. die invention provides isolated PR0220, PR0221 and PR0227 polypeptides. In particular, provided herein is the isolated native: sequence for the PR0220 polypeptde, which in one embodiment, includes an amino acid sequence comprising residues I to 708 of Figure 26 (SEQ ID NO:69). Additionally provided herein is the isolated native seqz=c for the PR0221 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 259 of Figure 28 (SEQ ID NO:71). Moreover, provided herein is the isolated native sequence for the PR0227 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 620 of Figure 30 (SEQ ID NO:73).

12. PRB28 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to CRTAM and poliovinis receptor precursors, wherein the polypeptide is designated in the present application as "PR0258".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO258 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO258 polypeptide having amino acid residues I to 398 of Figure 32 (SEQ ID NO:84), or is complementary to such encoding nucleic acid sequence, and remains statly bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO258 polypeptide. In particular, the invention provides isolated native sequence PR0258 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 398 of Figure 32 (SEQ ID NO:84). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0258 polypcptide.

13. PRO2 Applicants have identified a cDNA clone that encodes a novel polypeptide. wherein the polypeptide is 15 designated in the present application as "PR0266".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0266 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0266 polypeptide having amino acid residues 1 to 696 of Figure 34 (SEQ ID NO:91), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0266 polypeptide. In particular, the invention provides isolated native sequence PR0266 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 696 of Figure 34 (SEQ ID NO:91).

25 14. PRO269 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as PR0269.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0269 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO269 polypeptide having amino acd residues I to 490 of Fig. 36 (SEQ ID NO:96). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0269 polypeptide. In particular, the invention provides isolated native sequence PR0269 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 490 of Figure 36 (SEQ ID NO:96). An additional embodiment of the present invention is directed to an isolated extraccllular domain of a PR0269 polypeptide.

I

PRQ287 Applicants have identified a cDNA done that encodes a novel polypeptidc, wherein the polypeptide is designated in the present application as "PR0287".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO287 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0287 polypeptide having amino acid residues I to 415 of Fig. 38 (SEQ ID NO:104), or is complementary to such enooding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO287 polypcptidc. In particular, the invention provides isolated native sequence PRO287 polypeptide, which in one embodiment, includes an amino acid sequence S comprising residues I to 415 of Figure 38 (SEQ ID NO: 104).

16. PR0214 Applicants have identified a cDNA clone that encodes a novel polypeptide, designated in the present application as "PRO214".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO214 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO214 polypeptide of Fig. 40 (SEQ ID NO: 109). or is complementary to such encoding nucleic acid sequence, and remains stably bound C. to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the invention provides a nucleic acid comprising the coding sequence of Fig. 39 (SEQ ID NO: 108) or its complement. In another aspect, the invention provides a nucleic acid of the full length protein of clone DNA32286-1191. deposited with ATCC under accession number ATCC 209385.

In yet another embodiment, the invention provides isolated PRO214 polypeptide. In particular, the invention provides isolated native sequence PR0214 polypeptide, which in one embodiment, includes an amino acid sequence comprising the residues of Figure 40 (SEQ ID NO:109). Alternatively, the invention provides a polypeptide encoded by the nucleic acid deposited under accession number ATCC 209385.

17. PR0317 Applicants have identified a cDNA clone that encodes a novel polypcptide, designated in the present application as "PR0317".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding PR0317 polypeptide. In one aspect, the isolated nucleic acid comprises DNA (SEQ ID NO:113) encoding PR0317 polypeptide having amino acid residues I to 366 of Fig. 42, or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO317 polypeptide. In particular, the invention provides isolated native-sequence PR0317 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 366 of Figure 42 (SEQ ID NO:114).

In yet another embodiment, the invention supplies a method of detecting the presence of PRO317 in a sample, the method comprising: a) contacting a detectable anti-PRO3 17 antibody with a sample suspected of containing PRO3 17; and b) detecting binding of the antibody to dhe sample. wherein the sample is selected from the group consisting of a body fluid, a tissue sample. a cell extract. and a cell culture medium.

In a sut further embodiment a method is provided for determining the presence of PR0317 mRlIA in a sample, the method comprising: a) contacting a sample suspected of containing PR0317 mRNA with a detectable nuclekt acid probe that hybridizes under moderate to stringent conditions to PR0317 mRNA; and b) detecting hybridization of the probe to the samnple.

Preferably, in this method the sample is a tissue sample and the detecting step is by In situ hybridization, or th apei eletataddtcini yNrhr nlss Further. the invention provides a method for treating a PRO3 17-associated disorder comprising administering to a mammial an effece amount of die MR0317 polypeptide or a composition thereof containing a carrier, or with an effective amount ofla PR0317 agonist or PR0317 antagonist. such as an antibody which binds specifically to PROMl.

18 Applicants have identified a eDNA clone (DNA40628-12 16) that encodes a novel polypeptide. designated in the present application as PR0301.

In one embodiment, the invention provides an isolated nucleic acid molecule having at least about sequence identity to a DNA molecule encoding a PRO301 polypeptide comprising the sequence of amino acids 28 to 258 of Fig. 44 (SEQ ID NO:1 19). or the complement of die DNA molecule of The sequence identity prcferably is about 85 more preferably about 90%, most preferably about 95 In one aspect, the isolated nuclic adid has at leas about 80%, preferably at least about 85%. more preferably at least about 90%, and most preferably at least about 95% sequence identity with a polypeptide having amino acid residues 28 to 258 of Fig. 44 (SEQ ID NO:119). Preferably, the highest degree of sequence: identity occurs within dhe extracellujar domains (amino acids to 25 28 to 258 of Fig. 44, SEQ ID NO 19). Ina frther embodiment. the isolated nucleic acid molecule comprises DNA encoding a PRO301 polypeptide having amino acid residues 28 to 299 of Fig. 44 (SEQ ID NO:119), or is complenientaty to such encoding nuecic acid sequence, and remains stably bound to It under at least moderate, and *550..optionally, unde high stringency conditions. In another aspect, the invention provides a nucleic acid of the full length protein of clone DNA40628-l216, deposited with the ATCC under accession number ATCC 209432, alternatively the coding sequence of cdone DNA40628-1216. deposited under accession number ATCC 209432.

In yet another embodiment, the invention provides isolated PRO301 polypeptide. In particular, the invention provides Isolated native sequence PRO301 polypeptide, which in one embodiment, includes an amino acid sequence comprising the extracellular domain residues 28 to 258 of Figure 44 (SEQ ID NO: 119). Native PRO301 polypeptides wiho ihu h aiesga eune(mn cd 1 to 27 in Figure 44 (SEQ ID NO: 119), and with or without the initiating methionine are specifically included. Additionally, the sequences of the invention may also comprise the transniembrane domain (residues 236 to about 258 in Figure 44; SEQ ID NO: 119) and/or the intracellular domain (about residue 259 to 299 in Figure 44; SEQ ID .NO:l 19). Alternatively. the invention provides a PRO301 polypeptide encoded by the nucleic acid deposited under accession number ATCC 209432.

19. PRO224 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PRO224".

In one embodiment, the invention provides an isolated nucleic acid molecule comprisingDNA encoding a PRO224 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0224 polypeptide having amino acid residues I to 282 of Figure 46 (SEQ ID NO:127), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at Ieast moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO224 polypeptide. In particular, the invention provides isolated native sequence PRO224 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 282 of Figure 46 (SEQ ID NO:127).

20. PR0222 15 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PRO222'.

,In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO222 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO222 polypeptide having amino acid residues 1 to 490 of Fig. 48 (SEQ ID NO:132), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO222 polypeptide. In particular, the invention provides isolated native sequence PRO222 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 490 of Figure 48 (SEQ ID NO:132).

C. C 21. PRQ234 Applicants have identified a cDNA clone that encodes a novel lectin polypeptide molecule, designated in the present application as "PRO234".

SIn one embodiment, the invention provides an isolated nucleic acid encoding a novel lectin comprising DNA encoding a PR0234 polypeptide. In one aspect, the isolated nucleic acid comprises the DNA encoding PRO234 polypeptides having amino acid residues I to 382 of Fig. 50 (SEQ ID NO:137), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the invention provides an isolated nucleic acid molecule comprising the nucleotide sequence of Fig. 49 (SEQ ID NO:136).

In another embodiment, the invention provides isolated novel PR0234 polypeptides. In particular, the invention provides isolated native sequence PR0234 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 382 of Figure 50 (SEQ ID NO:137).

In yet another embodiment, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nuclcotide sequences.

22. PRO231 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to a putative acid phosphatase, wherein the polypeptide is designated in the present application as 'PRO231".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0231 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0231 polypepide having amino acid residues I to 428 of Fig. 52 (SEQ ID NO. 142). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0231 polypeptide. In particular, the invention provides isolated native sequence PR0231 polypcptide. which in one embodiment, includes an amino acid sequence comprising residues I to 428 of Figure 52 (SEQ ID NO:142).

23. PR0229 15 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to scavenger receptors wherein the polypeptide is designated in the present application as "PR0229".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0229 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO229 polypeptide having amino acid residues I to 347 of Figure 54 (SEQ ID NO:148), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO229 polypeptide. In particular, the invention provides isolated native sequence PRO229 polypcptidc, which in one embodiment, includes an amino acid sequence comprising residues 1 to 347 of Figure 54 (SEQ ID NO:148).

24. PRO238 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to reductase, S* wherein the polypeptide is designated in the present application as "PR0238".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0238 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0238 polypeptide having amino acid residues 1 to 310 of Figure 56 (SEQ ID NO:153), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO238 polypeptide. In particular, the invention provides isolated native sequence PRO238 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 310 of Figure 56 (SEQ ID NO:153).

PR0233 Applicants have identified a cDNA clone that encodes a novel polypeptide. wherein the polypeptide is designated in the present application as "PRO233'.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0233 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0233 polypeptide having anmin acid residues 1 to 300 of Figure 58 (SEQ ID NO:159), or is complementary to suclencoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0233 polypeptide. In particular, the invention provides isolated native sequence PRO233 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 300 of Figure 58 (SEQ ID NO:159).

26. PRO23 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to serine carboxypeptidase polypeptides, wherein the polypeptide is designated in the present application as "PRO223".

15 In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO223 polypcptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0223 polypeptide .having amino acid residues 1 to 476 of Figure 60 (SEQ ID NO:164). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0223 polypeptide. In particular, the invention provides isolated native sequence PR0223 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 476 of Figure 60 (SEQ ID NO:164).

27. EPRO2 25 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PRO235".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a oo PRO235 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0235 polypeptide having amino acid residues 1 to 552 of Figure 62 (SEQ ID NO: 170), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0235 polypeptide. In particular, the invention provides isolated native sequence PRO235 polypcptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 552 of Figure 62 (SEQ ID NO:170).

28. PR0236 and PR0262 Applicants have idenified cDNA clones that enode novel polypeptides having homology to P-galactosidasc, wherein those polypeptides are designated in the present application as 'PR0236* and IPR0262'.

In on embaodiment, the invention provides an isolated nuclicc acid molecule comprising DNA encoding a PR0236 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0236 polypeptide havingamino acid residues I to 636 of Figure 64 (SEQ ID NO: 175), oris coplementary to suchencoding nucleic acid sequence. and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment. t invention provides an isolated nucleic; acid molecule comprising DNA encoding a PR0262 polypeptide. In one aspect. the isolated nucic acid comprises DNA encoding the PR0262 polypeptide having amino acid residues 1 to 6.54 of Figure 66 (SEQ ED NO: 177). or is complemenatary to such encoding nucleEc acid sequence. and remains stably bound to it under at least moderate, and optionally. under high stringency conditions.

In another emibodiment, the invention provides isolated PR0236 polypeptide. In particular. the invention provides isolated native sequence PR0236 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 636 of Figure 64 (S EQ ID NO: 175).

In another embodiment, the invention provides isolated PR0262 polypeptide. In particular. the invention provides isolated native sequence PR0262 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 654 of Figure 66 (SEQ ID NO: 177).

20 29. PRO739 Applicants have identified a cDNA done that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0239".

P0239In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR23 polypeptide. In one aspect. the isolated nucleic acid comprises DNA encoding the PR0239 polypeptide having amnino acid residues 1 to 501 of Figure 68 (SEQ ID NO- 18S). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at leat moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0239 polypeptide. In pa rticular, the invention provides isolated native sequence PR0239 polypeptide, which in one em~bodinment. includes an amino acid sequence comprising residues I to 501 of Ftgure 68 (SEQ ID NO: 185).

PRO27 Applicants have identified a cDNA done that encodes a novel polypeptide, wherein the polypcptide is designated in the present application as 'PR07".

In one embodimeni, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0257 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0257 polypeptide having amino acid residues I to 607 of Figure 70 (SEQ ID NO: 190). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO257 polypeptide. In particular, the invention provides isolated native sequence PR0257 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 607 of Figure 70 (SEQ ID NO:190). An additional embodiment of the present invention is directed to an isolated extracllular domain of a PR0257 polypeptide.

31. PR0260 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is S designated in the present application as "PRO260'.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0260 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0260 polypeptide having amino acid residues I to 467 of Figure 72 (SEQ ID NO: 195). or is complementary to such encoding nucleic Sacid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

15 In another embodiment, the invention provides isolated PR0260 polypeptide. In particular, the invention provides isolated native sequence PR0260 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 467 of Figure 72 (SEQ ID NO:195).

32. PR0263 20 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to CD44 antigen, wherein the polypeptide is designated in the present application as "PRO263".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO263 polypcptidc. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0263 polypeptide S- having amino acid residues 1 to 322 of Figure 74 (SEQ ID NO:201). or is complementary to such encoding nucleic 25 acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0263 polypcptide. In particular, the invention provides isolated native sequence PR0263 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 322 of Figure 74 (SEQ ID NO.201). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0263 polypeptide.

33. PRo270 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0270".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO270 polypeptide. In one aspect, the isolated nucleic acid comprises DNA whivch includes the sequence encoding the PRO270 polypeptide having amino acid residues I to 296 of Fig. 76 (SEQ ID NO:207), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0270 polypcptidc. In particular, the invention provides isolated native sequence PR0270 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 296 of Figure 76 (SEQ ID NO:207).

34. PR0271 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to the protcoglycan link protein, wherein the polypepdde is designated in the present application as "PRO271".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO271 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO271 polypeptide having amino acd residues I to 360 of Figure 78 (SEQ ID N0113), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0271 polypeptide. In particular, the invention provides isolated native sequence PRO271 polypcptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 360 of Figure 78 (SEQ ID NO:213).

PRO72 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is 20 designated in the present application as *PRO272".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0272 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0272 polypeptide having amino acid residues I to 328 of Figure 80 (SEQ ID NO:221), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency 25 conditions.

In another embodiment, the invention provides isolated PR0272 polypeptide. In particular, the invention provides isolated native sequence PRO272 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 328 of Figure 80 (SEQ ID NO-211).

36. PR0294 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0294".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0294 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0294 polypeptide having amino acid residues I to 550 of Figure 82 (SEQ ID NO:227), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

48 In another embodiment, the invention provides isolated PRO294 polypeptide. In particular, the invention provides isolated native sequence PR0294 polypeptide, which in one embodiment, includes an amino acid sequence ccrprising residues 1 to 550 of Figure 82 (SEQ ID NO:227) 37. PRO295 Applicants have identified a cMNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0295".

In one embodiment, the invention provides an isolated nucleic acid molecule ccoprising DNA encoding the PR0295 polypeptide. In one aspect, the isolated nucleic acid ccnprises CNA encoding the PR0285 polypeptide having amino acid residues 1 to 350 of Figure 84 (SEQ ID N0:236), or is ccuplementary to such encoding nucleic acid sequence, and 15 remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0295 polypeptide. In particular, the invention provides isolated native sequence PR0295 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 350 of Figure 84 (SEQ ID NO:236).

.e 38. PRO293 Applicants have identified a cEA clone that encodes a novel human neuronal leucine rich repeat polypeptide, wherein the polypeptide is 25 designated in the present application as "PRO293".

In one embodiment, the invention provides an isolated nucleic acid molecule ccnprising UNA encoding a PR0293 polypeptide. In one aspect, the isolated nucleic acid ccnprises IEA encoding the PR0293 polypeptide having amino acid residues 1 to 713 of Figure 86 (SEQ ID NO:244), or is ccnplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO293 polypeptide. In particular, the invention provides isolated native sequence PRO293 polypeptide, which in one embodiment, includes an amino acid sequence ccprising residues 1 to 713 of Figure 86 (SEQ ID NO:244).

An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0293 polypeptide.

49 39. PRO247 Applicants have identified a cEM clone that encodes a novel polypeptide having leucine rich repeats wherein the polypeptide is designated in the present application as P24' In one embo~diment, the invention provides an isolated nucleic acid molecule comprising WA encoding a PR0.247 polypeptide.. In. one aspect, the isolated nucleic acid ccuprises =1 encoding the PR0247 polypeptide having amino acid residues 1. to 546 of Figure 88 (SBQ ID NO:249), or is ccxplementary to such encoding nucleic acid sequence, and remiin stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO247 polypeptide. In particular, the invention provides isolated native sequence PR0247 polypeptide, which in one etbodimit, includes an amino acid sequence comprising residues 1 to 546 of Figure 88 (SBQ ID NO:249).

An additional effbodiment of the present invention is directed to an isolated extracellular domrain of a PRO247 polypeptide.

PRO302, PPD303, EW~3O4, PRO307 and PR~O343 Aplicants have identified cflM clones that encode novel *:polypeptides having hcffiology to various proteases, wherein those polypeptide are designated in the present application as -PRO3 02", -PR0303-, -PR0304-, "PRO307"' and 'PRO343- polypeptides.

in one embodiment, the invention provides an isolated nucleic acid molecule ccxtprising MA~ encoding a PR0302 polypeptide. In one aspect, the isolated nucleic acid comrprises MA encoding the PR0302 polypeptide having amino acidresidues 1 to 452 of Figure 90 (SE)Q ID IND:254), or is caxplementary to such encoding nucleic acid sequence, and rema.ins stably bound to it under at least imderate, and optionally, under high stringency conditions.

In another emibodiment, the invention provides an isolated nucleic acid molecule comprising EVA encoding a PR0303 polypeptide. In one aspect, the isolated nucleic acid cconprises £XN.A encoding the PR0303 polypeptide havingj aino acid residues 1 to 314 of Figure 92 (SD 2 ID INK):256), or is ccnplementaxy to such encoding nucleic -acid sequence, and renains stably bound .to it under at least ntderate, and optionally, under high stringency conditions.

In yet another entodiment, the invention provides an isolated nucleic acid inolecule comprising EMU encoding a PR0304 polypeptide. In one 50 aspect, the isolated nucleic acid molecule corprises [NA encoding a PR0304 polypeptide having amino acid residues 1 to 556 of Figure 94 (SEQ ID NO:258), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another erbodiment, the invention provides an isolated nucleic acid molecule ccnprising ENA encoding a PRO307 polypeptide. In one aspect, the isolated nucleic acid conprises EA encoding the PRO307 polypeptide having amino acid residues 1 to 383 of Figure 96 (SBQ ID NO:260), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another entiodinent, the invention provides an isolated nucleic acid nolecule ccnprising ENA encoding a PRO343 polypeptide. In one 15 aspect, the isolated nucleic acid ccnprises INA encoding the PRO343 polypeptide having amino acid residues 1 to 317 of Figure 98 (SEQ ID NO:262), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO302 polypeptide. In particular, the invention provides isolated native sequence PR0302 polypeptide, which in one embodiment, include an amino acid sequence comprising residues 1 to 452 of Figure 90 (SEQ ID N3:254).

In another enbodiment, the invention provides isolated PRO303 25 polypeptide. In particular, the invention provides isolated native sequence PR0303 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 314 of Figure 92 (SEQ ID ND:256).

In another embodiment, the invention provides isolated PR0304 polypeptide. In particular, the invention provides isolated native sequence PRO304 polypeptide, which in one embodiment, includes an amino acid sequence corprising residues 1 to 556 of Figure 94 (SEQ ID ND:258).

In another erbodiment, the invention provides isolated PRO307 polypeptide. In particular, the invention provides isolated native sequence PRO307 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 383 of Figure 96 (SEQ ID NO:260).

In another embodiment, the invention provides isolated PR0343 polypeptide. In particular, the invention provides isolated native sequence PR0343 polypeptide, which in one enbodximent, includes an amino acid sequence carprising residues 1 to 317 of Figure 98 (SEQ ID N0:262).

51 41. PR0328 Applicants have identified a cENA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PRO328".

In one embodinent, the invention provides an isolated nucleic acid molecule comprising ENA encoding a PR0328 polypeptide. In one aspect, the isolated nucleic acid conprises ENA encoding the PR0328 polypeptide having amino acid residues 1 to 463 of Figure 100 (SEQ ID NO:284), or is complementary to such encoding nucleic acid sequence, and retains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO328 1 5 polypeptide. In particular, the invention provides isolated native sequence PRO328 polypeptide, which in one embodiment, includes an amino acid sequence coaprising residues 1 to 463 of Figure 100 (SEQ ID N0:284).

An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0306 polypeptide.

42. PR0335, PR0331 and PR0326 Applicants have identified three cEA clones that respectively encode three novel polypeptides, each having leucine rich repeats and homology to LIG-1 and ALS. These polypeptides are designated in the present application as PRO335, PRO331 and PRO326, respectively.

25 In one embodimment, the invention provides three isolated nucleic acid molecules camprising IIA respectively encoding PR0335, PRO331 and PRO326, respectively. In one aspect, herein is provided an isolated nucleic acid carprising EA. encoding the PRO335 polypeptide having amino acid residues 1 through 1059 of Figure 102 (SEQ ID ND:289), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. Also provided herein is an isolated nucleic acid catprises IA encoding the PRO331 polypeptide having amino acid residues 1 through 640 of Figure 104 (SEQ ID N0:291), or is ccaplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. Additionally provided herein is an isolated nucleic acid ccmprises EA% encoding the PRO326 polypeptide having amino acid residues 1 through 1119 of Figure 106 (SEQ ID NO: 293), or is complementary to such encoding nucleic acid sequence, and remains 52 stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0335, PR0331 and PR0326 polypeptides or extracellular domains thereof. In particular, the invention provides isolated native sequence for the PR0335 polypeptide, which in one embodiment, includes an amino acid sequence ccnprising residues 1 through 1059 of Figure 102 (SDEQ ID NO:289). Also provided herein is the isolated native sequence for the PR0331 polypeptide, which in one embodiment, includes an amino acid sequence corprising 1 through 640 of Figure 104 (SEQ ID NO: 291). Also provided herein is the isolated native sequence for the PR0326 polypeptide, which in one embtodiment, includes an amino acid sequence ccmprising residues 1 through 1119 of Figure 106 (SBQ ID N: 293).

43. PR0332 Applicants have identified a cDNA clone (IA40982-1235) that encodes a novel polypeptide, designated in the present application as "PR332".

In one embodiment, the invention provides an isolated nucleic acid nolecule corprising EA having at least about 80% sequence identity to a ENA molecule encoding a PR0358 polypeptide corprising the sequence of .amino acids 49 to 642 of Fig. 108 (SB ID NO:309), or the ccrplement of the UMA molecule of The sequence identity preferably is about 2 more preferably about 90%, most preferably about 95%. In one aspect, the **25 isolated nucleic acid has at least about 80%, preferably at least about more preferably at least about 90%, and most preferably at least about sequence identity with a polypeptide having amino acid residues 1 to 642 of Fig. 108 (SEQ ID N0:309). Preferably, the highest degree of sequence identity occurs within the leucine-rich repeat domains (amino acids 116 to 624 of Fig. 108, SEQ ID ND:309). In a further embodiment, the isolated nucleic acid nolecule comprises CNA encoding a PR0332 polypeptide having amino acid residues 49 to 642 of Fig. 108 (SB ID N0:309), or is ccmplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0332 polypeptides. In particular, the invention provides isolated native sequence PR0332 polypeptide, which in one embodiment, includes an amino acid sequence coprising residues 49 to 624 of Figure 108 (SEQ ID NO:309).

53 Native PR0332 polypeptides with or without the native signal sequence (amino acids 1 to 48 in Figure 108, SEQ ID NO: 309), and with or without the initiating methionine are specifically included.

44. PI0334 Applicants have identified a cENA clone that encodes a novel polypeptide having hamology to fibulin and fibrillin, wherein the polypeptide is designated in the present application as "PRO334".

In one embodinent, the invention provides an isolated nucleic acid molecule caoprising MNA encoding a PR0334 polypeptide. In one aspect, the isolated nucleic acid ccnprises ENA encoding the PRO334 polypeptide having amino acid residues 1 to 509 of Figure 110 (SEQ ID NO:314), or is ccaplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0334 polypeptide. In particular, the invention provides isolated native sequence PR0334 polypeptide, which in one etbodiment, includes an amino •acid sequence conprising residues 1 to 509 of Figure 110 (SEQ ID NO:314).

45. PR0346 Applicants have identified a cIEA clone (ENA44167-1243) that encodes a novel polypeptide, designated in the present application as "PR0346".

In one embodiment, the invention provides an isolated nucleic acid molecule having at least about 80% sequence identity to a CNA molecule encoding a PRO346 polypeptide ccnprising the sequence of amino acids 19 to 339 of Fig. 112 (SEQ ID NO:319), or the ccaplement of the EA molecule of SThe sequence identity preferably is about 85%, more preferably about most preferably about 95%. In one aspect, the isolated nucleic acid has at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity with a polypeptide having amino acid residues 19 to 339 of Fig. 112 (SEQ ID NO:319). Preferably, the highest degree of sequence identity occurs within the extracellular daonins (amino acids 19 to 339 of Fig. 112, SEQ ID NO:319). In alternative embodiments, the polypeptide by which the homology is measured ccaprises the residues 1-339, 19-360 or 19-450 of Fig. 112, SEQ ID ND:319). In a further embodiment, the isolated nucleic acid molecule carprises NA encoding a PRO346 polypeptide having amino acid residues 19 to 339 of Fig. 112 (SEQ ID NO: 319), alternatively residues 1-339, 19-360 54 or 19-450 of Fig. 112 (SEQ ID NO: 319) or is couplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the invention provides a nucleic acid of the full length protein of clone E1A44167-1243, deposited with the ATCC under accession number ATCC 209434, alternatively the coding sequence of clone 11A44167-1243, deposited under accession number ATICC 209434.

In yet another enbodiment, the invention provides isolated PR0346 polypeptide. In particular, the invention provides isolated native sequence PR0346 polypeptide, which in one embodinent, includes an amino acid sequence ccmprising residues 19 to 339 of Figure 112 (SEQ ID N:319).

Native PRO346 polypeptides with or without the native signal sequence (residues 1 to 18 in Figure 112 (SXQ ID NO: 319), with or without the initiating methinine, with or without the transmaTtrane docain (residues 15 340 to 360) and with or without the intracellular dcuain (residues 361 to 450) are specifically included. Alternatively, the invention provides a PR0346 polypeptide encoded by the nucleic acid deposited under accession number ATCC 209434.

46. PRO268 Applicants have identified a cEA clone that encodes a novel polypeptide having homology to protein disulfide isamerase, wherein the polypeptide is designated in the present application as "PRO268".

In one embodinent, the invention provides an isolated nucleic 25 acid molecule comprising EA encoding a PRO268 polypeptide. In one aspect, the isolated nucleic acid comprises I@M encoding the PRO268 polypeptide having amino acid residues 1 to 280 of Figure 114 (SEQ ID N:324), or is "i *ccnplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another enbodiment, the invention provides isolated PRO268 polypeptide. In particular, the invention provides isolated native sequence PRO268 polypeptide, which in one embodiment, includes an amino acid sequence ccuprising residues 1 to 280 of Figure 114 (SEQ ID NO:324).

An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0268 polypeptide.

47. PRO330 Applicants have identified a cIA clone that encodes a novel 54a polypeptide having hcaology to the alpha subunit of prolyl 4-hydroxylase, wherein the polypeptide is designated in the present application as "PR0330".

In one embodiment, the invention provides an isolated nucleic acid molecule ccaprising I~A encoding the PR0330 polypeptide. In one aspect, the isolated nucleic acid molecule carprising INA encoding a PR0330 polypeptide having amino acid residues 1 to 533 of Figure 116 (SEQ ID N0:331), or is carplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0330 polypeptide. In particular, the invention provides isolated native sequence PR0330 polypeptide, which in one embodiment, includes an amino acid sequence cacprising residues 1 to 533 of Figure 116 (SEB ID NO:331).

48. PR0339 and PRO310 Applicants have identified two cINA clones wherein each clone encodes a novel polypeptide having harology to fringe, wherein the polypeptides are designated in the present application as "PRO339" and "PRO310".

In one e~bodiment, the invention provides isolated nucleic acid molecules caoprising IEA encoding the PR0339 and/or a PRO310 polypeptide.

In one aspect, the isolated nucleic acid canprises ENA encoding the PR0339 polypeptide having amino acid residues 1 to 772 of Figure 118 (SEQ ID 25 N:338), or is carplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the isolated nucleic acid ccmprises ENA encoding the PRO310 polypeptide having amino acid residues 1 to 318 of Figure 120 (SBQ ID ND:340), or is camplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO339 as well as isolated PR0310 polypeptides. In particular, the invention provides isolated native sequence PR0339 polypeptide, which in one embodiment, includes an amino acid sequence carprising residues 1 to 772 of Figure 118 (SEQ ID NO: 338). The invention further provides isolated native sequence PRO310 polypeptide, which in one embodiment, includes an amino acid sequence ccrprising residues 1 to 318 of Figure 120 (SEQ ID KN: 340).

54b 49. PR0244 Applicants have identified a cINA clone that encodes a novel polypeptide, designated in the present application as "PR0244".

In one embodinent, the invention provides an isolated nucleic acid nolecule comprising IEA encoding PR0244 polypeptide. In one aspect, the isolated nucleic acid comprises INA encoding PRO244 polypeptide having amino acid residues 1 to 219 of Fig. 122 (SEQ ID ND:376), or is caplerentary to such encoding nucleic acid sequence, and remains stably bound to it under at last moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0244 polypeptide. In particular, the invention provides isolated native sequence PR0244 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 219 of Figure 122 (SEQ ID NO:376).

50. Additional bodiments In other embodiments of the present invention, the invention provides vectors coprising ENA encoding any of the above or below S described polypeptides. A host cell comprising any such vector is also provided. By way of example, the host cells may be CHO cells, E. coli, or Syeast. A process for producing any of the above or below described S" polypeptides is further provided and cmrprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide fron the cell culture.

25 In other embodiments, the invention provides chimeric molecules carprising any of the above or below described polypeptides fused to a heterologous polypeptide or amino acid sequence. An exanple of such a chimeric nolecule caorprises any of the above or below described polypeptides fused to an epitope tag sequence or a Fc region of an irmnnoglobulin.

In another embodinent, the invention provides an antibody which specifically binds to any of the above or below described polypeptides.

Optionally, the antibody is a nonoclonal antibody.

In yet other gbodiments, the invention provides oligonucleotide probes useful for isolating genanic and cN1A nucleotide sequences, wherein those probes may be derived from any of the above or below described nucleotide sequences.

54c For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequence PRO211 cDNA, wherein SEQ ID NO:1 is a clone designated herein as "UNQ185" and/or "DNA32292-1131".

Figure 2 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 shows a nucleotide sequence (SEQ ID NO:3) of 15 a native sequence PR0217 cDNA, wherein SEQ ID NO:3 is a clone designated herein as "UNQ191" and/or "DNA33094-1131".

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

Figure 5 shows a nucleotide sequence (SEQ ID NO:11) S* of a native sequence PR0230 cDNA, wherein SEQ ID NO:ll is a clone designated herein as "UNQ204" and/or "DNA33223-1136".

Figure 6 shows the amino acid sequence (SEQ ID NO:12) derived from the coding sequence of SEQ ID NO:ll shown in Figure 25 Figure 7 shows a nucleotide sequence designated herein as DNA20088 (SEQ ID NO:13).

Figure 9 shows a micleorde sequence (SEQ MD NO: 17) of a native sequence PR0232 cDNA. wherein SEQ ID NO: 17 is a clone designated herein as 'UJ1Q206 and/or -DNA34435-1140 Figure 9 shows the amino acid sequence (SEQ ID 140:18) derived from the coding sequence of SEQ ID NO: 17 shown in Figure 8.

Figure 10 shows a macleodde sequence (SE ID NO:22) of anative sequence PRO187 cD14A, wherein SEQ ID N0:22 is a clone designated herein as *U14Q161 *and/or "DNA27864-1155".

Figure I1I shows the amino acid sequence (SEQ ID NO:23) derived from the coding sequence of S EQ ID N4022 shown in Figure Figure 12 shows a awclotidec sequence (SEQ MD NO:27) of a native sequence PR0265 cd)NA, wherein SEQ ID 140:27 is a clone designated herein as NUNQ232' and/or "DNA36350-11S8'.

Figure 13 shows the amino acid sequence (SEQ ID 140:28) derived from the coding sequence of SEQ ID 140:27 shown in Figure 12.

Figures 14A-B show a nueleotde sequence (SEQ MD NO:33) of a native sequence PRO21 9 cDNA. wherein *.SEQ ID NO:33 is a clone designated herein as 'UNQ193* and/or 'D1A32290-IIW4.

Figure 15 shows the amino acid sequence (SEQ ID 140:34) derived from the coding sequence of SEQ ID 140:33 shown In Figures 14A-B.

Figure 16 shows a ,ualcoddc. sequence (SEQ ED 140-38 of a native sequence PR0246 cdNA, wherein SEQ ID 140:38 is a dlone designated herein as *U1Q220" and/or *DNA35639-1172*.

Figure 17 shows the amin acid sequence (SEQ ID NO:39) derived from the coding sequence of SEQ ID 140:38 shown in Figure 16.

Figure 18 shows a nudleonde sequence (SEQ ID 140:48) of a native sequence PR0228 cDNA. wherein SEQ *ID 144 is a clone designated herein as 'UNQ202" and/or *DNA33092-1202'.

Figure 19 shows the amino acid sequence (SEQ ID 140:49) derived from the coding sequence of SEQ MD 140:8 shown in Figure 18.

Figure 20 shows a nucleotide sequence designated herein as DNA21951 (SEQ ID Figure 21 shows a nudleodde sequence (SEQ MD 10:58) of a native sequence PR0533 cDNA, wherein S EQ ID NO:58 is a clone designated herein as "UNQ344* aund/or ODNA49435-12196.I Figure 22 shows the amino acid sequence (SE ID 140:59) derived from the coding sequence of SEQ MD 140:58 shown in Figure 21.

Figure 23 shows a asicleotide sequence (SEQ ID NO:63) of a native sequence PRO245 cDNA, wherein SEQ ID 140:63 is a clone designated herein as 'UNQ219' and/or "DNA35638.1141*.

Figure 24 shows the amino acid sequence (SEQ ID 140:64) derived from the coding sequence of SEQ ID 140:63 shown in Figure 23.

Figure 25 shows a miudotide sequence (SEQ ED NO:68) of a native sequence PRO220 cDNA. wherein SEQ 113 NO:68 is a clone designated herein as 'UNQ194' and/or "DNA32298-I 132".

Figure 26 Shows the amino acid sequence (SEQ ID 10:69) derived from the~ooding sequence of SEQ ID 140:68 shown in Figure .Figure 27 shows a nuelcotide sequence (SEQ ID NO:70) of a native: sequence PR0221 cDNA. wherein S EQ ID NO:70 is a clone designated herein as 'UNQ195* and/or *DNA33089-1 132*.

Figure 28 shows the amino acid sequence (SEQ ID NO:71) derived from the coding sequence of SEQ MD shown in Figure 27.

Figure 29 shows a nucleode sequaee (SEQ ID NO:72) of a native sequence PR0227 cDNA. wherein SEQ ID NO:72 is a clone designated herein as *UNQ2Or* and/or DNA33786-1 1320.

Figure 30 shows the amino acid sequence (SEQ ID NO:73) derived from the coding sequence or SEQ MD NO: 72 shown in Figure 29.

Figure 31 shows a micleotde sequnce (SEQ ID NO:83) of a native sequence PR0258 eDNA, wherein SEQ ID NO:83 is a clone designated herein as OUNQ225' and/or'DNA3591 8-1174'.

Figure 32 shows die amino acid sequence (SEQ ID NO:84) derived from the coding sequence of SEQ ID NO: M shown in Figure 3 1.

Figure 33 shows a micleouide: sequence (SEQ ID N:90) of a native sequence PR0266 eDNA. wherein SEQ :.ID NO:90 is a clone designated herein as "UNQ233y ankd/or'DNA37150-1178*.

*000 Figure 34 shows dhe amino acid sequence (SEQ ID NO:91) derived from the coding sequence of SEQ ID NO:9O shown in Figure 33.

Figure 35 shows a ileodde sequece= (SEQ ID NO:95) of a native sequence PR0269 cDNA. wherein SEQ *ID NO:95 is a clone designated herein as L1NQ236' and/or ODNA38260-1 180.

Figure 36 shows die amino acid sequence (SEQ ID NO:96) derived from the coding sequence of SEQ MD shown in Figure 20 Figure 37 shows a nucleatide sequence (SEQ ID NO:103) of a niative sequence PR0287 cDNA. wherein SEQ ID NO:103 is a clone designated herein as 'UNQ250' and/or *DNA39969-118S".

Figure 38 shows the amino acid sequence (SEQ ID NO: 104) derived from the coding sequence of S EQ ID :NO: 103 shown in Figure 37.

Figure 39 shows a nucleodde sequence (SEQ ID NO:108) of a native sequence PR0214 cDNA. wherein SEQ ID NO:108 is a clone designated herein as 'UNQ188' and/or "DNA32286-1 191".

Figure 40 shows the amino acid sequence (SEQ ID NO: 109) derived from the coding sequence of SEQ ID NO. 108 shown in Figure 39.

Figure 41 shows a nucleotide sequence (SEQ ID NO: 113) of a niative sequence PR0317 cDNA, wherein SEQ ID NO:1 13 is a clone designated herein as "UNQ278* and/or 'DNA33461-1 199*.

Figure 42 showsteamino acid sequence (SEQ ID NO: 114) derived from the coding sequence ofSEQ ID NO: 113 shown in Figure 41.

Figure 43 shows a nucleodde sequence (SEQ ID NO: 118) of a native sequence PRO301 cDNA, wherein SEQ ID NO: 118 is a clone designated herein as UUNQ264' and/or 0 DNA40628-1216*.

Figure 44 shows the amino acid sequence (SEQ ID NO: 119) derived from the coding sequence of SEQ ID NO:ll8 shown in Figure 43.

Figure 45 shows a nucleotide sequence (SEQ ID NO: 126) of a native sequence PR0224 cDNA, wherein SEQ ID NO: 126 is a clone designated herein as 'UNQI98' and/or *DNA33221-1133'.

Figure 46 shows the amino acid sequence (SEQ ID NO: 127) derived from dhe coding sequence of SEQ ID NO: 126 shown in Figure Figure 47 shows a nucleotide sequence (SEQ [I NO: 13 1) of a native sequence PR0222 cDNA, wherein SEQ ED NO: 131 is a clone designated herein as 'UNQ196" and/or "DNA33107-l135'.

Figure 48 shows the amino acid sequence (SEQ ID NO: 132) derived from dhe coding sequence of SEQ FD NO: 131 shown in Figure 47. L Figure 49 shows a suclootide sequence (SEQ ID NO: 136) of a native sequence PR0234 cDNA, wherein SEQ ID NO: 136 is a clone designated herein as 'UNQ2OS" and/or 6 DNA35557-113*.

Figure 50 show the atnino acid sequence (SEQ ID NO:137) derived from the coding sequence of SEQ ID NO: 136 shown in Figure 49.

Figure 51 shows a nucleotide sequence (SEQ MD NO: 141) of a native sequence PR0O231 cDNA, wherein SEQ ID NO: 141 is a clone designated herein as 'UNQ205S and/or IDNA34434.1139".

Figuze52 shows dx amin acid sequence (SEQ ID NO:142) derived from die coding sequence of SEQ ID 141 shown in Figure 5 Figure 53 shows a nucleotide sequence (SEQ ID NO: 147) of a native sequence PR0229 cDNA, wherein SEQ ID NO: 147 is a clone designated herein as *UNQ203 *and/or "DNA33100-1159".

Figure 54 shows the amino acid sequence (SEQ ID NO:148) derived from die coding sequence of SEQ ID NO: 147 shown in Figure 53.

Figure 55 shows a nucleotide sequence (SEQ ID NO: 152) of a native sequence PRO238 cDNA, wherein SEQ ID NO: 152 is a clone designated herein as 'UNQ212* and/or *DNA3S600-1162".

Figure 56 shows the ammio acid sequence (SEQ ID NO: 153) derived from the coding sequence of SEQ ID N:12shown in Figure Figure 57 shows a nucleotide sequence (SEQ ID NO: 158) of a native sequence PRO233 cDNA. wherein :SEQ ID NO:158 is a clone designated herein as 'UNQ2Y7* and/or

M

DNA34436-1238".

Figure 58 shows the amino acid sequence (SEQ ID NO: 159) derived from the coding sequence of. SEQ ID NO: 158 shown in Figure 57.

Figure 59 shows a nuclodde sequence (SEQ ID NO: 163) of a native sequence PR0223 cDNA, wherein *SEQ ID NO:163 is a clone designated herein as *UNQ19r and/or "DNA33206-116S'.

Figure 60 shows the amino acid sequence (SEQ ID NO:164) derived from the coding sequence of SEQ ID NO: 163 shown in Figure 59.

Figure 61 shows a nucteotide sequence (SEQ ID NO: 169) of a native sequence PR0235 cDNA. wherein SEQ ID NO: 169 is a clane designated herein as 'UNQ209' arid/or "DNA35558-1167'.

Figure 62 shows the amino acid sequence (SEQ ID NO: 170) derived from the coding sequence of SEQ ID NO: 169 shown in Figure 61.

Figure 63 shows a nucleotide sequence (SEQ ID NO: 174) of a native sequenc PRO236 cDNA, wherein SEQ ID NO: 174 is a clone designated herein as 'UNQ210" and/or ODNA35599-1168'.

Figure 64 shows the amino acid sequence (SEQ MD NO: 175) derived from die coding sequence of SEQ ID NO: 174 shown in Figure 63.

Figure 65 shows a nucleodde sequence (SEQ ID NO:176) of a native sequence PR0262 cDNA, wherin SEQ ID NO: 176 is a clone designated herein as *UNQ229 and/or *DNA36992-1 165"'.

Figure 66 shows the amino acid sequence (SEQ ID NO: 177) derived from the coding sequence of SEQ ID NO: 176 shown in Figure Figure 67 shows a nucicotide sequence (SEQ ID NO:184) of a native scquece= PR0239 cDNA, wherein SEQ ID NO: 1S4 is a clone designated herein as *UNQ2130 and/or *DNA34407-1169'.

Figure 68 shows the amino acid sequence (SEQ D 140:185) derived from the coding sequence of SEQ ID NO: 184 shown in Figure 67.

Figure 69 shows a nucleotide sequence (SEQ I) 140:189) of a native sequence PR0257 cDNAi wherein SEQ ID NO: 189 is a clone designated herein as "UNQ224* and/or "DNA35841-1 173"'.

Fgur 70 shows the amino acid sequence (SEQ D 140:190) derived from the coding sequenc of SEQ ID NO: 189 shown in Figure 69.

Figure 71 shows a nucleotide sequence (SEQ ID NO: 194) of a native sequne PR0260 cDNA, wherein .*SEQ ID NO: 194 is a clone designated herein as "UNQ227" and/or uDNA334704 175".

Figure 72 shows the amino acid sequence (SEQ ID NO:195) derived from the coding sequence of SEQ ID NO:l194 shown in Figure 71.

Figure 73 shows a aucocde sequence (SEQ ID NO:200) of a native sequence PR0263 cDNA, wherein -SEQ ID NO:.200 is a cdone designated herein as 'UNQ230" and/or "DNA34431-1 177'.

Figure 74 shows the amino acid sequence (SEQ ID NOM20) derived from the coding sequence of SEQ ID NO:200 shown in Figure 73.

Figure 75 shows a nuclcotidc sequence (SEQ ID NO :206) of a native sequence PR0270 cDNA. wherein SEQ ID NO:.206 is a clone designated herein as m UNQ237" and/or *DNA395IO-11IS1V.

Figure 76 shows the amino acid sequence (SEQ ID NO:207) derived from the coding sequence of SEQ ID NO:206 shown in Figure Figure 77 shows a nucleotide sequence (SEQ 11) 140:212) of a native sequence PR0271 cDNA. wherein SEQ ID NO:212 is a clone designated herein as "UN Q238" anid/or "DNA39423-1182".

Figure 78 shows the amino acid sequence (SEQ ID N40213) derived from the coding sequence of SEQ ID 140:212 shown in Figure 77.

Figure 79 shows a nuclearide sequence (SEQ ID NO0:220) of a native sequence PR0272 cDNA, wherein SEQ ID NO0M2 is a clone designated herein as "UNQ239" and/ar 'DNA40620-1183".

Figure 80 shows the amino acid sequence (SEQ ID NO:221) derived from the coding sequence of SEQ ID NO:220 shown in Figure 79.

Figure 81 shows a nucleotide sequence (SEQ ID NO:226) of a native sequence PR0294 cDNA, wherein SEQ ID NO:226 is a clone designated hercin as "UNQ257 *and/or "D14A40604-1187".

Figure 82 shows the amino acid sequence (SEQ ID NO:227) derived from dhe coding sequence of SEQ ID 140:226 shown in Figure 81.

Figure 83 shows a nucleotide sequence (SEQ ID NO:235) of a native sequence PR0295 cDNA, wherein SEQ ID 140:235 is a clone designated herein as "UNQ258' and/or "DNA38268-1188*.

59 Figure 84 shows the amino acid sequence (SEQ ID NO:236) derived frm the coding sequence SEQ ID No:235 shown in Figure 83.

Figures 85A-B show a nucleotide sequence (SEQ ID NO:243) of a native sequence PRO293 cA, wherein SEQ ID N0:244 is a clone designated herein as "UNQ256" and/or "IIA37151-1193".

Figure 86 shows the amino acid sequence (SEQ I-NO:244) derived fran the coding sequence of SEQ ID N0:244 shown in Figures Figures 89A-B show a nucleotide sequence (SEQ ID N:248) of a native sequence PRO247 cENA, wherein SEQ ID NO:248 is a clone designated herein as "UNI221" and/or "EA35673-1201".

Figure 88 shows the amino acid sequence (SEQ ID N0:249) derived fram the coding sequence of SEQ ID N0:248 shown in Figure 87.

Figure 89 shows a nucleotide sequence (SEQ ID NO:253) of a native sequence PR0302 c1NA, wherein SEQ ID NO:253 is a clone designated 15 herein as 'UNQ265" and/or '*NA40370-1217".

Figure 90 shows the amino acid sequence (SEQ ID NO:254) derived from the coding sequence of SEQ ID N0:253 shown in Figure 89.

Figure 91 shows a nucleotide sequence (SEQ ID ND:255) of a native sequence PR0303 cDNA, wherein SEQ ID 10:255 is a clone designated herein as "UNQ266" and/or "I'NA42551-1217".

.Figure 92 shows the amino acid sequence (SEQ ID NO:256) derived from the coding sequence of SEQ ID NM:255 shown in Figure 91.

Figure 93 shows a nucleotide sequence (SEQ ID NO:257) of a native sequence PR0304 cIMA, wherein SEQ ID N0:257 is a clone designated 25 herein as "UNQ267" and/or '"IA39520-1217".

Figure 94 shows the amino acid sequence (SEQ ID NO:258) derived frno the coding sequence of SEQ ID N0:257 shown in Figure 93.

Figure 95 shows a nucleotide sequence (SEQ ID N3:259) of a native sequence PR0307 cENA, wherein SEQ ID ND:259 is a clone designated herein as "UNQ270" and/or -E1A41225-1217".

Figure 96 shows the amino acid sequence (SEQ ID NO:260) derived fram the coding sequence of SEQ ID N0:259 shown in Figure Figure 97 shows a nucleotide sequence (SEQ ID ND:261) of a native sequence PRO343 cIA, wherein SEQ ID N0:261 is a clone designated herein as "UNQ302" and/or "NA43318-1217".

Figure 98 shows the amino acid sequence (SEQ ID N0:262) derived fran the coding sequence of SE2 ID NO:262 shown in Figure 97.

Figure 99 shows a nucleotide sequence (SEQ ID NO:283) of a native sequence PRO328 cINA, wherein SEQ ID N0:283 is a clone designated 60 herein as "UNQ289" and/or "I40587-1231".

Figure 100 shows the amino acid sequence (SEQ ID ND:284) derived from the coding sequence of SEQ ID NO:283 shown in Figure 99.

Figures 101A-B show a nucleotide sequence (SEQ ID N3:288) of a native sequence PR0335 c1MA, wherein SEQ ID NO:288 is a clone designated herein as "Ut287 and/or "IE41388-1234".

Figure 102 shows the amino acid sequence (SEQ ID N3:289) derived fromn the coding sequence of SEQ ID N:288 shown in Figures 103A-B.

Figure 103 shows a nucleotide sequence (SEQ ID ND:290) of a native sequence PRO331 c1A, wherein SEQ ID ND:290 is a clone designated herein as 'UNQ292" and/or "IN40981-1234".

Figure 104 shows the amino acid sequence (SEQ ID NO:291) derived from the coding sequence of SEQ2 ID NO:290 shown in Figure 103.

Figure 105A-B show a nucleotide sequence (SEQ ID 11:293) of a 15 native sequence PR0326 cE1A, wherein SEQ ID ND:293 is a clone designated herein as "UNQ287 and/or IEN37140-1234.

Figure 106 shows the amino acid sequence (SEQ ID NO:294) derived froman the coding sequence of SEQ ID NO:293 shown in Figures 105A-B.

Figures 107A-B show a nucleotide sequence (SEQ ID N:309) of a 20 native sequence PR0332 cmlA, wherein SEQ ID N0:309 is a clone designated herein as "UNQ293 or "INA40982-1235".

Figure 108 shows the amino acid sequence (SEQ ID NO: 310) derived franom the coding sequence of SEQ ID NO: 309 shown in Figure 107.

Figure 109 shows a nucleotide sequence (SEQ ID NO:314) of a native sequence PR0334 cl, wherein SEQ ID ND:314 is a clone designated herein as "UNQ295" or "IA41379-1236.

Figure 110 shows the amino acid sequence (SEQ ID NO:315) derived from the coding sequence of SEQ ID N:314 showna in Figure 109.

Figure 111 shows a nucleotide sequence (SEQ ID NO:319) of a native sequence PRO346 cIr, wherein SEQ ID NO:318 is a clone designated herein as "UNQ305" or "I 44167-1243".

Figure 112 shows the amino acid sequence (SEQ ID 13:319) derived frcm the coding sequence of SEQ ID ND:318 shown in Figure 111.

Figure 113 shows a nucleotide sequence (SEQ ID N0:323) of a native sequence PRO268 cIENA, wherein SEQ ID ND:323 is a clone designated herein as "'UNQ235" or 'INA39427-1179".

Figure 114 shows the amino acid-sequence (SEQ ID NO::324) derived fran the coding sequence of SEQ ID NO:323 shown in Figure 113.

Figure 115 shows a nucleotide sequence (SEQ ID No:330) of a 61 native sequence PRO330 cIA, wherein SEQ ID N:330 is a clone designated herein as "U290 or "IE40603-1232'.

Figure 116 shows the amino acid sequence (SEQ ID 0: 331) derived fram the coding sequence of SEQ ID NO:330 shown in Figure 115.

Figure 117 shows a nucleotide sequence (SEQ2 ID NJ:337). of a native sequence PRO0339 cEM, wherein SEQ ID N3:337 is a clone designated herein as "UNQ229" or "EI43466-1225".

Figure 118 shows the amino acid sequence (SEQ ID NO:338) derived froman the coding sequence of SEQ ID 1NO: 337 shown in Figure 117.

Figure 119 shows a nucleotide sequence (SEQ ID N1:339) of a native sequence PRO310 cEMA, wherein SpD ID ND:339 is a clone designated herein as "UQ273 or 43046-1225.

Figure 120 shows the amino acid sequence (SEQ ID N: 340) derived fran the coding sequence of SEQ ID N):339 shown in Figure 119.

Figure 121 shows a nucleotide sequence (SBQ ID 1D:375) of a Snative sequence PR0244 cn1A, wherein SE2 ID NO:375 is a clone designated herein as "UNQ218" or "IIN35668-1171".

i Figure 122 shows the amino acid sequence (SEQ ID N0:376) derived fran the coding sequence of SEQ ID NO:375 shown in Figure 121.

MMM EESCRIPTIN OF THE PREEERE EMBODnMS I. Definitions The tenms "PRO polypeptide" and 'PRO" as used herein and when imnuediately :followed by a numerical designation refer to various polypeptides, wherein 25 the caiplete designation PRO/number) refers to specific polypeptide sequences as described herein. The terms "PRO/number polypeptide" and "PRO/nuber" as used herein encarpass native sequence polypeptides and polypeptide variants (which are further defined herein). e PRO polypeptides described herein nmay be isolated fran a variety of sources, such as fran hnuman tissue types or fran another source, or prepared by reconbinant or synthetic rethods.

A "native sequence PRO polypeptide ccnprises a polypeptide having the sane amino acid sequence as the corresponding PRO polypeptide derived froman nature. Such native sequence PRO polypeptides can be isolated froan nature or can be produced by recabinant or synthetic neans. The term "native sequence PRO polypeptide" specifically encarpasses naturallyoccurring truncated or secreted forms of the specific PRO polypeptide and extracellular domain sequence), naturally-occurring variant forms of alternatively spliced forms) and naturally-occurring 61a allelic variants of the polypeptide. In various ebodiments of the invention, the native sequence PRO211 is a mature or full-length native sequence PRO211 polypeptide ccprising amino acids 1 to 353 of Figure 2 (SEQ ID NO:2), the native sequence PRO217 is a mature or full-length native sequence PR0217 polypeptide ccnprising amino acids 1 to 379 of Figure 4 (SEQ ID the native sequence PR0230 is a mature or full-length native sequence PR0230 polypeptide cceprising amino acids 1 to 467 of Figure 6 (SEQ ID N:12), the native sequence PR0232 polypeptide is a mature or fulllength native sequence PR0232 polypeptide carprising amino acids 1 to 114 of Figure 9 (SEQ ID N3:18), the native sequence PR0187 is a mature or fulllength native sequence PR0187 ccrprising amino acids 1 to 205 of Figure 11 (SEQ ID NO:23), the native sequence PR0265 polypeptide is a mature or fulllength native sequence PRO265 polypeptide is an extracellular domain of the full-length PR0265 protein, wherein the putative transmebrane domain of the full-length PR0265 protein is encoded by nucleotides beginning at nucleotide 1969 of SEQ ID NO:31, the native sequence PR0219 polypeptide is a mature or full-length native sequence PR0219 polypeptide cacprising amino acids 1 to 915 of Figure 15 (SEB ID N:34), the native sequence PR0246 is a mature or full-length native sequence PR0246 polypeptide ccuprising amino acids 1 to 390 of Figure 17 (SEQ ID NO:39) or the native sequence PRO246 polypeptide is an extracellular damain of the full-length PR0246 protein, wherein the putative transmambrane docmain of the full-length PR0246 protein is encoded by nucleotides beginning at nucleotide 855 as shown in Figure 16, the native sequence PRO228 polypeptide is a mature or full-length 25 native sequence PR0228 polypeptide carprising amino acids 1 to 690 of Figure 19 (SQ ID NO: 49) or the native sequence PRO228 polypeptide is an extracellular domain of the full-length PR0228 protein, the native sequence PR0533 is a mature or full-length native sequence PRO533 ccrprising amino acids 1 to 216 of Figure 22 (SEQ ID NO:59), with or without the N-terminal signal sequence, and with or without the initiating rethionine at position 1, the native sequence PR0245 polypeptide is a mature or full-length native sequence PRO245 polypeptide *conliisig amino acids 1 to 312 of Figure 24 (SEQ ID 14:64). the native sequence of each PR0220. PRO221 and PRO227 polypeptides; is a mature or full-lengt native sequence PR0220. PR0221 and PRO22 polypeptide comisng amino acids 1 through 708 of Figure 26 (SEQ ID NO:69), 1 through 259 of Figure 28 (SEQ ID NO:7 1).

and I through 62D of Figure 30 (SEQ ID NO.73), the native sequence PR0258 polypeptide is a mature or full-length native sequence PR0258 polypeptide comprising amino acids I to 398 of Figure 32 (SEQ ID NO:84) or the native sequence PR0258 polypeptide is an extracelu domain of the full-lenth PRO258 protein, wherein the putative Unsmembnane domain of the full-length PR0258 protein is ecowded by nucleoddes beginning at nucleotide 1134 of SEQ ID NO:83, the native sequence PR0266 polypeptide is a mature or full-legth native sequence PR0266 polypeptide comprising amino acids I to 696 of Figure 34 (SEQ ID NO:91) or the native sequence PR0266 polypeptide is an cxnrcclhilar domain of the fufl-length PR0266 protein, wherein the putative transmembrane domi of the MIlength PR0266 protein is encoded by rmceotides beginning at about zmclotide 2009 of SEQ ID NO: 104.

the native sequence PR0269 polypeptide is a mature or full-length native sequence PR0269 polypetide comprising amin acids I o 490 of Figure 36 (SEQ MD NO:96) or die native sequence PR0269 polypeptide is an extracellular domain of the full-length PR0269 protein, wherein the putative transmembrane domain of the full-length PR0269 protein is encoded by nucleotides beginning at nucleotide 1502 as shown in Figure 35, the native sequence PRO2W 15 polypeptide is a mature or fil-engh native seqm=u PR0287 polypeptide comprising amino acids I to 415 of Figure 38 (SEQ ID NO: 104). the native sequcnce PR0214 is a mature or full-length native sequence PR0214 comprising .amino acids I to 420 of Fig. 40 (SEQ ID NO: 109), the native-sequence PR0317 is a MtI-length native-pre-sequence PR0317 comprising amino acids 1 to 366 of Fig. 42 (SEQ WD NO: 114) or a mature native-sequence PR0317 comprising amino acids 19 to 366 of Fig. 42 (SEQ ID 110:114). the native sequence PRO301 is a mature or full- 20 length native sequence PRO301 comprising amino acids 1 to 299 of Fig. 4 (SEQ ID NO: 119). with or without the N-terminal signal sequence, with or without the initiating methionine at position 1. with or without the potential transmembrane domain at position 236 to about 258. and with or without the intracellular domain at about position 259 to 299, the native sequence PR0224 polypeptide is a mamtur or full-length native sequence PR0224 polypeptide comprising amino acids 1 to 282 of Figure 46 (SEQ ID NO:127), the native sequence PR0222 polypeptide is a mature or full-length native sequence PR0222 polypeptide comprising amino acids I to 490 of Figure 48 (SEQ ID NO:132), the native sequaxe PR0234 is a mainr or full-length native sequence novel lectin comprising amino acids I to 382 of Fig. 50O EQ ID NO: 137). the native sequence PR023 1 polypepuide is a mature or funl-length native 'sequence PR0231 polyptde comprising amino acids I to 428 of Figure: 52 (SEQ ID NO:142), the native sequence PR0229 polypeptide is a mature or full-length native sequence PR0229 polypepide comprising amino acids 1. to 347 of Figure 54 (SEQ ID NO:148), the native sequence PR0239 polypeptide is a mature or full-length native sequence PR0238 polypepd& comprising amino acids I to 310 of Figure 56 (SEQ MD NO: 153), te native sequence PR0233 polypeptide is a mature or full-length native sequence PR0233 polypeptide comprising amin acids 1 to 300 of Figure 58 (SEQ ED NO: 159), the native sequence PR0223 polypeptide is a mature or MulIlengtht native sequence PR0223 polypeptide comprising amin acids I to 476 of Figure 60 (SEQ ID 140:164), the native sequence PR0235 polypeptde is a mature or full-length native sequence PRO235 polypeptide comprising amino acids 1 to 552 of Figure 62 (SEQ ED NO: 170), the native sequence PR0236 polypepidec is a mature or ful-length native sequence PR0236 polypeptide comaprising amino acids 1 to 636 of Figure 64 (SEQ ID 140:175), the native sequence PR0262 63 polypeptide is a nature or full-length native sequence PRO262 polypeptide carprising amino acids 1 to 654 of Figure 66 (SEQ ID ND:177), the native sequence PR0239 polypeptide is a mature or full-length native sequence PR0239 polypeptide crprising amino acids 1 to 501 of Figure 68 (SE ID ND:185), the native sequence PRO257 polypeptide is a mature or full-length native sequence PR0257 polypeptide carprising amino acids 1 to 607 of Figure 70 (SEQ ID 10:190) or the native sequence PRO257 polypeptide is an extracellular dacmain of the full-length PRO257 protein, wherein the putative transrbrane damain of the full-length PR0257 protein is encoded by nucleotides beginning at nucleotide 2668 as shown in Figure 69, the native sequence PR0260 polypeptide is a mrature or full-length native sequence PRO0260 polypeptide carprising amino acids 1 to 467 of Figure 72 (SEQ ID ND:195), the native sequence PRO263.polypeptide is a mature or full-length native sequence PRO263 polypeptide carprising amino acids 1 to 15 322 of Figure 74 (SEQ ID N10:201) or the native sequence PRO263 polypeptide is an extracellular daain of the full-length PRO263 protein, wherein the ~putative transrcbrane danin of the full-length PRO0263 protein is encoded by nucleotides beginning at nucleotide 868 of SEQ ID ND:200, the native sequence PR0270 polypeptide is a mature or full-length native sequence PRO270 polypeptide cacrprising amino acids 1 to 296 of Figure 76 (SEQ ID N J*:207), the native sequence PR0271 polypeptide ccarprising amino acids 1 to 296 of Figure 76 (SEQ ID ND:207), the native sequence PR0271 polypeptide is a mature or full-length native sequence PRO271 carprising amino acids 1 to 360 of Figure 78 (SEQ2 ID ND:213), the native sequence PR0272 polypeptide is a mature or full-length native sequence PR0272 polypeptide ccwprising amino acids 1 to 328 of Figure 80 (SEQ ID ND:221), the native sequence PRO294 polypeptide is a mature or full-length native sequence PR0294 polypeptide carprising amino acids 1 to 550 of Figure 82 (SEQ ID ND:227), the native sequence PR0295 polypeptide is a mature or full-length native sequence PRO29S polypeptide ccrprising amino acids 1 to 350 of Figure 84 (SEQ ID NO:236), the native sequence PRO293 polypeptide is a mature or full-length native sequence PRO293 polypeptide ccarprising amino acids 1 to 713 of Figure 86 (SEQ ID N:244) or the native sequence PRO293 polypeptide is an extracellular daain of the full-length PRO293 protein, wherein the putative transa.3rane damain of the full-length PRO293 protein is encoded by nucleotides beginning at nucleotide 2771 of SED ID ND:243, the native sequence PR0247 polypeptide is a mature or full-length native sequence PRO247 polypeptide ccuprising amino acids 1 to 546 of Figure 88 (SEQ ID ND:249), the native sequence PRO302 polypeptide is a mature or full-length 64 native sequence PRO302 polypeptide ccuprising amino acids 1 to 452 of Figure 90 (SDEQ ID NO:254), the native sequence PR0303 polypeptide is a nature or full-length native sequence PR0303 polypeptide cc"prising amino acids 1 to 314 of Figure 92 (SEQ ID NO:256), the native sequence PM304 polypeptide is a nature or full-length native sequence PRO304 polypeptide ccnprising amino acids 1 to 556 of Figure 94. (SEQ32 ID the native sequence PR0307 polypeptide is a nmature or full-length native sequence PR0307 polypeptide ccaprising amino acids 1 to 383 of Figure 96 (SEQ ID NO:260), the native sequence PRO343 polypeptide is a nature or full-length native sequence PRO343 polypeptide carprising amino acids 1 to 317 of Figure 98 (SEQ ID ND:262), the native sequence PRO328 polypeptide is a nature or full-length native sequence PRO328 polypeptide ccarprising amino acids 1 to 463 of Figure 100 (SEQ ID N3:284) or the native sequence PR0306 polypeptide is an extracellular danain of the full-length PRO306 protein.

wherein the putative extracellular dClanrain of the full-length PRO306 protein, the native sequence PR0335 polypeptide is a nature or full-length native sequence PRO335 polypeptide carprising amino acids 1 through 1059 of Figure 102 (SEQ ID ND:289), the native sequence PR0331 polypeptide is a nature or full-length native sequence PR0331 polypeptide carprising amino acids 1 through 640 of Figure 104 (SE2 ID NO:291), the native sequence PRO326 polypeptide is a mature or full-length native sequence PR0326 polypeptide carprising amino acids 1 through 1119 of Figure 106 (SEQ ID NO:293), wherein additional embodirrents include wherein the transnmbane regions are deleted or the peptides are truncated, so as to not include the 25 transmanbrane regions for each of PR0335, PRO331, and PR0326, the native sequence PRO332 is a nature or full-length native sequence PR0332 carprising amino acids 49 to 642 of Figure 180 (SED ID N):309), with or without the N-terminal signal sequence, and with or without the initiating nethionine at position 1, the native sequence PRi334 polypeptide is a nature or full-length native sequence PR0334 polypeptide ccrprising amino acids 1 to 509 of Figure 110 (SED ID NO:314), the native sequence PRO346 is a nature or full-length native sequence PRO346 airprising amino acids 19 to 339 of Figure 112 (SEB ID NO:319), with or without the N-terminal signal sequence, with or without the initiating methionine, with or without the transnanrane drain at positions 340 to 360 and with or wiEhout the intracellular dcmain at positions 361 to 450, the native sequence PR0268 polypeptide is a nature or full-length native sequence PRO268 polypeptide ccuprising amino acids 1 to 280 of Figure 114 (SED ID NO:324) or the native sequence PRO268 polypeptide is an extracellular domain of the full-length 64a PRO268 protein, wherein the putative transmetbrane dcmain of the fulllength PR0268 protein is encoded by nucleotides beginning at nucleotide 559 as shown in Figure 113, the native sequence PR0330 polypeptide is a mature or full-length native sequence PRO330 polypeptide corprising amino acids 1 to 533 of Figure 116 (SEQ ID ND:331), the native sequence PRO339 polypeptide is a mature or full-length native sequence PR0339 polypeptide coanprising amino acids 1 to 772 of Figure 118 (SEC ID N0:338), the native sequence PRO310 polypeptide is a mature or full-length native sequence PRO310 polypeptide conprising amino acids 1 to 318 of Figure 120 (SEQ ID ND:340) and the native sequence PRO244 is a mature or full-length native sequence PR0244 conprising amino acids 1 to 219 of Figure 122 (SEQ ID N3:376), wherein the mature, full-length native sequence PRO244 protein ccnprises a cytoplasmic domain (about amino acid positions 1 to 20), a transmembrane dnomain (about amino acid positions 21 to 46), and an extracellular domain (about amino acid positions 47 to 219). Within the extracellular damain, the C-lectin daoain is between about amino acid positions 55 and about amino acid position 206. Native sequence PRO244 as shown in Figure 122 maps to chromosame 12, bands pl2-pl3.

"PRO polypeptide variant" means an active PRO polypeptide as 20 defined above or below having at least about 80% amino acid sequence identity with the full-length native sequence PRO polypeptide sequence as eo. disclosed herein. Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence.

25 Ordinarily, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity, with the amino acid sequence of the full-length native amino acid sequence as disclosed herein.

30 "PRO317 variants" or "PR0317 sequence variants" as defined herein mean biologically active PRO317s as defined below having less than 100% sequence identity with the PRO317 isolated frcn reconbinant cell culture or frao manmalian fetal kidney tissue having the deduced sequence described in Figure 42. Ordinarily, a biologically active PRO317 variant will have an amino acid sequence having at least about 70% amino acid sequence identity with the PRD317 of Figure 42, preferably at least about more preferably at least about 80%, still more preferably at least about 85%, even more preferably at least about 90%, and most preferably at least about 95% 65

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5555 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, and 95-100% sequence identity, respectively). These variants include covalently modified polypeptides, as well as PRO317 fragments and glycosylation variants thereof. PRO317 fragments have a consecutive sequence of at least 10, 15, 20, 25, 30, or 40 amino acid residues, preferably about 10-150 residues, that is identical to the sequence of the PR0317 shown in Figure 42.

Other preferred PRO317 fragments include those produced as a result of chemical or enzymatic hydrolysis or digestion of the purified PRO317.

A "chimeric PRO317" is a polypeptide comprising fulllength PR0317 or one or more fragments thereof fused or bonded to a second protein or one or more fragments thereof. The chimera will typically share at least one biological property in common with PR0317. The second protein will typically by a cytokine, growth factor, or hormone such as a neurotrophic or angiogenic factor such as GDNF or VEGF, or another member of the TGF-superfamily such as EBAF-1. Another exemplary preferred PR0317 chimera is a "domain chimera' that consists of the Nterminal residues substituted with one or more, but not all, of the residues of the human EBAF-1. In this embodiment, the PR0317 chimera would have individual or blocks of residues from the human EBAF-1 sequence added or substituted into the PR0317 sequence. For example, one or more of those segments of EBAF-1 25 that are not homologous could be substituted into the corresponding segments of PRO317. It is contemplated that this "PR0317-EBAF-1 domain chimera" will have an agonist biological activity.

"Percent amino acid sequence identity" with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in the PRO sequence which are identical with the amino acid residues in a candidate polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software 65a such as BLAST, ALIGN or Megalign (DNASTAR) software. The preferred software alignment program is BLAST. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

"Percent nucleic acid sequence identity" with respect to PRO-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in the PRO sequence of interest which are identical with the nucleotides in a candidate nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be .o achieved in various ways that are within the skill in the art, 15 for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in S. the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

"Isolated", when used to describe the various polypeptides disclosed herein, means polypeptide 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 typically interfere with 25 diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the polypeptide will be purified to a degree sufficient to 0900 obtain at least 15 residues of N-terminal or internal amino acid sequnce by use of a spinning cup scqunaror. or to homogeneity by SDS-PAGE under nonreducing or reducing conditions using Coomassic blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, sim at least one component of the PRO polypcptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

An "isolated" PRO polypeptide nucleic acid is a nucleic acid molecule that is identified and separated from at least one conaminint uleic acid molecule with which it is ordinarily associated in the natural s0urce of the PRO polypeptide nucleic acid. An isolated PRO polypeptide nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated PRO polypeptide nucleic acid molecules therefore are distinguished from the specific PRO polypeptide nucleic acid molecule as it exists in natural cells. However, an isolated PRO polypeptide mcleic acid molecule includes PRO polypeptide nucleic acid molecules contained in cells that ordinarily express the PRO polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

'Southem analysis" or "Southern blotting" is a method by which the presence of DNA sequences in a restriction cndorducase digest of DNA or a DNA-conaining composition is confirmed by hybridization to a known, labeled oligonucleotide or DNA fragment. Southern analysis typically involves elcctrophoretic separation of DNA S 15 digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nitrocellulose, nyloo, or another suitable membrane support for analysis with a radiolabeled. biotinylated, or enzymelabeled probe as described in sections 937-9.52 of Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

"Northern analysis' or 'Northern blotting" is a method used to identify RNA sequences that hybridize to a known probe such as an oligonucleode, DNA fragment, cDNA or fragment thereof, or RNA fragment. The probe is labeled with a radioisotope such as P, or by bioinylation. or with an enzyme. The RNA to be analyzed is usually clectrophoretically separated on an agarose or polyacrylamide gel. transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe, using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook a al. supra.

*25 The term 'control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, 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, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, 'operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The term "antibody" is used in the broadest sense and specifically covers single ant-PRO polypeptide monoclonal antibodies (icluding agonist, antagonist and neutralizing antibodies) and and-PRO polypeptde antibody compositions with polyepitopic specificity. The term "monoclonal antibody' as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.

"Active" or activity" for the purposes herein refers t form(s) of PRO polypeptide which retain the biologic and/or immunologic activities of the specific native or naturally-occurring PRO polypcptidc. The activity of a PRO332 polypeptide preferably involves the regulation of exracellular matrix, cartilage, or bone function.

"PRO317-associated disorder" refers to a pathological condition or disease wherein PRO317 is over- or underexpressed. Such disorders include diseases of the female genital tract or of the endomerium of a mammal, including hyperplasia, endomctriis. endometriosis, wherein the patient is at risk for infertility due to endomctrial factor, endomctrioma, and cndomeaial cancer, especially those diseases involving abnormal bleeding such as a gynecological disease. They also include diseases involving angiogenesis. wherein the anglogenesis results in a pathological condition, such as cancer involving solid tumors (the therapy for the disorder would result in decreased vascularization and a decline in growth and metastasis of a variety of tumors). Alternatively, the angiogenesis may 15 be beneficial, such as for ischemia, especially coronary ischemia. Hence, these disorders include those found in patients whose hearts are functioning but who have a blocked blood supply due to atherosclerotic coronary artery disease, and those with a functioning but underperfused heat, including patients with coronary arterial disease who S*:i are not optimal candidates for angioplasty and coronary artnry by-pass surgery. The disorders also include diseases involving the kidney or originating from the kidney tissue, such as polycystic kidney disease and chronic and acute renal failure.

"Treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures.

Those in need of treatment include those already with the disorder as well as those prone to have the disorder of those in which the disorder is to be prevented.

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans.

"25 domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, and the like.

**Preferably, the mammal herein is a human.

'Carriers" as used herein include pharmaccutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the *physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypcptidc; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycioc, glutamine.

asparagine, arginine or lysine: monosaccharides, disaccharides, and other carbohydrates including glucose, mannosc, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannltol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN, polyethylene glycol (PEG), and PLURONICS''.

The term "agonist" is used to refer to peptide and non-peptide analogs of the native PRO polypeptidcs (where native PRO polypeptide refers to pro-PRO polypeptide, pre-PRO polypeptide, prepro-PRO polypeptide, or mature PRO polypeptide) of the present invention and to antibodies specifically binding such native PRO polypeptides. provided that they retain at least one biological activity of a native PRO polypeptide. Preferably, dhe agonists of the present invention retain the qualitative binding recognition properties and receptor activation properties of the native PRO potypeptide.

Ilhc term *antagonist' is used to refer to a molecule inhibiting a biological activity of a nativec PRO polypeptide of the present invention wherein native PRO polypeptide refers to pro-PRO polypepide. pre-PRO polypeptide. prepro-PRO polypeptide, or mature PRO polypeptide. Preferably, the antagonists herein inhibit the binding of a native PRO polypeptide of the present invention. Preferred antagonists essentially completely block the binding of a native PR0317 polypptde to a PR0317 polypepdec receptor to which It otherwise binds. Such receptors may include the Type I and Type U. and possibly Type IM recetors identified for the TOP- superfamily.

KolodzieJezyk and Hall, stupra. A PRO polypeptide *antagonist* is a molecule which prevents, or interferes with, a PRO antagonist effector function a molecule which prevents or interferes with binding and/or activation of a PRO polypeptide receptor by PRO polypetde). Such molecules can be screened for their ability to competitively inhibit PRO polypeptide receptor activation by monitoring binding of native PRO polypeptide in the prtsecc and absence of the test antagonist molecule. for example. Examples of PR0317 polypptide antagonists include neutraizing antibodies against F-2. An antagonist of the invention also encompasses an antiense polynucleotide against the PRO polyetide gene. which aniisense polynuecotide blocks transcription or translation of the PRO polypetdec gene, thereby inhibiting its expression and biological activity.

*'Stringent conditions" means employing low ionic strength and high temperature for washing, for example. 0.015 sodium chlorideI0.0015 M sodium citrateff.l sodium dodecyl sulfate atSO0C, or(2) employing during hybridization a dentiauring agentI, such as formamide, for example. 50% (vol/vol) formamide with 0. 1% bovine :serum albwnin/0. I FicollIO.1 polyvinylpyrrolidone/5O nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42*C. Another example is use of 50% formamide, 5 x SSC (0.75 M NaCI. 0.075 M sodium citrate). 50 mM sodium phosphate (pH 0.1 sodium pyrophosphate, 5 x Denhardt's 25 solution, sonicated salmon sperm DNA (S0 pg/mIl). 0. 1% SDS, and 10% dextran sulfate at 420C. with washes at 2 42*Cin 0.2 xSSC and0.1% SDS. Yet another example is hybridization using a buffer of 10% dextran sulfate, 2 xSSC (sodium diloride/sodium ultrare) and 50% formantide at 55 followed by a high-stringency wash consisting of 0. 1 x SSC containing EDTA at 556C.

"Moderately stringent couitions* ame described in Sambrook et at.. supra, and include the use of a washing solution and hybridization conditions temperature, ionic strength, and %SDS) less stringent than described above. An example of moderately stringent conditions is a condition such as overnight incubation at 37'C in a solution comprising: 20% fornamide, S x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 5 x Denhardt's solution, 10% dextran sulfate, and 20 tnimL denatured sheared salmon sperm DNA, followed by washing the filters in I x SSC at about 37-50T. The slled artisan will recognize how to ajust the temperature, ionic strength. eic., as necessary to accommodate factors such as probe length and the like.

11. Compositions and Methods of die lnvewition- 1. Full-length PRO211 and PR0217 PolypentIdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to n th present application as PRO211 and PR0217. In particular, Applicants have identified and isolated cDNA encoding PRO211 and PR0217 polypeptides. as disclosed in further detail in the Examples below. Using BLAST (PastA format) sequence alignment computer programs. Applicanits found that cDNA sequences encoding full-length native sequence PRO211 and PR0217 have homologies to known proteins having EGF-like domains.

Specifically, the cDNA sequaee DNA32292-1 131 (Figure 1, ,SEQ ID NO:1) has 36% identify and a Blast score of 209 with PAC6 RAT and 31 identify and a Blast score of 206 with Fibulin-1, isoform c precursor. The cDNA sequence DNA33094-1131 (Figure 3. SEQ ID NO:3) has 36% identity and a Blast score of 336 with eastern newt senascin. anid 37% Identify and a Blast score of 331 with human tenascia-X precursor. Accordingly, it is presently believed that PRO211I and PR0217 polypeptides disclosed In the present application are newly identified members of the EGF-like family and possesses properties typical of the EGF-likc protein family.

Fufl4ength PRO230 Povuentides The present invention provides newly identified and isolated nuclcotide sequences encoding polypeptides referred to in the present application as PROM3. In particular, Applicants have identified and isolated eDNA encoding a PR0230 polypeptide, as disclosed in further detail in the Examples below. Using known programs such as BLAST uand FastA sequence alignment computer program Applicants found that a eDNA sequence encoding fulllength native sequence PR0230 has 48% amino acid identity with the rabbit tubulointerstitial nephritis antigen precursor. Accordingly. it is presently believed that PR0230 polypeptide disclosed in the present application is a newly identified member of the tubulointerstitial nephritis antigen family and possesses the ability to be recognized by human autoantibodies in certain forms of tuhulointerstitial nephritis.

Full-length PRO232 Polypetides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROM3. In particular. Applicants have identified and'isolated eDNA encoding a PR0232 polypeptide, as disclosed in futher detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that a portion of the ful-lngth native sequence PR0232 (shown in Figure 9 and SEQ ID NO:18) has 35% sequence identity with a stem cell surface antigen from Gallus gallus. Accordingly, it is presemly believed that the PR0232 polypeptide disclosed in the present application may be a newly identified stem cell antigen.

4. Full-length PRO187 Poyneptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0187. In particular, Applicants have identified and isolated cDNA encoding a PR0187 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequenice aligncrnt computer program, Applicants fWWm that a full-lagth native sequence PRO 187 (shown in Figure IS) has 74% amino acid sequence identity and BLAST score of 310 with various androgen-induced growth factors and PFF4. Accordingly, it is presently believed that PRO 187 polyetide disclosed in the present application is a newly identified mnember of the FGF-8 protein family and may possess identify activity or property typicai of the FGF-8-like protein family.

Full-lenrth PRO265 PolynetIdes The present invention provides newly Identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROM6. In particular, Applicants have identified and isolated cDNA encoding a PR0265 polypeptide, as disclosed in further detal i n the Examples below. Using programs such as BLAST and FastA sequence alignment computer programs, Applicants (ound that various portions of the PR0265 polypeptide have significant homology with the fibrozmdulin protein and fibromodulin precursor protein. Applicants have also found that the DNA encoding dhe PR0265 polypeptde has significant homology with platelet glycoprotein V. a 1 *er of the leucine ridh related protein family involved in sin and wound repair. Accordingly, it is presently believed that PR0265 polypeptide disclosed in the present application is a newly identified member of the leucine ::rich repeat family and possesses protein protein binding capabilities, as well as be involved in skin and wound repair as typical of this family.

6. Full-ensth PR0219 Potyptides T'Me present invention provides newly identified and isolated icleotide sequences encoding polypeptides referred to in the present application as PROM1. In particular, Applicants have identified and isolated cDNA encoding a PR0219 polypeptide, as disclosed in futrther detail in the Examples below. Using BLAST and FastA **:sequence alignment computer programs, Applicants found that various portions or the PR0219 polypeptide have significant homology with the mouse and human niatrilin-2 precursor potypeptides. Accordingly, it is presently believed that PR0219 polypeptide disclosed in the present application is related to the matrilin-2 precursor :polypeptide.

7. Full-length PROW4 Povpptdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0246. In particular. Applicants have identified and isolated cDNA encoding a PR0246 polypeptide, as disclosed in furthe detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that a portion of the PR0246 polypeptide has significant homology with the human cell surface protein HCAR. Accordigly, it is presently believed that PR0246 polypeptide disclosed in the present application may be a newly identified membrane-bound virus receptor or tumnor cell-specific antigen.

8. Full-length PRO228 PoDentides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0228. In particular, Applicants have identified and isolated cDNA encoding a PR0229 polypeptidc. as disclosed in further detail in die Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that variott portions of the PR0228 polypeptidc have significant homology with dhe EMRI protein. Applicants have also found that the DNA encoding the PR0228 polypeptide has significant homology with latrophilin, macrophage-restricted cell surface glycoprotein, B0457.1 and leucocyte antigen CD97 precursor. Accordingly, it is presently believed that PR0228 polypeptide disclosed in the present application is a newly identified member of the seven ftnsnembrane superfaniilyt'and possesses characteristics and functional propertics typical of this faily. In particular, It is believed that PR0228 is a new nmemiber of the subgroup within this family to which CD97 and EMRI belong.

9. Fulength PROS33 Polvoeptides ihe present invention provides newly identified and isolated nucleodde sequences encoding polypeptides referred to in the present application as PR0533. In particular, Applicants have identified and isolated cDNA encoding a PR0533 polypepide, as disclosed in further detail in the Examples below. Using BLAST-2 and PastA samuce alignmnt comur= Programs, Applicamt found that a full-length native sequence PRO533 (shown in Figure 22 and SEQ ID N0-59) has a Blast score of 509 and 53 amino acid sequence identity with fibroblast-growth factor Accordingly, It is presently believed that PR0533 disclosed in die present application is a newly identified member of the fibroblast growth factor family and may possess activity typical of such polypeptides.

10. Full-leng~th PRO24S Poy~eotldes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROM4. In particular. Applicants have identified and isolated cDNA encoding a PR0245 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that a portion of the amino acid sequence of the PR0245 polypeptide has 60% amino acid idcntity with the human c-snyb protein. Accordingly, it is presently believed that the PR0245 polypeptide disclosed in the preset application may be a newly identified member of the transmembrn protein tyrosine kinase family.

11. Fultlength PR0220. PRO221 and PROM2 Polynlentides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0220. PR0221 and PROW2. In particular, Applicants have identified and isolated cDNAs encoding a PR0220, PR0221 and PRO227 polypeptide. respectively, as disclosed in further detail in the Exampiles below. Using BLAST and FastA sequence aligniment computer programs, PR0220 has amn acid identity with the amino acid sequecec of a leticine rich pmrotin wherein the identity is 87 PR0220 additionally has amino acid identity with die ncutronal leucine rich protein wherein the identity is 55 The neuronal leucine rich protein is further described in Taguchi, etaat., Mol. Brin es., 35:31-40 (1996).

PR0221 has amino acid identity with the SLIT protein precursor, wherein different portions of these two proteins have the respective percent identities of 39%. 38%, 34%, 31 and 30 PR0227 has amino acid identity with the amino acid sequence of platelet glycoprotein V precursor. The same Meults were obtained for human glycoprotein V. Differen portions of amee two proteins show the following Percent identities of 30%, 28%. 28%. 31%. 35%. 39% and 27%.

Accordingly, it is presently believed that PR0220, PR0221 and PR0227 polypeptides disclosed in the present application are newly identified members of the leucine rich repeat protein superfamily a2d that each possesses protein-protein binding capabilities typical of dhe lceine rich repeat protein superfamnily. It is also believed that they have capabilities similar to those of SLIT, the leucine rich repeat protein and human glycoprotein V.

12. Full-ength PRO25S Polvueptides The Present invention provides newly identified and isolated nucleotidc sequences encoding polypeptides referred to in the present application as PR0258. In particular, Applicants have identified and isolated cDNA encoding a PR0258 polypeptide. as disclosed in further detail in the Examples below. Using BEAST and FastA sequence alignment computer program, Applicants found that various portions of the PR0258 polypeptde have significan homzology with the CRTAM and poliovinas; receptors. Accordingly, it is presently believed that PR0258 polypetide disclosed in the present application is a newly identified memtber of the Ig superfamily and possesses virus receptor capabilities or regulates imune function as typical of this family.

13. Full-leng~th PR0266 olvuentides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0266. In particular, Applicants have identified and isolated cDNA.

encoding a PR0266 polypeptide, as disclosed in fArter detail in t Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0266 polypeptide have significant homology with the SLIT protein from Drosophilia. Accordingly, it is presently believed that PR0266 polypeptide disclosed in the present application is a newly identified member of the leucine rich repeat family and possesses ligand-ligand binding activity and ricuronal development typical of thds family. SLIT has been shown to be useful in the study and treatment of Alzheinmer's disease, supra, and thus, PR0266 may have involvement in the study and cure of this disease.

14. Full-length PR0269 olyveptidesi The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0269. In particular, Applicants have identified and isolated cDNA encoding a PR0269 polypeptide, as disclosed in futher detail in the Examples below. Using BLAST. FastA and sequence alignment computer program, Applicants found that the amino acid sequence encoded by nucleotides 314 to 1783 of the full-length native sequene PR0269 (shown in Figure: 35 and SEQ ID NO:95) has significant homology to human urinary thrombomodulin and various thrombomnodulin analogues respectively, to which it was aligned.

Accordingly. it is presently believed that PR0269 polypeptik disclosed in the present application is a newly Identified member of the thrombomodulin family.

IS. ulleneth PR0297 Polvueptides The present invention provides newly idcntifid and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROW9. in particular, Applicants have identified and isolated cDNA encoding a PRO287 polylpeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that various portions of the PROW 7polypeptide have signficant homology with the tye I procoflagcn C-protentiase enhar protein precursor and type 1 procolbagen Cproteinase enhancer protein. Accordingly. it is presently believed tha PRO28 polypeptide disclosed in the present application is a newly identified member of the C-proteinase enhancer protein family.

16. Pull-length PRO214 Povne~tldes The present invention provides newly identified anid isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO2 14. In particular, Applicants have identified and isolated cDNA encoding a PR0214 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that a full-Lngth native sequence PR0214 polypeptide (shown in Figurt 40 and SEQ ID NO: 109) has 49% amino acid sequence identity with HT protein, a known member of the EG-farnily. The comparison resulted in a BLAST score of 920, with 150 matching nucleotides. Accordingly, it is presently believed that the PR0214 polypeptide disclosed in the present application is a newly identified member of the Lamily comprising EOF domains and may possess activities or properties typical of the BGF-domain containing family.

17. Full-length PR0317 Polvoepides; :The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROW1. hIparticular, cDNA encoding a PR0317 polypeptide has been identified and isolated, as disclosed in further detail in the Exanples below. Using BLASI' and FastA" sequence 25 alignmnt computer programs, it was found that a full-lengt native-sequence PR0317 (shown in Figure 42 and SEQ ID NO:114) has 92% amino acid sequence identity with EBAF-l. Further, it is closely aligned with many other members of the TGF- superfamily.

Accordingly, it is presently believed that PR0317 disclosed in the present application is a newly identified member of t TGP- superfamily and my possess properties that are therapeutically useful in conditions of uterine bleeding, etc. Hence, PR0317 may be useful in diagnosing or treating abnormal bleeding involved in gynecological diseases, for example, to avoid or lessen t need for a hysterectomy. PR0317 may also be useful as an agent that affects angiogenesis in general, so PR0317 may be useful in anti-tumor indications, or conversely, in treating coronary ischemic conditions.

Library sources reveal that ESTs used to obtain the consensus DNA for generating PR0317 primers and probes were found in normal tissues (uterus, prostate, colon, and pancreas), in several tumors (colon, brain (twice), 35 pancreas, anid mullerian cell), and in a heart with ischemia. PR0317 has shown up in several tissues as well, but it does look to have a greater concentration in uterus. Hence, PR0317 may have a broader use by the body than EBAF-l It is contemplated that, at least for some indications, PR0317 may have opposite effects from EBAF-1.- 1s. Full-length PRO301 Pol~'pentdg.

The present invention provides newly identified and isolated nucleotide sequenme encoding polypeptides referred to in the present application as PRO3OI. In particular. Applicants have identified and isolated cDNA encoding a.PR0301 polypeptde. as disclosed in further detail in die Examples below. Using BLAST and FascA sequexx aligvznent computer programus. Applicants fond that a full-length native sequence PRO301 (shown in Figure 44 and SEQ W NOAl19) has aBlast scom of 246 corresponin~gto 30% amino acid sequence identitywit uman A33 antigen precursor. Accordingly. it is-presently believed that PRO301 disclosed in the present application is a newly identified meber of the A33 antigen protein family and may be expressed in human neoplastic diseases such as colorctral cancer.

19. Full-lenoth PR10224 Polvpentides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides refermd to in the present application as PR0224. In particular, Applicants have identified and isolated cDNA encoding a PRO224 polypeptide, as disclosed in further detail in the Examples below. Using known programs such as BLAST and FastA sequence alignment computer programs, Applicants found that full-length native PR0224 (Fgur 46, SEQ ID NO: 127) has amino acid identity with apolipoprotein E receptor 2906 from homo sapiens. The alignments ofdifferent portions ofthese two polypeptides show amino acid identities of 37%. 36%, 30%, 44%, 44 and 28% respectively. Full-length native PR0224 (Figure 46, SEQ ID NO:127) also has amino acid identity with very low-density lipoprotein receptor precursor from gall. The alignments of different portions of these two polypeptides show amino acid identities of 38%, 37%, 42%, 33%, and 37% respectively. Additionally, full-length native PR0224 (Figure 46, SEQ ID NO: 127) has amino acid identity with the chicken oocyte receptor P95 from Gallus gallus. The alignments of different portions of these two polypeptdes show amino acid identities of 38 37%, 42%, 33%, and 37% respectively. Moreover, fall-length native PR0224 (Figure 46, SEQ ID) NO:127) has amino acid identity with vexy low density lipoprotein receptor short form precursor from humans. The alignments of different portions of these two polypeptides show aino acid identities of 32%, 38 34%, 45 and 31 respectively. Accordingly, it is presently believed that PR0224 polypeptide disclosed in the present application is *a newly identified member of the low density lipoprotein receptor family and possesses the structural characteristics required to have: the finctional ability to recognize and endocytose low density lipoproteins typical of the low density lipoprotn recoeptor family. (The alignments described above used the following scoring parameters: T=7, S S2= 36, Matrix: BLOSUM62.) Full-length PR10222 Poluentides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0222. In particular, Applicants have identified and isolated cDNA encoding a PR0222 polypeptide, as disclosed in farther detail in the Examples below. Using BLAST and PastA sequence alignment computer programs, Applicants found that a sequence encoding full-length native sequence PRO2 (shown in Figure 48 and SEQ ID NO: 132) has 25-26 amino acid identity with mouse complement factor h precursor, has 27-29% ano acid identity with complemnt receptor, has 25-47% amino acid identity with mouse coniplernnt COb receptor type 2 long form precursor, has 40% amino acid identity with human hypothcal protein kiaae247. Accordingly, it is presently believed that PR0222 polypeptide disclosed in the present application is a newly idetitfied memnber of the ~cmnt receptor family and possesses activity typical of the complement receptor family.

21. Fulleknth PRO234 Folvuetides The present invention provides newly identified and isolated nacleotide sequences encoding polypeptides referred to in the present application as PR0234. I particular, Applicants have identified and isolated cDNA encoding a PR0234 polypeptidc. as disclosed in fimlher detail in the ExaMples below. Using BLAST (FastA-format) sequence aligrnent computer programs. Applicants found that a cDNA sequence encoding full-length native sequence PR0234 has 31 identity and Blas score of 134 with E-Weectin precursor. Accordingly, it is presently believed that the PR0234 polypeptides disclosed in the present application are newly identified members of the lectin/selectin brmily and possess activity typical of die lectin/selectin family.

22. Fullenipth PR0231 Polvtdes *.15 Thec present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0231. I particular, Applicants have identified and isolated cDNA encoding. a PR0231 polypeptide, as disclosed in further detail in fte Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that the full-length native sequence PRCY231 polypeptide (shown inFigure52 and SEQID NO: 142) has30 and 31 amioacid identiy with human and rat prostatic acid phosphatase precursor proteins, respectively. Accordingly, it is presently believed that the PR0231 polypeptide :disclosed in dhe present application may be a newly identified member of the acid phosphatase protein family.

23. Full-Ie'nith PRO229 Polvnenlides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0229. I particular, Applicants have identified and isolated cDNA encoding a PR0229 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and PastA -sequence alignment computer programs, Applicants found that various portions of the PR0229 polypeptide have ~sigificant homology with antigen wcl M130 ansigeri, T cell surface glycoproccin CD6 and CD6. It also is related to Sp-alph. Accordingly, it is presently believed that PR0229 polypeptidc disclosed in the present application is a newly identified meber of the family containing scavenger receptor homology, a sequence motif fond in a number of proteins involved in immune function and thus possesses hmmune function and /or segments which resist degradation, typical of this funnily.

24. Full-lenpth PR=28 Polynegtides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0238. I particular, Applicants have identified and isolated cDNA encoding a PR0238 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence aligom nncomnputer program. Applicants found that various portions of the PR0238 polypeptide have significant homology with reductases. including oxidoreductase and ftty acyl-CoA reductase. Accordingly, it is presently believed that PRO238 polypeptide disclosed in the present application is a newly identified member of the reductase family and possesses reducing activity typical of the reductase family.

25. Full-lengh PRO233 Pollneutdes Thc present invention provides newly identified and isolated nucleotide sequences encooding polypeptides referred to in the present application as PR0233. In particular, Applicants have identified and isolated cDNA encoding a PR0233 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequenwealignment computer program, Applicants found that various portions of the PR0233 polypeptide have significant homology with the reductase protein. Applicants have also found that the DNA encoding the PR0233 polypeptide has significant homology with proteins from Caenorhabditis elegans. Accordingly, it is presently believed that PR0233 polypeptde disclosed in the present application is a newly identified member of the reductase family and possesses the ability to effect the redox state of the cell typical of the reductase fanily.

26. Full-leng~th PR0223 Polvneotldes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0223. In particular, Applicants have identified and isolated cDNA encoding'a PR0223 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequenxealignnient computer programs, Applicants found that the PR0223 polypeptide has significant homology with various serine carboxypeptidase polypeptides. Accordingly, it is presently believed that PR0223 polypeptide disclosed in the present application is a newly identified serine carboxypeptidase.

27. Full-length PR0235Polvneptifts The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0235. In particular, Applicants have identified and isolated cDNA encoding a PR023S polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that variouts portions of the PRO235 polypeptide have significant homology with the various plexin proteins. Accordingly, it is presently believed that PR0235 polypeptde <a disclosed in the present application is a newly identified member of the plexin family and possesses cell adhesion properties typical of the plexin family.

28. Full-lenglh PR236 snd PRO26 Polvuntide The present invention provides newly identified and isolated nucleotide sequences encoding polyetides referred to in the present application as PR0236 and PR0262. In particular. Applicants have identified and isolated cDNA encoding PR0236 and PR0262 polypeptides, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that various portions of the PR0236 and PR0262 polypeptides have significant homology with vaious P-galactosidase and P-galactosidase precursor Polypcpddes. Accordingly. it bs prewend believed that dhe PR0236 and PR0262 polypeptides disclosed in the present application ame newly identified P-galactosidase homologs.

29. QuIlnt R239 Polynentides The present invention provides newly identified and isolated nucleotide sequences encloding polypeptides referred to in the present application as PR0239. In particular. Applicants have identified an&risolated cDNA' encoding a PR0239 polypeptdec, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0239 polypeptide have significant homology with denim proteins. Accordingly. it is ptesendly believed that PR0239 polypeptide disclosed inthe presetappliatonisawyidntied member of tedauin fy d possesses cll adhesion and the ability to effect synaptic processes as bs typical ofthde densin family.

Full-lenrth PR0257 Polveeptldes V The present invention provides newly identified and isolated nucleotdec sequences encoding polypeptides referred to in dhe present application as PROM5. In particular, Applicants have identified and isolated cDNA encoding a PRO2S7 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer program, Applicants found that various portions of the PR0257 polypeptide have significant homology with the ebricrin precursor and ebinerin protein. Accordingly, it is presently believed that PR0257 polypepidde disclosed in tde present application is a newly identified protein member which is related to the ebnerin protein.

Full-length PR260 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0260. In particular, Applicants have identified and isolated cDNA encoding a PR0260 polypeptide, as disclosed in further detail in the Examples below. Using programs such as BLAST and FastA sequence alignment computer programs. Applicants found that various portions of the PR0260 polypeptide have significant homology with the alpha-l-fucosidase precursor. Accordingly, it is presently believed that PR0260 polypeptide disclosed in the present application is a newly identified mnember of the facosidase family and possesses enzymatic activity related to fuIcose residues typical of the fiucosidase family.

32. Full-length PR-263 Polyeiddes The present invention provides newly identified and isolated nuceotide sequences encoding polypeptides referred to in the present application as PROW6. In particular, Applicants have identified and isolated cDNA encoding a PR0263 polyeptide, as disclosed in further detail in the'Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0263 Rolypeptide have significantl homology with the CD44 antigen and related proteins. Accordingly, it is presently believed that PR0263 polypeptide disclosed in the present application is a newly identified member of the CD44 antigen family and possesses at leas one of the properties associated with these antigen&, ie., cancer and HIV marker, cell-cell or cellmantrix interactions, regulating cell traffic, lymph node homing. transmission of growth signals. and presentation of chemoines and growth facors to traveling cells.

33. Fullength ER0270 Polyglgtides The present invention provides newly identified and isolated nucleotide sequences encoding polypcptidC3 referred to in the present application as PROM7. In particular. Applicants have identified and isolated cDNA encoding a PR0270 polypeptide. as disclosed in further detail in the Examples below. Using BLAST. FattA and sequence alignment comititer programs. Applicants found that that various portions of the PR0270 polypeptide have significant homology with various thioredoxin proteins. Accordingly. it is presently believed that PR0270 polypeptide disclosed in the present application is a newly identified member of the thioredoxin family and possesses the ability to effect reduction-oxidation (redox) state typical of the thioredoxin family.

34. Full-Icneth PR271 Polypentides The present invention provides newly identified and isolated nucleoidec sequences encoding polypeptides ***refcrred to in the present application as PR0271. In particular. Applicants have identified and isolated cDNA encoding a PR0271 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicanits found tht the PR0271 polypeptide has significant homology with various link proteins and precursors thereof. Accordingly, it is presently believed that PR0271 polypeptide disclosed in the present application is a newly identified link protein hosnolog.

35. Full-length PR272 Povoensides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides refred to in the present application as PROM7. In particular, Applicants have identified and isolated cDNA encoding a PR0272 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment comnputer programs, Applicants found that various portions of the PR0272 polypeptide have significant homology with the human reticulocalbin protein arnd its precursors. Applicants have also found that the DNA encoding die PR0272 polypepside has significant homology with the mouse reticulocaibin precursor protein.

Accordingly, it is presently believed that M0O272 polypeptde disclosed in the present application is a newly identified member of dhe resiculocalbin family and possesses the ability to bind calcium typical of the reticulocalbin family.

36. Full-length PR294 Poly=otdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROM9. in particular, Applicants have identified and isolated cDNA encoding a PR0294 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found thac various portions of the PR0294 polypeptide have significant homology with the various portions of a nmnte of collagen proteins. Accordingly. it is presently believed that PR0294 polypeptide disclosed in the present application is a newly identified member of the collagen family.

37. Full-length PRO29S Polypeptdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0295. In particular. Applicants have identified aid isolated cDNA encoding a PRO295 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA scquence alignment computer programs. Applicants found that various portions of the PR0295 polypeptide have significant homology with integrin picins. Accordingly. it is presently believed that PR0295 polypptde disclosed in the present application is a newly identified member of the integrin family and possesses cell adhesion typical of the integrin family.

38. Full-length PRO293 Poftspfnides The present invention provides newly identified and isolated nucleodde sequences ceoding polypeptides referred to in the present application as PR0293. In particular, Applicants have identified and isolated cDNA encoding a PR0293 polypeptide, as disclosed in Further detail in ft Examples below. Using BLAST and FastA *.**seupiee alignent computer program., Applicants found that portions of the PR0293 polypeptide have significant to.. homology with the neuroml leucine rich repeat proteins 1 and 2. (NLRR-l and NLRR-2), particularly NLRR-2.

Accordingly. it is preseutly believed that PR0293 polypeptide disclosed in the present application is a newly identified menmber of the neuronal leuicine rich repeat protein family and possesses ligand-ligand binding activity typical of the NRLL prti family.

39. Full-leneth PR247 Polvnentdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROW4. In particular, Applicants have identified and isolated eDNA encoding a PR0247 polypeptide, as disclosed in further detail in the Ex amples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0247 polypeptide have :significant homology with densin. Applicants have also found that the DNA enicoding the PR0247 polypeptide has significant homology with a number of other proteins, including KAO231. Accordingly, it is presently believed tho PR0247 polypeptide disclosed in the present application is a newly identified member of the leucine rich repeat family and possesses ligand binding abilities typical of this family.

040. Full-length PR0302. PROM,3 PR0304. PRO307 and PRO343 PolvoentIdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0302, PR0303. P10304. PR0307 and PR034. In particular, Applicants have identified and isolated cDNA encoding PR0302, PR0303, PR0304, PR0307 and PR0343 polypeptides, as disclosed in furftTe detail in the Examles below. Using BLAST and FastA sequence alignment computer programs, Applicants fount that various portions of the PR0302. PR0303. PR0304, PR0307 and PR0343-polypeptides have significant homology with various protease: proteins. Accordingly, it is presently believed that the PR0302, PR0303, PR0304. PR0307 and PR0343 polypeptides disclosed in the present application are ncwly identified protease proteins.

80 41. Fu11-Length PR0328 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0328. In particular, Applicants have identified and isolated cENA encoding a PR0328 polypeptide, as disclosed in further detail in the Exanples below. Using BLAST and FastA sequence alignment cacputer programs, Applicants found that various portions of the PR0328 polypeptide have significant horology with the human glioblastcra protein ("GLIP").

Further, Applicants found that various portions of the PR0328 polypeptide have significant hcnology with the cysteine rich secretory protein ("CRISP") as identified by BLAST hmrology E(CCRISP3_I, S68683, and CRS3I_HMAN]. Accordingly, it is presently believed that PR0328 polypeptide disclosed in the present applicaticn is a newly identified member of the GLIP or CRISP families and possesses transcriptional regulatory activity 15 typical of the GLIP or CRISP families.

6SO@ 42. Full-Length PR0335, PR0331 and PR0326 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO335, PR0331 or PR0326. In particular, Applicants have identified and isolated cENA encoding a PRO335, PR0331 or PRO326 o polypeptide, as disclosed in further detail in the Examples below. Using e*e*c BLAST and FastA sequence alignment ccmputer programs, Applicants found that various portions of the PR0335, PR0331 or PR0326 polypeptide have 25 significant hrrology with LIG-1, ALS and in the case of PR0331, Se additionally, decorin. Accordingly, it is presently believed that the PRO335, PR0331 and PRO326 polypeptides disclosed in the present application are newly identified mnabers of the leucine rich repeat superfamily, and particularly, are related to LIG-1 and possess the biological functions of this family as discussed and referenced herein.

43. Full-Length PR0332 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO332. In particular, Applicants have identified and isolated cEDNA encoding PR0332 polypeptides, as disclosed in further detail in the Exanples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that a full-length native sequence PR0332 (shown in Figure 108 and SED ID N0:309) has about 30-40% amino acid sequence Boa identity with a series of )mow proteoglycari seuences, including, for exanple, f ibramoduiin and f ibrcmodulin. precursor seuences of various species (RO BOVIN, EMELDSHICK, FVDEjRAT, FMELMMXSE, FMEDJ1UW, PJU36773), osteczrodulin seuences (ABOO114_., AB007848_1), decorin sequences (CFU83141-1, OCU03394-1, PR42266, P._R42267, P -7 R42260, p _R89439), keratan sulfate proteoglycans (mIU48360 1, AF022890L1), corneal proteoglycan (AF022256-1), and kxone/cartilage proteoglycans and proteoglycane precursors (PCSl.BOV3h, P3S2_M:VUSE, PGS2..jUNN).- Accordingly, it is presently believed that PR0332 disclosed in the present application is a new proteoglycan-type molecule, and mray play a role in regulating extracellular mvatrix, cartilage, and/or inne function- V 44. FuILnl-rh PR0334 PoVlvnenlfs The present invention provides newly identified amd isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0334. In particular. Applicants have identified and isolated cDNA encoding a PR0334 polypeptide. as disclosed in further detail in dhe Examples, below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of dhe PR0334 polypeptide have significant homology with fibulin and flbrillin. Accordingly, it is presently believed that PR0334 polypcptide disclosed in the present application is a newly identified member of the epidermal growth factor family and possesses properties and activities typical of this family.

Flll-length PRO346 PolXVeetde The present invention provides newly identified and isolated mudceotide sequences-encoding polypeptides referred to in the present application as PR0346. In particular. Applicants have identified and isolated cDNA encoding a PR0346 polypeptide. as disclosed in furthe detail in die Examples below. Using BLAST and PastA sequence alignment computer program Applicants found that a full-length native sequence PR0346 (shown in Figure 112 and SEQ ID NO:320) has 28 amino acid sequence identity with carcinocmbryonic antigen. Accordingly, it is presently believed that PR0346 disclosed in the present application is a newly identified member of the carcinoembzyonic protein family and may be expressed in association with neoplastie tissue disorders.

46. Fullnt R0268 Pollnptltde The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in die present application as PR0268. In particular. Applicants have identified and isolated cDNA encoding a PR0268 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that portions of the PR0268 polypeptide have significantm homology with the various protein disulfide isomerase proteins. Accordingly, it is presently believed that PR0268 polypeptide disclosed in the present application is a hornolog of the protein disulfide isomerase p5 protein.

47. Full-lenpth PR330 Polvuentides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0330. In particular. Applicants have identified and isolated cDNA encoding a PR0330 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and PastA sequence alignment computer programs, Applicants found that various portions of the PR0330 polypeptide have significant homology with the murine prolyl 4-hydroxylase alpha-Il subunit protein. Accordingly, it is presently believed that PR0330 polypeptide disclosed in the present application is a novel prolyl 4-hydroxylase subunit polypeptide.

48. R1ll-ength 039an R310 Polypentides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0339 and PRO3 10. In particular, Applicants have identified and isolated 82 cENA encoding a PR0339 polypeptide, as disclosed in further detail in the Examples below. Applicants have also identified and isolated cENA encoding a PRO310 polypeptide, as disclosed in further detail in the Exanples below.

Using BLAST and FastA sequence alignment ccmputer programs, Applicants found that various portions of the PRO339 and PR0310 polypeptides have significant homology with small secreted proteins from C. elegans and are distantly related to fringe. PRO339 also shows hoaology to collagen-like polymers. Sequences which were used to identify PRO310, designated herein as EIA40533 and ENA42267, also show honology to proteins from C. elegans.

Accordingly, it is presently believed that the PRO339 and PR0310 polypeptides disclosed in the present application are newly identified menber of the family of proteins involved in development, and which may have regulatory abilities similar to the capability of fringe to regulate serrate. 49. Full-Length PRO244 Polypeptidss The present invention provides newly identified and isolated nucleotide sequences encoding C-type lectins referred to in the present application as PR0244. In particular, applicants have identified and isolated cENA encoding PR0244 polypeptides, as disclosed in further detail in the Exanples below. Using BLAST and FastA sequence alignment conputer programs, Applicants found that a full-length native sequence PRO244 (shown in Figure 122 and SEQ ID N0:376) has 43% amino acid sequence identity with the hepatic lectin gallus gallus (LECH-CHICK), and 42% amino acid sequence identity with an HIV gpl20 binding C-type lectin (A46274). Accordingly, it is presently believed that PRO244 disclosed in the present application is a newly identified member of the C-lectin superfamily and may play a role in imnune function, apoptosis, or in the pathogenesis of atherosclerosis. In addition, PR0244 may be useful in identifying tuwor-associated epitopes.

PRO Polypeptide Variants In addition to the full-length native sequence PRO polypeptides described herein, it is conterplated that PRO polypeptide variants can be prepared. PRO polypeptide variants can be prepared by introducing appropriate nucleotide changes into the PRO polypeptide NA, or by synthesis of the desired PRO polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

82a Variations in the native full-length sequence PRO polypeptides or in various domains of the PRO polypeptides described herein, can be made, for exanple, using any of the techniques and guidelines for conservative and non-conservative nutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codns encoding the PRO polypeptide that results in a change in the amino acid sequence of the PRO polypeptide as cnapared with the native sequence PRO polypeptide. Optionally the variation is by substitution of at least one amino acid residue with any other amino acid in one or nore of the domains of the PRO polypeptide. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by ccxparing the sequence of the PRO polypeptide with that of hcnologous known protein molecules and minimizing the number of amino acid sequence changes 15 made in regions of high horology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may opdortally be in the range of I to 5 amino acids.

The variation allowed way be determined by systelmaaly making insertions. deletions or substitions of amino acids in the sequence and testing the resulting variants for activity i the in uitro assay described in the Examples below.

7be variations can be made using methods known in the at such as oligomicleotdec-mediatcd (site-directed) mutagenesis. alanine scanning. and PCR mutageneis. Site-directed niutagenesis [Carner et al., MCI.Acid Re 1l.4331 (1986). Zollcr et al., kNucL-A.idc Re-t. 1:6497 (19M],j cassette mutagenesis [Wells et al., fl= .14:315 (19851,restriction selectoaionutagenss[Wells eta!.. Ptilos-Trnt R-Soc. London SerA, 312:415 (1986)J or othe known techniques can be performed on the cloned DNA to produce the desired PRO polypeptide variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids iluealanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alan;n is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton. 7Thcroins. Freeman Co., Chothia. L.

Md. li], 150:1 (1976)). If a lanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

51. Modifications or PRO Polyneptides Covalent miodifications of PRO polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of the PRO polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO polypeptide. Derivatination with bifunctional agents is useful, for instance, for crosslinkinig a PRO polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO polypeptide antibodies, and vice-versa. Commonly used crosstinking agents include, 1, 1-bis(diazoacetyl)-2-phenylcthane. glutaraldchyde, N-hydroxysuccinunide esters, for example. esters with 4-azidosalicylic acid, homobifanctional irnidoesters, including disuccinimidyl esters such as 3,3-dithiobis(succinimidylpropionate), bifunctional maleimides such as his-Ninaleimido-1,8-ocaie and agents such as methyl-34[(p-azido phenyodithio)propioimidatc.

Other modifications include deamidation of glutaminyl and asparaginyl residues to thc corresponding glutamnyl and aspartyl residues, respectively. hydroxylation of praline and lysine, phosphorylation of hydroxyl groups of seryl or threoyl residues, methylation of the at-amino groups of lysine, arginine, and bistidine side chains IT.E.

Creighton, Proteins- Sructure and Molecular Propertie, W.H. Freeman Co., San Francisco. pp. 79-86 (1983)).

acetylation of the N-terminal am='e, and ainidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO polypeptides included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the native glcosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in a native sequence PRO polypeptide. and/or adding one or more glycosylation sites that are not present in the native sequence PRO polypeptide.

Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amaino acid sequence. The alteration may be made, for example, by the addition of. or substitution by. one or morm serine or threonine residues to the native sequence PRO polypeptide (for 0-linked glycosylation sites). The PRO polypeptidc amino acid sequene may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polpetide at preselected bases such that codons are generated that will translate into the desired amino adds.

Another means of increasing the mnumber of carbohydrate moieties on the PRO polypeptide polypeptide is by chemical or enzymatic coupling of glycosidcs to the po~ypcptide. Such methods are described in the art, in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 4 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or en zymatcafly or by mutational substitution of codons encoding for amino acid residues that serve as- targets for glycoylaion. Chemical deglyensylazlon tchdniques are bkwn in the art and described, for instance, by Hakimuddin.

et al., Arch. llochem- Rionhs W ~:52 (1987) and by Edge cc al., Aral. iochem.. 11.8:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo- .15 glycosidases as described bylboctakura et al., Meth. Elymo. 13:350 (1987).

Another type of covalent modification of PRO polypeptides of the invention comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes. in the manner set forth in U.S. Patent Nos. 4,640,835; 4.496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179.337.

The PRO polypeptides of the present invention tny also be modified in a way to form a chimeric molecule :comprising a PRO polypepide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises-a fusion of the PRO polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amnino- or carboxyl- terminus of the PRO polypeptide. The presence of such epitope-4agged forms of the PRO polypeptde can be detected using an artibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO polypeptide to be reail purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO polypeptide with an umnmoglobulin or a particular region of an immunoglobulin. For a bivalent form of ***4the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.

Various tag polypeptides and their respective antibodies are well kniown in the art. Examples include polyhistidinc (poy-bis) or poly-histidine-glycine (poly-bis-gly) tags; the flu HA tag polypeptide and its antibody 12CAS (Field et al., Mol. Cell. fIM., 1:2159-2165 (1988)]; the c-rnyc tag and the 81F9. 3C7, 6EI0, G4, B7 and 91110 antibodies thereto [Evan et al., Molecular and Cellular Biolozy, 5:3610-3616 (1985)]; and the Herpes Simplex. virus glyitoprotein D (gD) tag and its antibody IPaborsky et al., Protein Engiingerin, 1(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptdec (H-opp et al., Bio~echnology, 6: 1204-1210 (1988)]; the KT3 epitope peptide (Martin et al., Sce= 92-194 (1992)]; an a-tubulin epitope peptdec (Skinner et al., L. iol- 2k:15163- 15166 (1991)]; and the T7 geme 10 protein peptide tag (Liitz-Freyernmth ct al., Proc. Nai. Acad. Sci. USA, 12:6393- 6397 (1990)].

52. Modification of PR0317 Amino acid sequence variants of PRO317 are prepared by introducing appropriate nucleotide changes into the PRO317. DNA. or by in vitro synthesis of the desired PR0317 polypeptide. Such variants include, for example.

deletons from or insertions or substitutions of. residues within the amino acid sequence shown fothuman PR0317 in Figure 42. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter posttranslational processes of the PR0317. such as changing the number or position of glycosylation sites. Moreover, like most mammalian genes, PRO317 is presumably encoded by multi-exon genes. Alternative mRNA constructs which may be attributed to different mRNA splicing events following transcription, and which share large regions of identity with the cDNAs claimed herein, are considered to be within the scope of the present invention.

For the design of amino acid sequence variants of PRO317. the location of the mutation site and the nature of the mutation will depend on the PR0317 charactcristic(s) to be modified. For example, candidate PR0317 antagonists or agonists will be initially selected by locating sites that are identical or highly conserved among 15 PR0317, EBAF-I. LEFTY, and other members of the TGF- superfamily. The sites for mutation can be modified individually or in series, by substituting first with conservative amino acid choices and then with more radical selection depending upon the results achieved. deleting the target residue, or inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.

A useful method. for identification of certain residues or regions of the PR0317 polypeptide that are preferred locations for mutagenesis is called alanine scanning nmnagenesis. as described by Cunningham and Wells.

Science, 24: 1081-1085 (1989). Here, a residue or group of target residues are identified charged residues S such as arg, asp. his, lys, and glu) and replaced by a neutral or negatively charged amino add (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions then are refined by ,25 introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example.

to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at :the target codon or region and the PR0317 variants produced are screened for the optimal combination of desired activity.

There are two principal variables in the construction of amino acid sequence variants: the location of the mutation site and the nature of the mutation. These are variants from the Figure 42 sequence, and may represent naturally occurring alleles (which will not require manipulation of the PR0317 DNA) or predetermined mutant forms made by mutating the DNA, either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the PR0317 characteristic to be modified.

Amino acid sequence deletions generally range from about I to 30 residues, more preferably about 1 to residues, and typically are contiguous. Contiguous deletions ordinarily are made in even numbers of residues, but single or odd numbers of deletions are within the scope hereof. Deletions may be introduced into regions of low homology among PRO317. EBAP-1, and other members of the TGF- superfamily which share the most sequence identity to the human PR0317 amino acid sequence to modify the activity of PR0317. Deletions from PRO317 in areas of substantial homology with one of the receptor binding sites of other members of the TGF- superfamily will be more likely to modify the biological activity of PRO317 more significantly. The number of consecutive deletions will be selected so as to preserve the tertiary structure of PR0317 in the affected domain, beta-pleated sheet or alpha helix.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypcptidcs containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions insertions within the mature PR0317 sequence) may range generally from about I to 10 residues, more preferably 1 to 5, most preferably 1 to 3. Insertions are preferably made in even numbers of residues, but this is not required. Examples of terminal insertions include mature PR0317 with an N-terminal methionyl residue, an artifact of the direct production of mature PR0317 in recombinant cell culture, and fusion of a heterologous N-terminal signal sequence to the N-terminus of the mature PRO317 molecule to facilitate the scretion of mature PR0317 from recombinant hosts. Such signal sequences may be obtained from, and thus homologous to, the intended host cell species, but also may be from other members of the TGF- 15 superfamily. Suitable sequences include STI or Ipp for E. coli. alpha factor for yeast, and viral signals such as herpes gD or the native EBAF-1 sequence for mammalian cells.

Other insertional variants of the PRO317 molecule include the fusion to the N- or C-terminus of PRO317 of immunogenic polypeptides, bacterial polypeptides such as beta-lactamase or an enzyme encoded by the E.

co trp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobulin constant regions (or other immunoglobulin regions), albumin, or ferritin, as described in WO 89/02922 published 6 April 1989.

A third group of variants are amino acid substitution variants. These variants have at least one amino acid residue in the PR0317 molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as the active site(s) of PRO317 and sites where the amino acids 25 f ou d in the known analogues are substantially different in terms of side-chain bulk. charge, or hydrophobicity, but Swhere there is also a high degree of sequence identity at the selected site within various animal PRO317 species, or where the amino acids found in known members of the TGF- superfamily and novel PR0317 are substantially :different in terms of side-chain bulk. charge, or hydrophobicity, but where there also is a high degree of sequence identity at the selected site within various animal analogues of such members among all the animal EBAF-l molecules). This analysis will highlight residues that may be involved in the modulation of endometrial tissue or angiogenesis, and therefore, variations at these sites may affect such activities.

Other sites of interest are those in which particular residues of the PR0317 obtained from various species are identical among all animal species of PR0317 and other members of the TGF- superfamily, this degree of conservation suggesting importance in achieving biological activity common to these cytokines. These sites, especially those falling within a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table I under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table or as t~irthcer described below in reference to amino acid classes.

are introduced and the products screened.

Jible I Otiginal Ala (A) Arg (R) Asa (M Asp (D) Cys (C) Gin (Q) Ghs (E) Gly (G) His (H) le (1) Leu(L) 20 Lys (K) Met (M) Plie (F) Pro (P) Ser (s) Thr (1) Trp (W) TyrmY Val (V) Exemplary Subsitutions Preferred Substitutions Val; lea; Hle In; gin; asn his. lys; arg gln scr an asp pro; ala sn: gln. lys; arg leu; val; miet. ala; phe; norleucane norleucine; ile; val;, met; ala; phe arg; gln; asn leu; phe;ile leu; val; ile; ala; tyr ala thr ser tyr; phe trp; phe; dir; ser ile; lIU; met. phe; ala; norleucine Substantial modifications in function or immological identity of the PRO317 are accomplished by selecting substitutions that differ significatly in their effect on maintaining the structure of the polypeptidi backbone in die area of the substitution. for example. as a sheet or helical conformation, the charge or hydrophobicity of the 35 molecule at the target site, or the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: hydrophobic: norleucine, met, ala. val. leu, B~e; neutral hydrophilic: cys, ser, thr; acidic: asp, Stu; basic: asn, gin, his, lys. arg; residues that influence chain orientation: gly, pro; and aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of diese classes for another class.

Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into die remaining (non-conserved) sites.

In one enmbodiment of the invention, it is desirable to inactivate one or more protease cleavage sites that are present in the mnolecule. These sites are identified by inspection of the encoded amino acid sequence, in the case of Iypsin, for an arginyt or lysinyl residue. When protease cleavage sites are identified, they are rendered inactive to proteolytic cleavage by substituting the targeted residue with another residue, preferably a basic residue such as glutamine or a hydrophilic residue such as serine; by deleting the residue; or by insening a prolyl residue immediately after the residue.

Iq another embodiment, any mcthionyl residues other than the starting methionyl residue of the signal sequence, or any residue located within about three residues N- or C-terminal to each such methionyl residue, is substituted by another residue (preferably in accord with Table 1) or deleted. Alternatively, about 1-3 residues are inserted adjacent to such sites.

Any cysteine residues not involved in maintaining the proper conformation of PR0317 also may be substituted, generally with scrine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

Nucleic acid molecules encoding amino acid sequence variants of PR0317 are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonuclcotide-mediated (or site-directed) nmtuagensis. PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of PRO317.

15 Oligonucleoide-mediated mutagenesis is a preferred method for preparing substitution, deletion, and insertion variants of PR0317 DNA. This technique is well known in the art as described by Adelman er al.. DA, 2:183 (1983). Briefly, PR0317 DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of PRO317. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the PR0317 DNA.

Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template 25 molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea t Proc. Natl. Acad. Sci. USA, 75: 5765 (1978).

The DNA template can be generated by those vectors that are either derived from bacteriophage M13 vectors (the commercially available Ml3mpl8 and Ml3mpl9 vectors are suitable), or those vectors that contain a single-stranded phage origin of replication as described by Vicra e al. Meth. Enzmol. 153: 3 (1987). Thus, the DNA that is to be mutated may be inserted into one of these vectors to generate single-stranded template. Production of the single-stranded template is described in Sections 4.21-4.41 of Sambrook e at., supra: Alternatively, single-stranded DNA template may be generated by denaturing double-stranded plasmid (or other) DNA using standard techniques.

For alteration of the native DNA sequence (to generate amino acid sequence variants, for example), the oligonucleoide is hybridized to the single-stranded template under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonuclcotide as a primer for synthesis. A heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of PRO317, and the other strand (the original template) encodes the native, unaltered sequence of PR0317. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JMIO1. After the cells are grown, they are plated onto agarose plates and screened using the oligotucleotide primer radiolabeled with "P to identify the bacterial colonies that contain the mutated DNA. The mutated region is then removed and placed in an appropriate vector for protein production, generally an expression vector of the type typically employed for transformation of anappropriate host.

The method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s). The modifications are as follows: The single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP).

is combined with a modified thio-dcoxyribocytosine called dCTP-(aS) (which can be obtained from the Amersham Corporation). This mixture is added to the template-oligonucleotidc complex. Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated. In addition, this new strand of DNA will contain dCP-(aS) instead ofdCTP, which serves to protect it from restriction endonuclease digestion.

15 After the template strand of the double-stranded hcteroduplex is nicked with an appropriate restriction enzyme. the template strand can be digested with Exom nuclease or another appropriate nuclease past the region that contains the sitc(s) to be mutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four dcoxyribomncleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell such as E. coli JM101. as described above.

DNA encoding PR0317 mutants with more than one amino acid to be substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated 0sinaultancously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than about ten amino adds), it is more S 25 difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.

In the first method, a separate oligonuclcotide is generated for each amino acid to be substituted. The oligonuclcotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single nutants: wild-type DNA is used for the template, an oligonuclcotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonuclcotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on.

PCR mutagenesis is also suitable for making amino acid variants of PRO317. While the following discussion refers to DNA, it is understood that the technique also finds application with RNA. The PCR technique generally refers to the following procedure (see Erlich, PCR Technology, (Stockton Press. NY, 1989). the chapter by R. Higuchi, p. 61-70): When small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. For inroduction of a mutation into a plasmid DNA, one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a stretch of sequence of the opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 200 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced. PCR amplification uing a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.

Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells 15 et al., Ge ne4: 315 (1985). The starting material is the plasmid (or other vector) comprising the PRO317 DNA to be mutated. The codon(s) in the PR0317 DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the PR0317 DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonuclcotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are Ssynthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3' and 5' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated 25 PR0317 DNA sequence.

Covalent modifications of PRO317 are also included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of the PR0317 with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO317. Derivatization with bifunctional agents is useful, for instance, for crosslinking PRO317 to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO317 antibodies, and vice-versa. Commonly used crosslinking agents include, l.l-bis(diaoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid. homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis- (succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l.8-octane, and agents such as methyl-3- ((p-azidophenyl)dithio)propioimidate.

Other modifications include deamidation of glutaminyl and asparaginyl residues to ihe corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the "-amino groups of lysine. arginine. and histidine side chains (T.E.

Creighton, Proteins: Strucmmr and Molecular Ptroprtes, W.H. Freeman Co.. San Francisco. pp. 79486 (1983)), acetylation of the N-terminal amine. and amidation of any C-terminal carboxyl group.

Another type of covalent modification of dhe PRO317 polypeptdec included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptidc. 'Altering dt native glycosylauion pattern* is intended for purposes herein to mean deleting one or more carbohydrate moieties found in nativc-sequence; PRO polypeptide. and/or adding one or more glycosyatuon sites that are not present in the naie-sequence PRO polypeptide. The deduced aminoacid sequence of PR0317 shown in Figure 42 (SEQ ID NO: 114) has one predicted N-linked glycosylation site at residue 160.

Addition of glycosylauion sites to the PR0317 polypeptide may be accomplished by altering the amino acid sequence. The alteration may be nude, for example, by the addition of, or substitution by. one or more scrine or direonine residues to the inative-sequence PR0317 (for 0-inked glycosylation sites). The PR0317 amino acid sequece may optionally be altered through changes at the DNA evel, particularly by mutating dhe DNA encoding the PR0317 polypeptide at preselected. bases such that codons are generated that will translate into the desired amino acids.

:6 Another mens of increasing the number of carbohydrate moieties on the PR0317 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art in WO 97/05330 published I1I September 1987. and in Aplin and Wriston, CRC Crit. Rev. Biochem.. pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PR0317 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that serve as targets for gl~voylaion. Chemical deglycosylation techniques ame known in the art and described, for instance. by Hakimuddin.

er al, Arch. Biochem. Biophvs 252:52 (1987) and by Edge etoal.. Anal. Biochem., JLBf: 131 (198 Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exoglycosidases as described by Thotalwra aal., Meth. EnZYnxtL. Ml.:350 (1987).

Another type of covalent modification of PR0317 comprises linking the PR0317 polypeptide to one of a variety of nonproteinaceous polymers. polyethylene glycol. polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4.496.689; 4.301.144; 4.670,417; 4.791.192 or 4.179,337.

The PR0317 of the present invetiuonniay also be modiified in a way to form a chimeric molecule comprising PR0317 fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of the PR0317 with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. Tn epitope tag is genetally placed at the amino- or carboxyl- termin of the PR0317.

The presence of such epitope-tagged form of the PR0317 can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PR0317 to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. in an alternative embodiment, the chimeric molecule may comprise a fusion of the PR0317 with an immunoglobulin or a particular region of an inmiunoglobulin. For a bivalet form of the chimieric molecule, such a fusion could be to the Fc region of an lgG molecule.

Various tag polypeptides andI their respective antibodies are well known in the art. Examples include polyhistidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 (Field ai al., Mol Cf Bo I 1:2159-2165 (1988)): the c-myc tag and the SF9, 3C7. 6E10, G4, B7. and antibodies thereto (Evan et al., Molecular and Celhular Biolorv. 5:3610-3616 (1985)); and die Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al..Ppti Ejgdfieg, 2(6):547-S53 (1990)). other tag polypeptides include the Flag-pepride (Hopp et al.. BigM2cchmolog., f:1204-l210 the K73 epitope peptide (Martn ez al. 5cie .:92-194 (1992)); an '-tuhilin epitope peptide (Skinner cra!.. L il-Che~m 2U: 15163- 15166 (1991)); and dhe T7 gene 10 protein peptide tag (Lxz-Frcyruth e al., Proc. Hadl. Acad. Scf. USA, Z2:6393,- 6397 (1990)).

S3. Preparation of PRO PolynepIdes The description below relates primarily to production of PRO polypeptides by culturing cells transformed or transfected with a vector containing the desired PRO polypeptide nucleic acid. It is. of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare the PRO polypeptide. For instance die PRO polypepaide seq er, or portions thereof, may be produced by direct peptide synthesis using solidphase techniques [see. Stewart ct al.. Solid-Phase Penride Synthesis, W.H. Freeman Co.. San Francisco. CA (1969); Merrifield, Am. Chem. Soc., U5:2149-2154 (1963)1. In viro protein, synthesis may be performed using manual techniques or by automation. Autmated synithesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City. CA) using manusfacturer's instructions. Various portions of the desired PRO polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to *produce the fifflecngth PRO polypeptide.

A. Isolation of DNA Encoding PRO Pohpcutides DNA encoding PRO polypeptides may be obtained from a eDNA libraty prepared from tissue believed to Possess the desired PRO polypepdde mRNA and to express it at a detectable level. Accordingly, human PRO polypeptde DNA can be conventently obtained from a cDNA library prepared from human tissue, such as described in the Examples. The PRO polypeptide-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis.

libraries can be screcried with probes (such as antibodies wo the desired PRO polypeptide or oligonucleotides of at least about 20-80 bases) designed to identifyr the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A L*boratorv Manrual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding the desired PRO polypeptide is to use PCR methodology (Sambrook et al.. UMgg; Dieffenbach et al., PCR Prinmer.A Laboratory Manual (Cold Spring Harbor Laboratory Press. 1995)].

The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling arc well known in the art, and include the use of radiolabels like 31P-labeled ATP.

biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency. are provided in Sambrook et al.. gmwx.

Sequences identified in such library screening mnethods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases.

Sequence identity (at either the amino adid or nueleotde level) within defined regons of the molecule or across the ful-length sequence can be determined through sequence alignment using computer software programs such as BlAST, AUGN. DNAstar, and INHERIT which employ various algorithms to measure homology.

Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using dhe deduced amino acid sequence disclosed herein for the first timc, and, if necessary, using* ccntina primer extension procedures as described in Sambrook et al.. s* a, to detect precursors and processing intermediates of roRNA that may not have been reverse-tanscribed into cDNA.

B. Selection and Transformation or Host Cells Host cells are transfectod or transformed with expression or cloning vectors described herein for PRO polypeptide production and cultured in conventional nuttrient media modified as appropriate for inducing promoters.

0 scectng transformanss, or amplifying the genes encoding the desired sequences. The culture conditions, such as I.L e 15 media, temperature. pH and the like, can be selected by the skilled artisan without undue experimentation. In gercral, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian -Cell Biotecmology: a Practical Approac, M. Butler, ed. (IRL Press, 1991) and Sambrook et al..

Ssup Methods of =rasfction are known to the ordinarily skilled artisan, for example, CaPO, and electroporation.

Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells.

The calcium treatment employing calcium chloride, as described in Sambrook et al., a= or electroporation is **generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with Agrobadteriwn nefaciens Is used for transformation of certain plant cells, as described by Shaw et al., r&W 23:315 (1983) and WO 89/05859 published 29 June 1989. For miammalian cells without such cell walls, the calcium phosphate *:25 precipitation method oftGrahiam and van der Eb. Virology. 5.2:456-457 (1978) can be employed. General aspects of mammaian cell host system transformations have been described in U.S. patent No. 4,399,216. Transformations into yeast are typically carried out a crdiuig to the method of Van Solingen et J. Bact., M~:946 (1977) and Hsiao.

et Pro- NaI. Aca! Si (MSA), 2f3M2 (1979. However, other methods for introducing DNA into Cells, such as by nuclear aieroinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g..

polybVAIC polyomnithine, may also be used. For various techniques for transforming mammialian cells, see Keown et Methods in E0ZwMolnev 18f5:527-537 (1990) and Mansour ct al..1 "tr, 2:34-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are nwt limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactediaeae such as E. co/i. Various E. coil strains are publicly available, such as E. cvii K12 strain MM294 (ATCC 31.446); E. co/i X1776 (ATCC 31,537); E. co/i strain W3 110 (ATCC 27,325) and K5 77 (ATCC 53,635). Other suitable prokaryotic host cells include Entcrobacteriaceac such as Erciieridzia, E. coff, EF uerobacrtf,'Erwnia, lebriella, Proteus, Sabnoneia, Sabnwnella rjphimurium, Sefrraa, Se &nia mxrecu, and Shgella. as well as Bacilli such as B. subdIji and B. liclzemformis B.

Iiclteniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruganosa, and &repomyr~ Various E. coil strains are publicly available, such as E. coil K12 strain MM294 (ATCC 31.446); E.

coli X 1776 (ATCC 31,537); E. cold strain W31 10 (ATCC 27,325); and K5 772 (ATCC 53.635). These examples are illustra4ve rather than limiting. Sain W3110 is one particularly preferred host or parent host because'it is a common lost stwain (or recombinan DNA product fermentations. Preferably, the host cell secretes aninimal-amounts of proteolytic enzymes. For example. strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. cold W31 10 strain IA2, which has the complete genotype tonA E. coli W31 10 strain 9E4. which has the complete genotype ronA ptr3; E. coil W31 10 strain 27C7 (ATCC 55.244), which has the complete genotype lonA ptr3 phoA EI5 (argF-iac))69 degP cwpTknn'; E. CONi W31 10 strain 37D6. which has the complete genotype tonA ptri phoA E15 (argF-lac)169 degP oWipr bs7itvG kamf; E. coff W31 10 strain 40B4, which is strain 37D6 with a non~kanamycin resistant degP deletion mtation; and an E. cofd strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 *e Atugust 1990. Alternatively, in vito methods of cloning. PCR or other ncleic acid polymerae reactions, are suitable.

O. .015 In addition to prokaryotes, eukaryotic microbes such as filanienous fungi or yeast arc suitable cloning or expression hosts for PRO polypeptide-encoding vectors. Sacclzaronryces cerevisiae is a conmmonly used lower eukaryotuc host Microorganism. Others include Schizosaceharomyces pombe (Beach and Nurse, 1N1fflg. 22Q- 140 [1981]: EP 139.383 published 2 May 1985); Kiryveromyces hosts Patent No. 4,943,529; Fleer er aL.

BjojtgphIoZ2: :968-975 such as. K. lacris (MW98-8C, CBS683, CBS4574. Lauvencourt a aL, L BacteioL., 737 11983D), X. fragilis (ATCC 12.424). K. bulgarics (ATCC 16,045), K. ickeramli (ATCC 24.178).

K. wasi (ATCC 56,500). K. drosophilarum (ATCC 36,906; Van den Berg ej at., Rio/Technolonv, 1: 135 (1990)).

thennoeolerans, and K. marziauws yarrowia (EiP 402,226); Piciapastoris (EiP 183,070; Sreekrishna ea aL, L.

'.meBasic Microbiol., 2: 265-278 11988]); Candida, Tichderyma reesia (EP 244,234); Neurospora crassa (Case er aL.

Proc. Nall Acad. Sci, USA, 5259-5263 (1979]); Schuwawaomyces such as Schimaniomyces occidentalis (EiP 394,538 published 31 October 1990); and filament=u fungi such as, Neurospora. Penicillium. Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulais (Ballance ar at. Biochm.

Biophyr Res. Como=i, flk. 284-289 (1983]; Tilburn etl.. 2m 205-221 11983]; Yclton aal. Pmc Nail Acad. SL.JUSA. flI: 1470-1474 11984]) and A. niger (Kelly and Hynes, EMBOLL, 4: 475-479 11985]).

.me. Methylotropic yeasts are suitable herein and include, but arc not limited to, yeast capable of growth on methanol selected frm the genera consisting of Hansenud, Candida, Kloeckera. Picza, Sacchiaromyces, Tondlopsis, and Rlzcdotoruda. A list or specific species that arc exemplary of this class of yeasts may be found in C. Anthony. Mbe Biochemistry of Methylotrous, 269 (1982).

Suitable host cells for the expression of glycosylated PRO polypcptides are derived from mutlticellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 andi Spodoptera SM. as well .as plan cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS Cells.

More specific examples include motnkey kidney CVl line tanformed by SV40 (COS-7. ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subeloned for growth in suspension culture, Graham et al., LGI.YirL.

36:59 (1977)): Chinese amster ovary cels/-DHFR (CHO. Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 27:4216 (1980)): mouse scrtoli cells (TM4. Mather, Biol Renrod. 21:243-251 (1980)); human lung cells (W138, ATCC CCL human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriate host cell is deemed to be within the skill in the an.

C. Selection and Use of a Replicable Vector The nucleic acid cDNA or genomic DNA) encoding a desired PRO polypeptide may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available.

The vector may. for example, be in the form of a plasmid, cosmid. viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general. DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but arc not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

The PRO polypcptide of interest may be produced recombinantly not only directly, but also as a fusion 15 polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a componen of the vector, or it may be a pan of the PRO polypeptide DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase.

penicillinase, Ipp, or heat-stable enterotoxin nI leaders. For yeast secretion the signal sequence may be, the 20 yeast invcrtase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the latter described in US. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamnylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal •sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.

25 Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2i plasmid origin is suitable for yeast, and various viral orgins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker.

Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, ampicillin, neomycin, methotrexate, or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media, the gene encoding D-alanine racemase for Bacilli.

An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO polypeptide nucleic acid, such as DHFR or thymidine kinasc. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub ct al.. Proc. Natl. Acad Sd. USA, 22:4216 (1980). A suitable selection gene for use in yeast is the upl gene present in the Yeat Plasmid YRp7 [Stinchcoalb ct al.- Nlature. M:39 (1979); Kingsmn et al., C= 2:141 (1979); Tschenaper et al.. Us V-.157'(1980)). The Inp1 gene provides a selection marker for a muitant strain of yeast lackig the ability to grow in Utpophari, for example. ATCC No. 44076 or PEP4- I (Jones, Genetics, U:12 (1977)].

Expresi ion and cloning vectors usually contain a promoter operably linked to the PRO polypetide nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells arewcU known.

Promoters suitable for usC with prokarryotic hosts include the P-lartainase and lactose promoter systems [Chang ct NLature. =m.615 (1978); Goeddel et al., Na1u, 2U:544 alkcaline phosphatasc, a tryptophan (trp) promoter system [Goeddel, NulcLAcid Re., 1:4057 (1980); EP 36.7761, and hybrid promoters such as the tac promoter (deBoer ct al., Proc- Nail Aad, Si. UiSA, -fl:21-25 (1983)]. Promoters for use in bacterial systems also [0 wWl contain a Shine-Dalgarno sequence operably linked to the DNA encoding the desired PRO polypeptide.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3pbosphoglyct kinase (Hitrman etal.. 1. Iiol. C. =:2073 (19M) or other Slycolytic enzy mes; (Hess et al., Ady. Enzm Reg., 149 (1968); Holland, Bjidbcm Lstu. 12:4900 (1978)], such as enolasc, glyccraldehyde-3phosphate dchydrogenase, hexoinse. pyruvate decarlboxylase phosphoftuctokinase. glucose-6-phosphate isomera se, 3-phesphoglycerate inutase, pyruvatc kirimse ujosephosphatc isomerase. phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription .*:controlled by growth conditions, are the promoter regions for alcohol dchydrogenase 2, isocytochrome C, acid phosphatase. degradative enzymnes associated with nitrogen metabolism, metailothionein. glyceraldehydc-3-phospbatc dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and p romoters for 20 use in yeast expression are further described in EP 73.657.

PRO polypeptide transcription from vectors in mammalia host cells is controlled, for example, by prooters obtained from the genomcs of viruses such as polyoma virus, fowlpox virus (UK 2211,504 published July 1989). adcntovirus (such as Adenovirus bovine papiloma virus, avian sarcoma virus, cytomegaloviflis, a rervi us hepatits-B virus and Simian Virus 40 (SV40), from heterologous mamnmalian promoters. dite actin promoter or an imniunoglobulin promoter. and from heat-shock promoters, provided such promoters arc compatible with the host cell systems.

Transcription of a DNA encoding the desired PRO polypeptide by higher eukarYotes may be increasd by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from to 300 bp, that act on a promoter to increase its trancription. Many enhancer sequences are now knsown from mammialian genes (globin, elastase, albumin, ot-fetoprotein. and insulin). Typically, however, one will use an enhancer from a eulcryotic: cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-70), the cytomegaloviiizs early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the PRO polypeptide coding sequence, but is preferably located at a site 5' from the promoter.

Expression vectors used in eukaryotic host Ccls (yeast, fungi, insct, plant, animal, huma, or nucleated cells from other multicellular organism) will also contain sequences necessary for the termination of transcription andl for stabilizing the znRNA. Suc~h sequences are coimmonly available from the 5S and, occasionally 3Y, untranslated regions of eukarycuic or viral DNAs or cDNAs. Thbese regions contain nucleotide segments transcribed as Polyadenylated fragments in dhe untranslated Portion Of the tnRNA encoding PRO polypeptides.

Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO polypeptides in recombinant vertebrate cell culture are described in Gething et al.. N~ature, M:620,625 (1981); Mantei et al., Nature, 2U:40-46 (1979); EP 117.060; and EP 117,058.

D. Detecting Gene ArnulficationtErrssion Gene amplification and/or expression may be measured in a samnple directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of niRNA r~homas, Proc. Nad. Acad. Sd. USA, 77:5201-5205 (1980)1 dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe.

based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes. and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

:::.Gene expression, alternativel y, may be measuzrcd by inmmunological methods, such as isumunobistochemical V.600 15 staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for *-nuwhistodlianicai staining and/or assay of sample fluids may be either mnonoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO polypeptidc or against a synthetic peptide based'on the DNA sequences provided herein or against exogenous sequence fused to a PRO polypeptide DNA and encoding a specific antibody epitope.

E. Purification or Polynentide *Forms of PRO polypeptides may be recovered from culture medium or from host cell lysates. If membranebound, it can be released from the membrane using a suitable detergent solution Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PRO polypeptides can be disrupted by various physical or chemical *25 means, such as freeze-thaw cycling. sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO polypeptdecs from mecombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; ebromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex protein A Sepharose columns to remove contaminants such as IgG; and metal chelatin columns to bind epitopetagged forms of ibc PRO polypeptide. Various methods of protein purification may be employed aiid such mnethods are known in the art and described for example in Deutscher, Methods in EzM~ology, 1B2 (1990); Scopes, Protin Purification: Principles and Pratic, Springer-Verlag, New York (1982). The purification step(s) -selected will depend, for example, on the nature of the production process used and the particular PRO polypeptidt: produced.

54. Uses for PRO Polvpentides Nucleodde sequences (or their complement) encoding the PRO polypeptides of the present invention have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA. PRO polypcptid-encoding nucleic acid will also be useful for the preparation of PRO polypeptides by the recombinant techniques described herein.

The full-lagth native sequence PRO polypeptide-encoding nucleic acid or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length PRO polypeptide gene or to isolate still other genes (for instance, those encoding naturally-occurring variants of the PRO polypcptide or PRO polypeptides from other species) which have a desired sequence identity to the PRO polypeptide nucleic acid sequences. Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from the nucleotide sequence of any of the DNA molecules disclosed herein or from genomic sequences including promoters, enhancer elements and intons of native sequence PRO polypeptide encoding DNA. By way of example, a screening method will comprise isolating the coding region of the PRO polypeptide gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as "P or" S, or enzymatic labels such as alkaline phosphatase coupled to the probe via 15 avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the specific PRO polypeptide gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.

The ESTs disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.

Thie probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related PRO polypeptide sequences.

Nucleotide sequences encoding a PRO polypeptide can also be used to construct hybridization probes for mapping the gene which encodes that PRO polypeptide and for the genetic analysis of individuals with genetic S* 25 disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.

The PRO polypeptide can be used in assays to identify its ligands. Similarly, inhibitors of the Seceptor/ligand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Screening assays can be designed to find lead compounds that mimic the biological activity of a native PRO polypeptide or a ligand for the PRO polypeptide. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.

Nucleic acids which encode a PRO polypcptide or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of theraputically useful reagents. A transgenic animal a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a trangenic animal develops. In one embodiment, cDNA encoding a PRO polypeptide of interest can be used to clone genomic DNA encoding the PRO polypeptide in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding the PRO polypeptide. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4.736.866 and 4.870,009. Typically, particular cells would be targeted for PRO polypeptide transgene incorporaion with tissuespecific enhancer. Transgenic animals that include a copy of a transgene encoding a PRO polypeptide introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding the PRO polypeptide. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its ovcrexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced 15 incidence of the pathological condition, compared to untreated animals bearing the ransgenc, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO polypeptides can be used to construct a PRO polypeptide "knock out" animal which has a defective or altered gene encoding the PRO polypeptide of interest as a result of homologous recombination between the endogenous gene encoding the PRO polypeptide and altered genomic DNA encoding the PRO polypeptide introduced into an embryonic cell of the animal. For example, cDNA encoding a PRO polypeptide can be used to clone genomic DNA encoding the PRO polypeptide in accordance with established techniques. A portion of the genomic DNA encoding a PRO polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see Thomas and 25 Capecchi l, 51503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal a mouse or rat) to form aggregation chimeras [see Bradley, in Terntocardnomar and Embryonic Stem Cells: A Practical Approach, J. Roberton. ed. (IRL, Oxford, 1987); pp. 113-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the PRO polypeptide.

With regard to the PRO211 and PR0217 polypeptide, therapeutic indications include disorders associated with the preservation and maintnance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions cntarocolits. Zollinger-Ellison syndrome, gastrointestinal ulceration and congenital microvillus atrophy), skin diseases associated with abnormal keratinocyte differentiation psoriasis, epithelial cancers such as hmg squamous cell carcinoma, epidermoid carcinoma of the vulva and gliomas.

Since the PRO232 polypeptide and acleic acid encodig it possess sequence homology to a ccU surface stem cell antigen and its encoding nucleic acid. probes based upon the PR0232 nucleotide sequence may be employed to identify other novel stem cell surface antigen proteins. Soluble forms of the PRO232 polypeptide may be employed as antagonists of membrane bound PRO232 activity both in vitro and in vivo. PRO232 polypeptides may be employed in screening assays designed to identify agonists or antagonists of the native PRO232 polypeptide, wherein such assays may take the form of any conventional cell-type or biochemical binding assay. Moreover, the PR0232 S polypeptide may serve as a molecular marker for the tissues in which the polypeptide is specifically expressed.

With regard to the PR0187 polypeptides disclosed herein, FGF-8 has been implicated in cellular differentiation and embryogenesis, including the paerning which appears during limb formation. FGF-8 ahd the PR0187 molecules of the invention therefore are likely to have potent effects on cell growth and development.

Diseases which relate to cellular growth and differentiation are therefore suitable targets for therapeutics based on functionality similar to FGF-8. For example, diseases related to growth or survival of nerve cells including 15 Parkinson's disease, Alzheimer's disease, ALS, neuropathies. Additionally, disease related to uncontrolled cell growth, cancer, would also be expected therapeutic targets.

With regard to the PR0265 polypeptides disclosed herein, other methods for use with PR0265 are described in U.S. Patent 5,654,270 to Ruoslahti et al. In particular, PR0265 can be used in comparison with the fibromodulin disclosed therein to compare its effects on reducing dermal scarring and other properties of the fibromodulin described therein including where it is located and with what it binds and does not.

The PRO219 polypeptides of the present invention which play a regulatory role in the blood coagulation cascade may be employed in vivo for therapeutic purposes as well as for in vitro purposes. Those of ordinary skill in the art will well know how to employ PRO219 polypeptides for such uses.

'he PR0246 polypeptides of the present invention which serve as cell surface receptors for one or more 25 viruses will find other uses. For example, extracellular domains derived from these PR0246 polypeptides may be employed therapeutically in vio for lessening the effes of viral infection. Those PR0246 polypeptides which serves as tumor specific antigens may be exploited as therapeutic targets for anti-tmor drugs, and the like. Those of ordinary skill in the an will well know how to employ PR0246 polypeptides for such uses.

Assays in which connective growth factor and other growth factors are usually used should be performed with PRO261. An assay to determine whether TOF beta induces PRO261, indicating a role in cancer is performed as known in the art. Wound repair and tissue growth assays are also performed with PRO261. The results are applied accordingly.

PRO228 polypeptides should be used in assays in which EMRI, CD97 and latrophilin would be used in to determine their relative activities. The resiats can be applied accordingly. For example, a competitive binding assay with PRO228 and CD97 can be performed with the ligand for CD97, Native PRO533 is a 216 amino acid polypeptide of which residues 1-22 are the signal sequence. Residues 3 to 216 have a Blast score of 509, corresponding to 53% homology to fibroblast growth factor. At the nucleotide level, DNA47412. dhe EST frm which I'CR oligos were generated to isolate dhe full length DNA49435-1219, has been observed to map tolIlpI5. Sequence homology to de IlpI5 locus would indicate that PR0533 may have utility in the treatmnt of Usher Syndrome or Atrophia areata.

As menfioned previously, fibroblast growth fators can act upon cells in both a mitogenic and non-itogenic manner. Thee factors are mitogenic for a wide variety of nornmal diploid miesoderm-derived and neural crest-derived cells, inducing grnimlosa cells, adrenal cortical cells, chrondrocytes. myoblasts. cornea! and vascular endothelial cells (bovine or human), vascular smooth muscle cells. lem., retina and prostatic epithelial cells, oligodendrocytes, astrocytes, chrondocytes, myoblasts and osteoblasts.

Non-mitogenic actions of fibroblast growth factors include promotion of cell migration into a wound area (chemoaxis). initiation of new blood vessel fornuilation (aggnesis), modulation of nerve regeneration and survival (neurotrophism). modulation of endocrine functions, and stiulation Or suppresion of specific cellular protein expression. extracellular matrix production and cell survival. Baird, A. Boblen, Handbook of Ep. Phnnacol.

369-418 (199). hese properties provide a basis for using fibroblast growth factors In therapeutic approaches to accelerate wound healing, nerve repair, collateral blood ve-ssel formation, and die lik. For example. fibroblast growth factors, have: been suggested to minimize rnyocardium damage in hert disease and surgery (U.S.P.

4,378.437).

Since the PR0245 polypeptide and nuclec acid encoding it possess seqence homology to a tratismcmbranec protein tyrosine kInase protein and its encoding nucleic: acid, probes based upon the PRO245 nucleotide sequence may be employed to identify other novel transmembrane tyrosine kinase proteins. Soluble forms of the PR0245 polypeptide may be employod as antagonists of membre bound PR0245 activity both in if and in vv. PR0245 polypeptides may be employed in screening assays designed to identify agonists or antagonists of the native PR0245 polypeptide, wherein such assays ay take the form of any conventional cell-type or biochemical binding assay.

Moreover, the PR0245 polypeptide may serve as a molecular marker for the tissues in which the polypeptide is specifically expressed.

PR0220, PR0221 and PR0227 all have leucine rich repeats. Additionally, PROM2 and PR0221 have homology to SLIT and leucine rich repeat protein. Therefore, these proteins are useful in assays described in the literature, supr, wherein the SLIT and leucine rich repeat protein are used. Regarding the Sll' protein, PR0227 ca eue in an assay to determine the affect of PR0227 on neurodegenerative disease. Aditionally; PR0227 has homology to human glycoprotein V. In the case of PR0227. this polypeptide is used in an assay to determrine its affect on bleeding, cloning, tissue repair and scarring.

The PR0266 polypeptide can be used in assays to determine if it has a role in neurodegenerative diseases or their reversal.

PR0269 polypeptides and portions thereof which effect the activity of thrombin may also be useful for in w therapeutic purposes, as well as for various in vWir applications. In addition, PR0269 polypeptides and portions thereof may have therapeutic use as an antidiroinbotic agent with reduced risk for hemorrhage as compared with heparin. Peptides having homology to thrombomodulin are particularly desirable.

PR0287 polypeptides and portions thereof which effect the activity of bone morphogenic protein "BMI/procollagen C-proteinase (PCP) may also be useful for in vivo therapeutic purposes, as well as for various in vitro applications. In addition, PRO287 polypeptides and portions thereof may have therapeutic applications in wound healing and tissue repair. Pcptides having homology to procollagen C-proteinase enhancer protein and its precursor may also be used to induce bone and/or cartilage formation and are therefore of particular interest to the scientific and medical communities.

Therapeutic indications for PRO214 polypeptides include disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions enterocolitis.

Zollinger-Elison syndrome, gastrointestinal ulceration and congenital microvillus atrophy), skin diseases associated with abnormal keratinocyte differentiation (e.g. psoriasis, epithelial cancers such as lung squamous cell carcinoma.

cpidermoid carcinoma of the vulva and gliomas.

Studies on the generation and analysis of mice deficient in members of the TGF- superfamily are reported in Mazuk, Trends in Endocrinol. and Metabol. 6: 120-127 (1995).

The PR0317 polypeptide, as well as PR0317-specific antibodies, inhibitors, agonists, receptors, or their analogs, herein are useful in treating PRO317-associated disorders. Hence, for example, they may be employed in modulating endometrial bleeding angiogenesis, and may also have an effect on kidney tissue. Endometrial bleeding can occur in gynecological diseases such as endometrial cancer as abnormal bleeding. Thus, the compositions herein I15 may find use in diagnosing and treating abnormal bleeding conditions in the endometrium, as by reducing or ^eliminating the need for a hysterectomy. The molecules herein may also find use in angiogenesis applications such as anti-tumor indications for which the antibody against vascular endothelial growth factor is used, or, conversely, ischemic indications for which vascular endothclial growth factor is employed.

Bioactive compositions comprising PR0317 or agonists or antagonists thereof may be administered in a suitable therapeutic dose dectrmined by any of several methodologies including clinical studies on mammalian species to determine maximal tolerable dose and on normal human subjects to determine safe dose. Additionally, the bioactive agent may be complcxed with a variety of well established compounds or compositions which enhance stability or pharmacological properties such as half-life. It is contemplated that the therapeutic, bioactive composition may be delivered by intravenous infusion into the bloodstream or any other effective means which could be used for 25 treating problems of the kidney, uterus, endomtrium, blood vessels, or related tissue, in the heart or genital tract.

Dosages and administration of PRO317. PR0317 agonist, or PR0317 antagonist in a pharmaceutical composition may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics.

See. for example, Mordeni and Rescigno, Pharmaceutical Research. 2:17-25 (1992); Morenti a al., Pharaccutical Research. :1351-1359 (1991); and Mordenti and Chappell, "The use of interspecies scaling in toxicokinetics in Toxicokinetics and New Drul Develonment. Yacobi a al. (cds) (Pergamon Press: NY, 1989), pp. 42-96. An effective amount of PR0317, PR0317 agonist, or PR0317 antagonist to be employed therapeutically will depend, for example, upon the therapeutic objectives, the mute of administration, and the condition of the mammal.

Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 10 ng/kg to up to 100 mg/kg of Id mannmal's body weight or more per day, preferably about 1 pg/kglday to 10 mg/kg/day. Typically, the clinician will administer PRO317. PR0317 agonist, or PR0317 antagonist, until a dosage is reached that achieves the desired effect for treatmen of the above mentioned disorders.

PR0317 or an PR0317 agonist or PR0317 antagonist may be administered alone or in combination with another to achieve the desired pharmacological effect. PR0317 itself, or agonists or antagonists of PR0317 can provide different effects when administered therapeutically. Such compounds for treatment will be formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8. more preferably 6 to 8, although the pH may vary according to the characteristics of the PRO317, agoist, or antagonist being formulated and the condition to be treated. Characteristics of the treatment compounds include solubility of the molecule, half-life, and antigenicityrimmunogenicity; these and other characteristics may aid in defining an effective carrier.

PR0317 or PR0317 agonists or PR0317 antagonists may be delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol, transdermal patch and bandage; injectable, Intravenous, and lavage formulations; and orally administered liquids and pills, particularly formulated to resist stomach acid and enzymes. The particular formulation, exact dosage, and route of administration will be determined by the attending physician and will vary according to each specific situation.

Such determinations of administration are made by considering multiple variables such as the condition to 15 be treated, the type of mammal to be treated, the compound to be administered, and the pharmacokirntic profile of the particular treatment compound. Additional factors which may be taken into account include disease state (e.g.

severity) of the patient, age, weight, gender, diet, time of administration, drug combination, reaction sensitivities, and tolerance/response to therapy. Long-acting treatment compound formulations (such as liposomally encapsulated PR0317 or PEGylated PR0317 or PR0317 polymeric microspheres, such as polylactic acid-based microspheres) might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular treatment compound.

Normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 g/kg/day to 10 mg/kg/day, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos.

4,657,760; 5.206344; or 5.225,212. It is anticipated that different formulations will be effective for different treatmet compounds and different disorders, that administration targeting the uterus, for example, may necessitate Sdelivery in a manner different from that to another organ or tissue, such as cardiac tissue.

SWhere sustained-release administration of PR0317 is desired in a formulation with release characteristics *suitable for the treatment of any disease or disorder requiring administration of PRO317, microencapsulation of PR0317 is contemplated. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2, and MN rgpl20. Johnson er at., Nt.aM 2:795-799 (1996); Yasuda. Biomcd. Ther., 22:1221-1223 (1993); Hora et Bio/Technoloy. 8: 755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems." in Vaccin Desin: The Submit and Adant Aproach Powell and Newman. eds, (Plenum Press: New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S Pat. No. 5,654,010.

The sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body. Moreover, the dcgradability of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactidc/glycolide polymer," in: M. Chasin and R. Langer (Eds.), Bioderadable Polymers s Drue Delivery Systems (Marcel Dekker: New York. 1990). pp. 1-41.

For example, for a formulation that can provide a dosing of approximately 80 g/kg/day in mammals with a maximum body weight of 85 kg. the largest dosing would be approximately 6.8 mg PRO317 per day. In order to achieve this dosing level, a sustained- release formulation which contains a maximum possible protein loading wlw PRO317) with the lowest possible initial burst is necessary. A continuous (zero-order) release of PR0317 from microparticles for 1-2 weeks is also desirable. In addition, the encapsulated protein to be released should maintain its integrity and stability over the desired release period.

It is contemplated that conditions or diseases of the uterus, endometrial tissue, or other genital tissues or cardiac tissues may prcipitae damage that is treatable with PR0317 or PR0317 agonist where PR0317 expression is reduced in the diseased state; or with antibodies to PR0317 or other PR0317 antagonists where the expression of i. PR0317 is increased in the diseased state. These conditions or diseases may be specifically diagnosed by the probing tests discussed above for physiologic and pathologic problems which affect the function of the organ.

15 The PR0317. PR0317 agonist, or PR0317 antagonist may be administered to a mammal with another biologically active agent, either separately or in the same fomnulation to treat a common indication for which they are appropriate. For example, it is contemplated that PR0317 can be administered together with EBAF-1 for those indications on which they demonstrate the same qualitative biological effects. Alternatively, where they have opposite effects, EBAF-1 may be administered together with an antagonist to PRO317. such as an anti-PRO317 antibody.

Further. PR0317 may be administered together with VEGF for coronary ischemia where such indication is warranted, or with an anti-VEGF for cancer as warranted, or, conversely, an antagonist to PRO317 may be administered with VEGF for coronary ischemia or with anti-VEGF to treat cancer as warranted. These administrations would be in effective amounts for treating such disorders.

:Native PRO301 (SEQ ID NO:119) has a Blast score of 246 and 30% homology at residues 24 to 282 of 25 Fgure 44 with A33 HUMAN, an A33 antigen precursor. A33 antigen precursor, as explained in the Background is a tuaor-specific antigen, and as such, is a recognized marker and therapeutic target for the diagnosis and treatment of colon cancer. The expression of tumor-specific antigens is often associated with the progression of neoplastic tissue disorders. Native PRO301 (SEQ ID NO:119) and A33 HUMAN also show a Blast score of 245 and homology at residues 21 to 282 of Fig. 44 with A33_HUMAN, the variation dependent upon how spaces are inserted into the compared sequcnces. Native PRO301 (SEQ ID NO:119) also has a Blast score of 165 and 29% homology at residues 60 to 255 of Fig. 44 with HS46KDA_1. a human coxsackie and adenovirus receptor protein, also known as cell surface protein HCAR. This region of PRO301 also shows a similar Blast score and homology with HSU90716_1. Expression of such proteins is usually associated with viral infection and therapeutics for the prevention of such infection may be accordingly conceived. As mentioned in the Background, the expression of viral receptors is often associated with neoplastic tumors.

Therapeutic uses for the PRO234 polypeptides of the invention includes treatments associated with leukocyte homing or the interaction between leukocytes and the endothelium during an inflammatory response. Examples include asthma, rheumatoid arthritis, psoriasis and multiple sclerosis.

Since the PR0231 polypcpide and nucleic acid encoding it possess sequence homology to a putative acid phosphatase and its encoding nucleic acid, probes based upon the PR0231 nucleotide sequence may be employed to identify other novel phosphatase proteins. Soluble forms of the PR0231 polypeptd may be employed as antagonists of membrane bound PRO231 activity both in vitro and in vise. PR0231 polypeptdes may be employed in screening assays designed to identify agonists or antagonists of the native PR0231 polypeptide, wherein suchlssays may take the form of any conventional cell-type or biochemical binding assay. Moreover, the PR0231 polypeptide may serve as a molecular marker for the tissues in which the polypcptide is specifically expressed.

PR0229 polypeptides can be fused with peptides of interest to determine whether the fusion peptide has an increased half-life over the pcptide of interest. The PR0229 polypeptides can be used accordingly to increase the half-life ofpolypeptides of interest. Portions of PR0229 which cause the increase in half-life are an embodiment of the invention herein.

PRO238 can be used in assays which measure its ability to reduce substrates, including oxygen and Aceyl- CoA, and particularly, measure PRO238's ability to produce oxygen free radicals. This is done by using assays which have been previously described. PR0238 can further be used to assay for candidates which block, reduce 15 or revere it reducing abilities. This is done by performing side by side assays where candidates are added in one assay having PRO238 and a substrate to reduce, and not added in another assay, being the same but for the lack of the presence of the candidate.

PRO233 polypcptides and portions thereof which have homology to reductase may also be useful for in vv therapeutic purposes, as well as for various other applications. The identification of novel reductase proteins and related molecules may be relevant to a number of human disorders such as inflammatory disease, organ failure, atherosclerosis, cardiac injury, infertility, birth defects, premature aging, AIDS, cancer, diabetic complications and mutations in general. Given that oxygen free radicals and antioxidants appear to play important roles in a number of disease processes, the identification of new reductase proteins and reductase-like molecules is of special importance in that such proteins may serve as potential therapeutics for a variety of different human disorders. Such polypeptides may also play important roles in biotechnological and medical research, as well as various industrial applications.

As a result. there is particular scientific and medical interest in new molecules, such as PR0233.

The PR0223 polypcptides of the present invention which exhibit serine carboxypeptidease activity may be *oc* employed in viv for therapeutic purposes as well as for in vitro purposes. Those of ordinary skill in the art will well Sknow how to employ PR0223 polypeptides for such uses.

PR0235 polypeptides and portions thereof which may be involved in cell adhesion are also useful for in vive therapeutic purposes, as well as for various in vitro applications. In addition, PR0235 polypeptides and portions thereof mayhave therapeutic applications in disease states which involve cell adhesion. Given the physiological importance of cll adhesion mechanisms in vivo, efforts are currently being under taken to identify new. native proteins which are involved in cell adhesion. Therefore, peptidcs having homology to plexin are of particular interest to the scientific and medical communities.

Because the PR0236 and PR0262 polypeptides disclosed herein are homologous to various known 3galactosidase proteins, the PR0236 and PR0262 polypeptides disclosed hercin will find use in conjugates of monoclonal antibodies and the polypcptide for specific killing of tumor cells by generation of active drug from a galactosylaed prodrug the genration of -fluorouridinc from the prodrug P-D-galactosyl-5-fluorouridine). The PR0236 and PR0262 polypcptides disclosed herein may also find various uses both in vivo and in vitro, wherein those uses will be similar or identical to uses for which P-galactosidase proteins are now employed. Those of ordinary skill in the art will well know how to employ PR0236 and PR0262 polypeptides for such uses.

PR0239 polypeptides and portions thereof which have homology to densin may also be useful for in vivo therapeutic purposes, as wll as for various in vtro applications. In addition. PR0239 polypeptides and portions thereof may have therapeutic applications in disease states which involve synaptic mechanisms, regeneration or cell adhesion.

Given the physiological importance of synaptic processes, regeneration and cell adhesion mechanisms in vio, efforts are currently being under taken to identify new, native proteins which are involved in synaptic machinery and cell adhesion. Therefore, peptides having homology to dcnsin are of particular interest to the scientific and medical communities.

The PR0260 polypeptides described herein can be used in assays to determine their relation to fucosidasc.

S**s In particular, the PRO260 polypeptides can be used in assays in determining their ability to remove fucose or other sugar residues from protcoglycans. The PRO260 polypcptides can be assayed to determine if they have any 15 functional or locational similarities as fucosidase. The PRO260 polypeptides can then be used to regulate the systems in which they are integral.

*too PR0263 can be used in assays wherein CD44 antigen is generally used to determine PR0263 activity relative to that of CD44. The results can be used accordingly.

PRO270 polypcptides and portions thereof which effect reduction-oxidation (redox) state may also be useful for in vim therapeutic purposes, as well as for various in vitro applications. More specifically, PRO270 polypeptides may affect the expression of a large variety of genes thought to be involved in the pathogenesis of AIDS. cancer, atherosclerosis, diabetic complications and in pathological conditions involving oxidative stress such as stroke and inflammation. In addition, PR0270 polypeptides and portions thereof may affect the expression of a genes which have arole in apoptosis. Therefore, peptides having homology to thioredoxin are particularly desirable to the scientific and 25 medical communities.

PR0272 polypeptides and portions thereofwhich possess the ability to bind calcium may also have numerous in vivo therapeutic uses, as well as various in vitro applications. Therefore, peptides having homology to reticulocalbin are particularly desirable. Those with ordinary skill in the art will know how to employ PR0272 polypeptides and **portions thereof for such purposes.

PR0294 polypeptides and portions thereof which have homology to collagen may also be useful for in vivo therapeutic purposes, as well as for various other applications. The identification of novel collagens and collage-like molecules may have relevance to a number of human disorders. Thus, the identification of new collagens and collage-like molecules is of special importance in that such proteins may serve as potential therapeutics for a variety of different human disorders. Such polypeptides may also play important roles in biotechnological and medical research as well as various industrial applications. Given the large number of uses for collagen, there is substantial interest in polypeptides with homology to the collagen molecule.

107 PRO295 polypeptides and portions thereof which have homology to integrin may also be useful for in vivo therapeutic purposes, as well as for various other application. The identification of novel integrins and integrin-like molecules may have relevance to a number of human disorders such as modulating the binding or activity of cells of the immune system.

Thus, the identification of new integrins and integrin-like'miolecules is of special inportance in that such proteins may serve as potential therapeutics for a variety of different human disorders. Such polypeptides may also play important roles in biotechnological and medical research as well as various industrial applications. As a result, there is particular scientific and medical interest in new molecules, such as PRO295.

As the PRO293 polypeptide is clearly a leucine rich repeat polypeptide hanologue, the peptide can be used in all applications that the known NLRR-1 and NLRR-2 polypeptides are used. The activity can be campared between these peptides and thus applied accordingly.

The PRO247 polypeptides described herein can be used in assays in which densin is used to determine the activity of PR0247 relative to densin or these other proteins. The results can be used accordingly in diagnostics and/or therapeutic applications with PR0247.

20 PR0302, PRO303, PRO304, PRO307 and PRO343 polypeptides of the present invention which possess protease activity may be enployed both in ~vivo for therapeutic purposes and in vitro. Those of ordinary skill in the art will well know how to enploy the PR0302, PRO303, PRO304, PRO307 and PRO343 polypeptides of the present invention for such purposes.

PRO328 polypeptides and portions thereof which have homology to GLIP and CRISP may also be useful for in vivo therapeutic purposes, as well as for various other applications. The identification of novel GLIP and CRISP-like molecules may have relevance to a nutmer of human disorders which involve transcriptional regulation or are over expressed in human tumors. Thus, the identification of new GLIP and CRISP-like molecules is of special inportance in that such proteins may serve as potential therapeutics for a variety of different human disorders. Such polypeptides ay also play inportant roles in biotechnological and medical research as well as in various industrial applications. As a result, there is particular scientific and medical interest in new molecules, such as PR0328.

Uses for PRO335, PRO331 or PR0326 including uses in competitive assays with LIG-1, ALS and decorin to determine their relative activities.

The results can be used accordingly. PR0335, PRO331 or PRO326 can also be 107a used in assays where LIG-I would be used to determine if the same effects are incurred.

PRO332 contains GAG repeat (GKEK) at amino acid positions 625- 628 in Fig. 108 (SEQ ID ND:309). Slippage in such repeats can be associated with human disease. Accordingly, PR0332 can use useful for the treatment of such disease conditions by' gene therapy, i e. b~y introduction of a gene containing the correct GKEK sequence ot if other uses of PR0334 include use in assays in which fibrillin or fihilin would be used to determine the relative activity of PR0334 to fibrillin or fibulin. In particular, PR0334 can be used in assays which require the mechanisms inparted by epidenral growth factor repeats.

Native PR0O346 (SE2 ID NO~:319) has a Blast score of 230, corresponding to 27% hcralogy between amino acid residues 21 to 343 with residues 35 to 1040 CXG46jUMAN, a carcinoeitbryonic antigen cgm6 precursor.

This harrology region include nearly all but 2 N-terminal extracellular dcmain residues, including an inmnuogloblin superfamily hcnuology at residues 148 to 339 of PRO346 in addition to several tas~i~ae residues (340.343). Carcinoembryonic Anige precuror, as explained in the Background is a tumor-spocific antigen.

and as such, is a recogni zed marker and therapeutic target for the diagnosis and treatment of colon cancer. The expression of tumor-specific antigens is often associated with the progrssion of neoplastic timse disorders. Native PR0346 (SEQ MD N0320 and P -W06874. a human Carcin~embriic antigen CEA-d have a Blast score of 224 and homology of 28% between residues 2 to 343 and 67 to 342, respectively. This homology- includes the entire extracellujar domain residues of native PR0346. minus die initiator methionine (residues 2 to 18) aik well as several tranasmembrane residues (340-343).

PRO268 polypeptides which have protein disulfide isomerase activity will be useful for many applications where protein disulfide isomerase activity is desirable inciding, for example, for use in promoting proper disulfide bond formation in aucombinantly produced proteins so as to increase the yield of correctly folded protein. Those of ordinary skill in dhe art will readily know how to employ such PR0268 polypeptides for such purposes.

PR0330 polypcptides of the present invention which possess biological activity related to that of the prolyl 4-hydroxylase alpha subunit protein may be employed both in vivo for therapeutic purposes and in vitro. Those of ordinary skill in the art will well know how to employ the PR0330 polypeptides of the present invention for such purposes.

Anti-PRO Polype~tide Antibodies The present invention further provides anti-PRO polypeptide antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific. and heteroconjugate antibodies.

*:20 A. Polvelonal Antibodies Th anti-PRO polypeptde antibodies may comprise polyckxial antibodies. Methods of preparing polyclonal antibodies are known to die skilled artisa. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the maimmal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the PRO polypeptide or a fusion protein thereof. It mnay be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins mnclude but ame not limited to keyhole limpet hemocyanin. serum albumin, bovine thyroglobulin. and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trebalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

B. Monoelonal Antibodie The anti-PRO polypptde antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybtidoma methods, such as those described by Kohler and Milstein, Nature, =495 (1975).

In a hybridoma. method, a mouse, hamster, or other appropiate host animal, is typically immunized with an inmmizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes mnay be imnmunized in vitro.

The immunizing agent will typically include the PRO polypeptide of interest or a fusion protein thereof.

Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells arc used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell lin using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding.

Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly mycloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.

For example, if the parental cells lack the enzyme hypoxanthin guanine phosphoribosyl transferase (HGPRT or HPRT), the culnure medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of Santibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, 15 Maryland. Human myeloma and mouse-laumn heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor. J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York. (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the PRO polypeptide of interest. Preferably, the binding specificity of 20 monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro Sbinding assay, such as radiaina ay (RIA) or enzyme-linked immuoabsorbent assay (EUSA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 10Z:220 (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, suDn]. Suitable culture media for this purpose include, for example, Dulbcco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in US. Patent No. 4,816.567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which arc thn tansfected into host cells such as simian COS cells. Chinese hamster ovary (CHO) cells, or mycloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the necombinam host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences Patent No. 4.816,567; Morrison ct al., augal or by covalently joining to the immnoglobulin coding sequence all or pan of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigencombining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is tuncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods ar also suitable for preparing monovalen antibodies. Digestion of antibodies to produce S* fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

C. Humanized Antibodies 15 The anti-PRO polypeptde antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab. Fab', or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region 20 (CDR) of the recipient ar replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances. Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially al of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region typically that of a human immunoglobulin [Jones e al., Nature, 21: 522-525 (1986); Ricchmann et al., Naure. 32:323-329 (1988); and Presta. Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acd residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.

Humanization can be essentially performed following the method of Winter and co-workers [Jones e al., Nature, 321: 522-525 (1986); Riechmann a Nature, 32:323-327 (1988); Vcrhocycn ec al.. Science. 232:1534-1536 (1988)].

by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized' antibodies are chimeric antibodies Patent No. 4,816,567). wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice. humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues art substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various technques known in the art. including phage display libraries [Hoogenhoomaund Winter, J. Mo!. Biol,m2:381 (1991); Marks et al.. J. Mo!. BioL, 222:591 (1991)]. The techniques of Cote et al. and Boerner a al. are also available for dhe preparation of human monoclonal antibodies (cote et aL, Monodonal Amgbodier and Cancer 7thempqy. Alan R. Lism, p. 77(1985) and Boerner er al.. J. ImmunoL., 1417_W:86-95 (1991)].

D. Bisneific Antibodies Bispecific antibodies are monoclonal. preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificies is for the PRO polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.

Metiodis for nmaking bispecific antibodies ame known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expresion of two immunoglobulin heavy-chainfligt-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello. Nature, 2-0:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridotnas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has dhe correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 199, and in Traunecker et EMBO ]Q:3655-3659 (1991).

Aiuibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Thr fusion preferably is with an unmunoglobulin heavy-chain constant domain, comprising at least part of the hinge CH2, andi C113 regions. It is preferred to have the first heavychain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.

encoding the immunoglobulin heavy-chain fusions and., if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see. for example, Suresh et al.. Metody in EnzMwooy, 121:210 (1986).

E. Heteroconiugate Antbodies Heteroconjugate antibodies are also within dhe scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells Patent No. 4,676.980], and for treatmnent of HrW infection (WO 91/)0360; WO 92120073; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known metlxds in synthetic protein chemistry. including those involving crosslinlting agents. For example, immunotoxins may be constructed using a disulfide ec~hange reaction or by forming a thioetber bond. Examples of suitable reagents for ti purpose include imnothiolate and methyl-4-mercaptobutyrinidate and those disclosed, for example, in U.S.

Patent No. 4,676,980.

56. Uses for Anti-Pro Polvoetide Antibodies The anti-PRO polypeptide antibodies of the invention have various utilities. For example, anti-PRO polypeptide antibodies may be used in diagnostic assays for a PRO polypeptide, detecting its expression in specific cells, tissues. or scrum. Various diagnostic assay techniques known in the an may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases (Zola Monoclonal Antibodies: A Manml of Techina, CRC Press, Inc.

(1987) pp. 147-158]. The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing. either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as S, ot I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodanine, or luciferin, or an enzyme, such as alkaline phosphatase.

beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the artibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature. 144:945 (1962); David et al., Biochemisry, 13:1014 (1974); Pain et aL, J. Immunol. Meth.. 40:219 (1981); and Nygren. J.

Hlstochem. and Cytochem., 3Q:407 (1982).

Anti-PRO polypeptide antibodies also arc useful for the affinity purification of PRO polypeptide from recombinant cell culture or natural sources. In this process, the antibodies against the PRO polypeptide are immobilized on a suitable support, such a Sephadcx resin or filter paper, using methods well known in the art. The Simmobilized antibody then is contacted with a sample containing the PRO polypeptide to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the PRO polypeptide, which is bound to the immobilized antibody. Finally, the support is washed with another suitable 20 solvent that will release the PRO polypeptide from the antibody.

With regard to PRO211 and PRO217, therapeutic indications include disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions enterocolitis. Zollinger-Ellison syndrome, gastrointestinl ulceration and congenital microvillus atrophy), skin diseases associated with abnormal keratinocyte differentiation psoriasis, epithelial cancers such as lung squamous cell carcinoma, cpidermoid carcinoma of the vulva and gliomas.

With regard to anti-PRO187 antibodies. FGF-8 has been implicated in cellular differentiation and embryogenesis, including the patterning which appears during limb formation. FGF-8 and the PRO187 molecules of the invention therefore are likely to have potent effects on cell growth and development. Diseases which relate to cellular growth and differentiation are therefore suitable targets for therapeutics based on functionality similar to FGF-8. For example, diseases related to growth or survival of nerve cells including Parkinson's disease, Alzheimer's disease. ALS, neuropathies. Additionally, disease related to uncontrolled cell growth, cancer, would also be expected therapeutic targets.

Native PR0533 is a 216 amino acid polypeptide of which residues 1-22 are the signal sequence. Residues 3 to 216 have a Blast score of 509, corresponding to 53% homology to fibroblast growth factor. At the nucleotide level, DNA47412, the EST from which PCR oligos were generated to isolate the full length DNA49435-1219, has been observed to map to llplS. Sequence homology to the 1 lp15 locus would indicate that PR0533 may have utility in the treatment of Usher Syndrome or Atrophia areata.

As mentioned previously, fibroblast growth factors can act upon cells in both a mitogenic and non-mitogenic manner. These factors are mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, inducing gramulosa cells, adrenal cortical cells, chrondrocytes, myoblasts, corneal and vascular endothelial cells (bovine or human), vascular smooth muscle cells, lens, retina and prostatic epithelial cells, oligodendrocytes, astrocytes, chrondocytes, myoblasts and ostcoblasts. Antibodies to these factors can be generated to modulate such effects.

Non-mitogenic actions of fibroblast growth factors include promotion of cell migration into a wound area (c emotaxis). initiation of new blood vessel formulation (angiogenesis), modulation of nerve regeneration and survival (ncurotrophism), modulation of endocrine functions, and stimulation or suppression of specific cellular protein expression, exrraoellular matrix production and cell survival. Baird, A. Bohlen, Handbook of Exp. Phrmacol.

369.418 (1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healing, nerve repair, collateral blood vessel formation, and the like. For example, fibroblast Sgrowth factors, have been suggested to minimize myocardium damage in heart disease and surgery (U.S.P.

-4,378,437). Antibodies to these factors can be generated to modulate such effects.

Therapeutic indications for PR0214 polypeptides include disorders associated with the preservation and 15 maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions enterocolitis, Zollinger-Elison syndrome, gastrointestinal ulceration and congenital microvillus atrophy), skin diseases associated with abnormal keratinocyte differentiation psoriasis, epithelial cancers such as lung squamous cell carcinoma, epidermoid carcinoma of the vulva and gliomas.

Anti-PR0317 antibodies find use in anti-tumor indications if they are angiostatic, or in coronary ischemic indications if they are angiogenic.

Native PRO301 (SEQ ID NO:119) has a Blast score of 246 and 30% homology at residues 24 to 282 of Fig.

44 with A33_HUMAN, an A33 antigen precursor. A33 antigen precursor, as explained in the Background is a tumor-specific antigen, and as such. is a recognized marker and therapeutic target for the diagnosis and treatment of Scolon cancer. The expression of tumor-specific antigens is often associated with the progression of neoplastic tissue disorders. Native PRO301 (SEQ ID NO:119) and A33_HUMAN also show a Blast score of 245 and 30% homology at residues 21 to 282 of Fig. 44 with A33_HUMAN, the variation dependent upon how spaces are inserted into the compared sequences. Native PRO301 (SEQ ID NO:119) also has a Blast score of 165 and 29% homology at residues to 255 of Fig. 44 with HS46KDA 1, a human coxsackie and adenovirus receptor protein, also known as cell surface protein HCAR. This region of PRO301 also shows a similar Blast score and homology with HSU90716_1.

Expression of such proteins is usually associated with viral infection and therapeutics for the prevention of such infection may be accordingly conceived. Accordingly, antibodies to the above identified antigens and receptors have therapeutic potential as diagnostic and treatment techniques.

Therapeutic uses for the PRO234 polypeptides of the invention includes treatments associated with leukocyte homing or the interaction between leukocytes and the endothelium during an inflammatory response. Examples include asthma, rheumatoid arthritis, psoriasis and multiple sclerosis.

Cancer-associated or specific antigens permit the creation of tumor or cancer specific monoclonal antibodies (mAbs) which are specific to such tumor antigens. Such mAbs, which can distinguish between normal and cancerous cells art useful in dhe diagnosis, prognosis and treatment of the disease.

Cancer specific monoclonal antibodies (niAbs) which are specific to tumor antigens. Such nlAbs. which can distinguish between normal and cancerous cells are useful in the diagnosis, prognosis and twreanent of the disease. Particular antigens are known to be associated with meoplastic diseases, such as colorectal and breast cancer.

Since colon cancer is a widespread disease. early diagnosis and treatment is an important medical goal. Diagnosis and treatment of cancer can be implemented using monoclonal antibodies (mAbs) specific therefore having fluorescent, nuclear magnetic or radioactive tags. Radioactive genes, toxins and/or drug tagged mAbs can be used for treatment in situ with minimlal patient description.

Thie following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

All patent and literature references cited in die present specification are hereby incorporated by reference in their entirety.

Commercially available reagents referred to in the examples were used according to manufacture's instructions unless otherwise indlicated. The source of those cells identified in the following examples, and throughout the specification. by ATCC accession numbers is the An~rican Type Culture Collection, Rockville, Maryland.

EXAMLF,: Extracellular Domain Homology Screeimi to ldenitify, Novel Polypeprdes and eDNA Encoding 20 The extraccelar domain (ECD) sequences (including the secretion signal sequence. if any) from about 950 known secreted proteins from die Swiss-Prot public database were used to search EST databases. The EST databases included public databases Dayboff. Getillank), and proprietary databases LIFESEQI., Incytc Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Ailschul, anti Gish, Methods in EUzMology 26: 460-80 (1996); http://blast.wust edu/blast/README.html) as a comnparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons with a Blast score of 70 (or in souie cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap' (Phil Green. University of Washington, Seattle, WA; O(rup: /tbozomaamnbt.washntoiLeduphrapdocsphrapbl).

Using this extracellular domain homology screen, consensus DNA sequences were assembled relative to the other identified EST sequence. In addition, the consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.

Based upon the consensus sequences obtained as described above, oligonucleotides were then synthesized and! used to identify by PCR a eDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide. Forward and reverse PCR primers generally range fronm 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about i-1 .Skbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PC R amplification, as per Ausubel et al., Curren Protocol-, in Molecular BiolnZX, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligoraicleodde and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commerciafly available reagents such as those from Invitrogen. San Diego, CA. The cDNA was primed with oligo tiT containing a Nodi site, linked with blunt to Sail hemikinased adaptors, cleaved with Nod, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as VRKB or pRKD; pRKSB is a precursor of pRKSD that does not contain the Sf1l site; see. Holmes et al., Science. LU:1278-1280 (1991)) in the unique Xbol and Nod sites.

EXAMPE&2: Isolation of cDNA Clones Fncoding PRO2I I and PRO217 Consensus DNA sequences were assembled as described in Example 1 above an were designated as DNA2873 and DNA28760. respectively. Based on these consensus sequences, oligonucleotides w ere synthesized and used to identif by PCR a cDNA library that contained the sequences of interest and for use as probes to isolate a clone of the fuletngh coding sequence for the PRO211 and PR0217 polypeptides. The libraries used to isolate DNA32292-1131 and DNA33094-1131 were fetal lung libraries.

cDNA clones were sequenced in their entirety. The entire nucleotide sequences of PRO211 (DNA32292- 1131; UNQISS) and PR0217 (UNQI9l; DNA33094-1 13 1) are shown in Figure 1 (SEQ ID NO; 1) and Figure 3 (SEQ HD NO:3), respectively. The predicted polypeptides are 353 and 379 amino acid in length, respectively, with respective molecular weights of approximately 38,190 and 41,520 daltons.

The oligonucleotide sequences used in the above procedures wcre the following- 2 9 73 O.p (OUJ 516) (SEQ ID NO:S) 5'-AGGGAGCACGGACAGTGTGCAGATGTGGACGAGTGCCACAGCA.3' 28730.f (OLI 517) (SEQ ID NO:6) 5,-AGAGTGTATCTCTGGCcTAcGC-3, 29730.r (OU 5.18) (SEQ ID NO:7) 5'-TAAGTCCGGCACATTACAGGTC--3* 2 876 0.p (OU 617) (SEQ ID NO: 9) 5.-CCCACGATGTATGAATGGTGGACTTTGTGTGACTCCrGGn-rCTGCATC-3' 297601f (OLI 618) (SEQ ID NO:9) 5'-AAAGACGCATCTGCGAGTGTCC-3' 28760.r (OLI 619) (SEQ ID 5:'-TGCTGATrTCACACrGCTCTCCC-3' EXAMPLE 3: Isolation of cDNA Clones Fiicodi Hmnan PRO23 A consensus DNA sequence was assembled relative to the other identified EiST sequences as described in Example I above, wherein the consensus sequence is designated herein as DNA30857. An EST proprietary to Genentech was employed in the consensus assembly. The EST is designated as DNA20088 and has the nucleotide sequence shown in Figur 7 (SEQ ID ?40:13).

Based on the DNA30857 consensus sequcoce, oligomatleotides were synthesized to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence foqr PROM3.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR prme S-TTCGAGGCCTCTGAGAAGTGGCCC-3' (SEQ ID NO:14) reverse PCR oRimer S'-GGCGGTATCFCrCTGGCCTcCCC.3* (SEQ ID NO: Additionally, a synthetic oigonucleotide hybridization probe was constructed from the consensus DNA30857 sequence which had the following nucleotide sequence hitbridization probe 5S.TFCTCCACAGCAGCTGTGGCATCCGATCGTATCCArCCAJ(J.3 (SEQ ID NO:46) In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amnplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0230 gente using the probe oligonucleotidc and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from humnan fetal lung tissue. DNAeqening the clones isolated as described above gave the full-length DNA sequence for PR0230 (herein designated as UNQ204 (DNA33223-1136)) and the derived protein sequence for PR0230.

The- entire nucleotide sequece of UNQ204 (DNA33223-l 136) is shown in Figure 5 (SEQ ID N40:1).

.Clone UNQ204 (DNA33m2-I 136) contains a single open reading frme with an apparent translational initiation site at nucleotide positions 100-103 and ending at the stop codon at nuclcotidc positions 1501-1503 (Figure SEQ ID NO:lt1). The predicted polypeptide precursor is 467 amino acids long (Figure 6).

.:EAMPLE 4: Isolation of cDNA Clones Encoding Human PRO232 A consensus DNA sequeoce was assembled relative to the other identified EST sequences as described in Example 1 above, wherein the consensus sequenice is designated herein as DNA30935. Based on the DNA30935 consensus sequentce, oligonucleotides were synthesized to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PR0232.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer 5'-TGCTGGCTACTCCGCAAAGCCC-3' (SEQ ID 140:19) reverse PCR primer 5'-TGCACAAGTCGGTGTCACAGCACG-3' (SEQ ID Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DN4A30935 sequence which had the folowing nuclcotide sequence 5S-AGCAACGAGGACTGCCTGCAGGTGGAGACTCACCCAGCTGGG-3. (SEQ ID N0:21) In order to sceenm several libraries for a source of a full-lenigth clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0232 gene using the probe oligonucleotidc and one of the PCR primers.

RNA (or construction of the cDNA libraries was isolated from human fe~tal kidney tissue.

DNA sequencing oft clones isolated as described above gave dhe full-length DNA sequence for PR0232 therein designated as UNQ206 (DNA34435-1140)] and the derived protein sequence for PROM3.

The entire nucleotide sequence of UNQ206 (DNA3443S-1140) is shown. in Figure 8 (SEQ ID NO: 17).

Clone UNq206 (D)NA34435-1 140) contains a single open reading frame with an apparent transatonal initiation site at icleootide positions 17-19 and ending at die stop codon at nucleotide posirions 359-361 (Fig. 9; SEQ ID NO: 17).

The predicted polypeptdec precursor is 114 amino acids long (Fig. Clone UNQ206 (DNA34435-1140) has been deposited with ATCC on September 16. 1997 and is assigned ATCC deposit no. ATCC 209250.

Analysis of the amino acid sequence of the full-length PR0232 suggests that it possesses 35% sequence identity with a stemn cell surface antigen from Gallus gallus.

EXAMPLj Isolation of cDNA Clone Encoding PRO187 A proprietary expressed sequence tag DNA database (UfESEQoh, Incyte Pharmaceuticals, Palo Alto, was searched and ant EST 0843193) was identified which showed homnology to fibroblast growth factor (FGF-8) also known as androgeninduced growth fictor. mRNA was isolated fromn human fetal bang tissue using reagents and 15 protocols from Invitroen. San Diego, CA (Fast Track The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents luvitrogen. San Diego, CA, Life Technologies, Gaithersburg. MD). The cDNA was primed with oligo dT containing a Notd site, linked with blunt to Sall heinikinaised adaptors, cleaved with Notd, sized appropriately by gel electrophoresis, and cloned in a defined orientation into the cloning vector pRK5D using reagents and protocols from LWe Technologies. Gaithersburg, MD 20 (Super Script Plasmid System). The double-stranded cDNA was sized to greater thani 1000 bp and the Sal/NodI linlcered eDNA was cloned into XhoIINocl cleaved vector. pRK5D) is a cloning vector that has an sp6 traniscription initiation site followed by an Sf1 restriction enzyme site preceding the XhoI/Notl cDNA cloning sites.

:Several libraries from various tissue sources were screened by PCR amplification with the following oligonucleotide probes: 1N843193.f (011315).(SEQ ID NO:24) 5CAGTACGTGAGGGACCAGGGCGCCATGA.3 IN843193.r (011 317) (SEQ ID 5S-CCGGTGACCTCACGTCTTGCA-3.

A Positive library was then used to isolate clones encoding the PRO 187 gene using one of the above oigonucleotides and the following oligonucleotide probe: 1N843193.p (011 316) (SEQ ID NO:26) A cDNA clone was sequenced in entirety. The entire nucleootde sequence of PRO0187 (DNA27864-1155) is shown in Figure 10 (SEQ ED NO:22). Clone DNA27864-l1155 contains a single open reading frame with an apparent trarisaional initiation site at ruricotide position 1 (Figure 10; SEQ ID NO:22). The predicted polypeptide precursor is 205 amino acids long. Clone DNA27864-1 155 has been deposited with the ATCC (designation: DNA2786-1 155) and is assigned ATCC deposit no. ATCC 209375.

Based on a BLAST midi PastA sequence alignment analysis (using tbce AUGN computer program) of the fulllength scquence. the PRO197 polypeptide shows 74% amino acid sequence identity (Blast score 310) (timman fibroblast growth factor-8 (androgeri-itduced growth factor).

EXAMPIL. 6. Isolation of cDNA Clones Fneoding PR9265 A consensus DNA sequence was assembled relative to other EST sequences as described in Example 1 above using phrap. This consensus sequence is herein designated DNA33679. Based on the DNA33679 consensus seqtence. oligonucleoudes were synthesized: 1)1to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the ful-length coding sequence for PR0265.

PCR primers (two forward and one reverse) were synthesized: forward PCR timner A- 5'-CGGrCTACCrGTATGGCAACC-3' (SEQ ID NO:29); forwadPCR timner B: SGCAGGACAACCAGATAAACCAC.3* (SEQ ID reverse PCR Mfgwir 5'-ACGCAGATrGiAGAAGGCrGTC.3* (SEQ 11D NO:31) Additionally, a synthetic olionucleotide hybridization probe was constructed from the consensus DNA33679 sequence which had the following nucleotide sequence hybridain grob STrCACGGGCTGCTCTGCCCAGCTGAAGC1YMAGAGCTGCAC- 3 .(SEQ ID NO:32) In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0265 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human a fetal brain library.

DNA sequencing of the clones isolated as described above gave the fu-length DNA sequence for PR0265 herin designated as UNQ232 (DNA36350-1 158) (SEQ ID NO:27) and die derived protein sequence for PROM6.

The entire nucleotide sequence of UNQ232 (DNA36350-1 158) is shown in Figure 12 (SEQ ID NO;27).

Clone UNQ232 (DINA36350-1L58) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 352-354 and ending at the stop codon at positions 2332-2334 (Figure 12). The predicted polypeptide precursor is 660 amino acids long (Figure 13). Clone UNQ232 (DNA36350-1158) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209378.

Analysis of the amino acid sequence of the full-length PR0265 polyetide suggests that portions of it possess significant homology to the fibromodulin and the fibroniodulin precursor. thereby indicating that PRO265 may be a novel member of the leucine rich repeat family. particularly related to fibrontodulin.

EAMPLE 7: Isolation of cQNA Clones Encoding Huma~n PR21 9 A consensus DNA sequence was assembled relative to other EST sequences wing phrap as described in Example 1 above. This consensus sequence is herein designated DNA28729. Based on the DNA29 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the fall-length coding sequence for PR0219.

A Pakr Of PCR primners (forward and reverse) were synthesized: frad C pie 5.GTGACCCTGGTTGTGAATACTCC-3- (SEQ MD reverse PCR primer 5'-ACAGCCATGGTCTATAG-17TGG-3* (SEQ ID NO:36) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28729 sequence which had the following nucleotide sequence byrdztimlrb 3 .(SEQ ID N 0:37) In order to screen several libraries for a source of a full-length clone, DNA from the libraries Was screened by PCR amuplification with the PCR primcr pair identified above. A positive library was then used to isolate clones encoding the PR0219 gene using the probe oigonucleotide and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0219 [herein designated as UNQ193 (DNA32290-1 164)1 (SEQ ID 140:33) and the derived protei sequence for PR0219.

The entire nucleotide sequence of LUNQ193 (DNA32290-1 164) is shown in Figures 14A-B (SEQ ID N0:33).

ClOne UNQ193 (DNA32290-1164) contains a single open reading frame with an apparent translationil initiation site at nucleockle positions 204-206 and ending at the stop codon at nucleotide positions 2949-2951 (Figures 14A-B). The predicted polypeptide precursor is 915 amino acids long (Figure 15). Clone UNQ193 (DNA32290-1164) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209384.

Analysis of the ammno acid sequence of the full-length PR0219 polypepide suggests that portions of it possess significant homology to the mouse and human niatrilin-2 precursor polypeptides.

EXAMPLE 8: Isolation of cDNA Clones Encodinp Hma PRO246 A consensus DNA sequence was assembled relative to oter EiST sequences using phrap as described in ExamplelIabove. This consensus sequence is herein designated DNA30955. Based on the DNA30955 consensus sequence. oigonuclootides were synthesized. 1) to identify by PCR a eDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length, coding sequence for PROM4.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer 5S-AGGGTCTCCAGGAGAAAGACC.3* (SEQ ID ?40:40) reverse PCR Rrimer S'-A1TGTGGGCCTTGCAGACATAUIAC.3- (SEQ ID NO:41) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA3O9SS sequence which had the following riucleotide sequence hbrzatin probe CrCGTC3(SEQ ID NO:42) In order to screen several libraries for a source of a fiul-ength clone. DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then use4 to isolate clones encoding the PR0246 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the CDNA libraries was isolated from human fetal liver tissue. DNAaTgmirg of the clones isolated as described above gave the ful-lngth DNA sequence for PR0246 [herein designated as UNQ220 (DNA35639-1 172)J (SEQ ID NO:39) and the derived protein sequence for PROM4.

The entire nucleotde sequence of UNQ220 (DNA35639-1 172) is shown in Figure 16 (SEQ ID NO:3 8).

Clow UNQ220 (DNA35639-i 172) contains a single open reading framne with an apparent translational initiation site at nucleotide positions 126-128 and ending at the stop codon at mieleotide positions 1296 1298 (Figure 16). The predicted pojypcptdec precursor is 390 amino acids long (Fgur 17). Clone UNQ220 (DNA35639-l 172) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209396.

Analysis of the amino acid sequence of die fulfl-length PR0246 polypeptide suggests that it possess significant homology to the human cell surface protein HCAR, thereby indicating that PR0246 may be a novel cell surface virus receptor.

EAM.ELEP 9: Isolation of edNA Clones Fmendj=n Human PRO228 A consensus DNA sequence was assembled relative to othe EST sequences using phrap as described in Example 1 above. This consenus sequece is herein designated DNA2S7SS. An EST proprietary to Genentech was employed in the consensus assembly. This EiST is shown in Figure 20 (SEQ ID NO:SO) and is herein designated as DNA21951.

Based on the DNA28758 consenus sequence. oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0228.

PCR primers (forward and reverse) were synthesized: forward PCR primer 5'-GGTAATGAGCrCCATI'ACAC.3' (SEQ ID NO:51) .20 forward PCR primer 5*-GGAGTAGAAAGCGCATGG-3' (SEQ ID NO:52) *..forward PCR primer 5*-CACCTGATACCATGAAT(JGCAG-3' (SEQ ID NO:53) reverse PrR primer 5*CGAGCTCGAATAAflC.3' (SEQ ID NO:54) :reverse PCR primer 5'GATCTCCTGAGCTCAGG-.3- (SEQ ED N0:55) reverse PCR mimer 5'-CCTAGTrGAGTGATCCT2GTAAG.3' (SEQ ID NO:56) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28758 sequence which had the folowing nucleotide sequence hybridization Drobe M O:7 In order to scteen several libraries for a source of a ful-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0228 gene using the probe oligonmeleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal Uincy tissue.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0228 [herein designated as UNQ202 (DNA33092-1202)] (SE M NO:48) and the derived protein sequence for PR0228.

The entire nucleotide sequec of UNQ202 (DNA33092-1202) is shown in Figure 18 (SEQ ID NO:48).

Clone UNQ202 (DNX33092-1202) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 24-26 of SEQ ID NO:48 and ending at the stop codon after micleotdec position 2093 of SEQ ID NO:48. The predicted polypeptide precursor is 690 amino acids long (Figure 19). Clone UNQ202 (DNA33092- 1202) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209420.

Analysis of the amino acid sequence of the ful-length PR0228 polypeptidc suggests that portions of it possess significant homology to the secreuin-related proteins CD97 and EMRI as well as the secretin member, latrophilin, thereby indicating that PR0228 may be a new miember of the secretin related proteins.

EXMLE 1: Isoation of cDNA Clone% Encodliw Human PR0533 The EST siquence accession numnber AF007268. a murine fibroblast growth factor (FGF- 15) was used to search various public EST databases Genflank, Dayhoff, etc.). The search was performed using the computer program BLAST or BLAS72 [Altschul et al.. Methods in Enaynolosy, 2k6i:460.480 (1996); http:/bLst st/edu/lblUA DMEIIiA as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. The search resulted in a hit with GcZRBankI EST AA220994, which has been identified as stratagenc NT2 neuronal precursor 937230.

.Base on the Genbank EST AA220994 sequence. ofigonucleotides were synthesized: 1) to identif by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the funl-length coding sequence. Forwa rd and reverse PCR primers may range from 20 to 30 nucleotides (typically about 24), and ane designed to give a PCR product of 100-1000 bp in length. The probe sequences are typically 40-55 bp (typically about 50) in length. In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology. with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonudeotide and one of the PCR prmers.

In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified below. A positive library was then used to isolate clones *encoding the PR0533 gene using the probe oligonrucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal retina. The cDNA libraries used to isolated the cDNA clones were constructed by standard methods using commercially available reagents Invilrogen, San Diego, CA; Clontech, etc.) The cDNA was primed with oligo dT containing a Notd site, linked with blunt to Sail hemilduased adaptors, cleaved with Notd, sized appropriately by gel electrophoresis, and cloned in a defined orientauion into a suitable cloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRK5D that does not contain the Sil site; see, Holmes et al., Science. Mf:278-1280 (199 in the unique XhoI and Nod sites.- A cDNA clone was sequenced in its entirety The ball length mucleotide sequence of PR0533 is shown in Figure 21 (SEQ ID NO:58). Clone DNA49435-1219 contains a single open reading frame with an apparent transilational initiation site at nucleotide positions 459-461 (Figure 21; SEQ ID NO:58). The predicted polypeptide precursor is 216 amino acids long. Clone DNA47412-1219 has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209480.

Based on a BLAST-2 and FastA sequence allgnmem analysis of the full-length sequence, PR0533 shows amino acid sequence identity to fibroblast growth factor The oligonucleotide sequences used in the above procedure wern the following: FCIF1S.forward: 5S-ATCCGCCCAGATGGCTACAATGTrGTA-3' (SEQ ID FGF1S.probe: 5'-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA..3 (SEQ ID NO:6 1); FGF1S.reverse: S'-CCAGTCCGGTGACAAGCCCMAA-3' (SEQ ID NO:62).

EXAMPLEI11: Isolation of eDNA Clones Encodin, Human PR04 A consensus DNA sequence was assembled relative to dhe other identified EST sequences as described in Example 1 above, wherein the consensus sequence is designated herein as DNA3O9S4.

Based on the DNA30954 consensus sequee. ofigomicleotide were synthesized to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PROM4.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer S'-ATCGTrGTGAAGTrAGTGCCCC-3' (SEQ ID S.*reverse PCR primer 5'-ACCTGCGATATCCAACAGAA1TG3- (SEQ ID NO:66) Additionally. a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30954 sequence which had the following nucotide sequence bybriffization orobe 5'-GGAAGAGGATACAGTCACTCTGGAAGTATAGTGGCT'CCAGCAGflCC-3' (SEQ ID NO:67) In order to screen severa libraries for a source of a full-length clone. DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0245 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. DNAsoqxring of the clones isolated as described above gave the full-length DNA sequence for PR0245 (herein designated as UNQ219 (DNA35638-l 141)] and the derived protein sequence for PR0245.

entire nucicotide sequence of UNQ219 (DNA35638-1 141) is shown in Figure 23 (SEQ ID NO:63).

Clone UJNQ219 (DNA35638-1 141) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 99-91 and ending at the stop codon at nucleotide positions I0MS-1027 (Fig. 23; SEQ ID NO:.63). The predicted polypeptide precursor is 312 amino acids long (Fig. 24). Clone UNQ219 (DNA35638-l 141) has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no. ATCC 209265.

Analysis of the amino acid sequence of the full-length PR0245 Suggests that a portion of it possesses 609'% amino acid identity with the humnan c-myb protein and, therefore, may be a new member of the transmrnmbrane protein receptor tyrosine kinase family.

EXAMPLE 12: Isolation of cDNA Clones Encoding Human PR0220, PR0221 and PR0227 PRO220 A consensus DNA sequence was assembled relative to dre other identified EST sequences as described in Example 1 above, wherein t consensus sequence is designated herein as DNA28749. Based on the DNA28749 consensus sequence, oligonucleotides were synthesized to identify by PCR a eDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PR0220.

A pair of PCR primers (forward and reverse) were synthesized: fonr PC rme .'TCACCGGAGCCTA7-JGC-3. (SEQ ID) NO:74) =vrs PC&R '-ATACCAGCTATAACCAGGCTGCG..3. (SEQ ID Additionally, a synthetic oigonucleotde hybridization probe was constructed from the consensus DNA28749 sequence which had the following nucleotide sequence: hybdztinrobe

ATGCTCAAATCTAGAGAATGAGG

3 (SEQ ID NO:76).

In order to screen several libraries for a source of a full-length clone. DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0220 gene using the probe oligonucleotdec and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from ]human fetal lung tisse. DNAseqxn*n of the clones isolated as described above gave the MIl-length DNA sequence for PRO220 (herein designated as UNQ194 (DNA32298-1132) and the derived protein sequence for PR0220.

entire nudeotide sequence of UNQ194 (DNA32298-l 132) is shown in Figure 25 (SEQ ID NO:68).

Clone UNQ194 (DNA32298-l 132) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 480-482 and ending at the stop codon at nucleotide positions 2604-2606 (Figure 25). The predicted polypeptide precursor is 708 amino acids long (Figure 26). Clone UNQ194 (DNA32298-1132) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209257.

Analysis of the amino acid sequence of the funl-length PR0220 shows it has homology to member of the leucine rich repeat protein superiiy, including the leucine rich repeat protein and the neuronal leucine-rich repeat protein 1.

PR-2 A consensus DNA sequence was assembled relative to the other identified EST sequences as described in Example 1 above, wherein the consensus sequence is designated herein as DNA287S6. Based on the DNA28756 consensus sequence. oligonucleotides were synthesized to identify by PCR a cDNA library that contained the of interest and for use as probes to isolate a clone of the fId-length coding sequence for PR0221.

pair of PCR primers (forward and reverse) were synthesized: forward PCR primer 5'-CCATGTGTCTCCTCCTACJAJAG-3' (SEQ ID NO:77) reverse PCR primer 5'-GGGAATAGATGGTTOAA1.IY 3 (SEQ ID NO:78) Additionally. a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28756 sequence which had the following nucleotide sequence: bfdization Rmkb ~G3(SEQ ID NO:79) In order to screen several libraries for a sourceof a fiul-lngth clone, DNA from the libraries was screened by PCR amplification with t PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO221 gene using the probe oligonucleotide and one of the PCR primecrs.

RNA for consauncton of the cDNA libraries was isolated from human fetal lung tissue. DNAseqxrg Of the clones isolated as described above gave the full-length DNA sequence for PR0221 [herein designated as UNQ195 (DNA33089-1 132) and the derived protein sequence for PR0221.

The entire nucleotide sequence of UTNQI95 (DNA33089-1 132) is shown in Figure 27 (SEQ ID NO' Clone UNQ 195 (DNA33089-1132) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 179-181 and ending at the stop codon at nucleotidc positions 956-958 (Figure 27). lhe predicted polypeptidc precursor is 259 amino acids long (Figure 28). PR0221 is believed to have a tlanstnembrane region at amino acids 206-225. Clone U1NQ195 (DNA33089.I 132) has been, deposited with ATCC and is assigned ATCC deposit no. ATCC 209262.

Analysis of dhe amin acid sequence of the full-length PR0221 shows it has homology to memnber of the leucine uich repeat protein superfamily, including the SLIT protein.

*060(C) PRO227 ec A consensus DNA sequence was assembled relative to the other identified EST sequences as described in Example 1 above, wherein the consensus sequence is designated herein as DNA28740. Based on the DNA28740 consensus sequence, oigonucleotides were synthesized to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length eoding sequence for PR0227.

00.0.A pair of PCR primers (forward and reverse) were synthesized: forward PCR nrimei S-AGCAACCGCCTGAAGCTCATCC..3' (SEQ ID reverse PCR prinler 5'-AAGGCGCGGTGAAAGATGTAGACJ.3' (SEQ ID NO:81) 0* :20 Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28740 sequence which had the following nucleotide sequence- GCA3(SEQ ED NO:82).

In order to screen several libraries for a sucof a MIl-length clone, DNA from the libraries was screened &see 25 by PCR amplification with the PCR primer pair identified above. A positive librAry was then used to isolate clones encoding the PRO22 gene using the probe oligonuclodde and one of the PCR primers.

00000RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. DNAsetquarig 0 of the clones isolated as described above gave the full-length DNA sequence for PR0227 [herein designated as UNQ201 (DNA33786-1132) and the derived protein sequence for PR0227.

The entire nucleotide sequence of UNQ201 (DNA33786-I 132) is shown in Figure 29 (SEQ MD NO:72).

Clone UNQ201 (DNA33786-1 132) contains a single open reading frame with an apparent translational initiation site at nucleotitic positions 117-119 and ending at the stop codon at nucleotide positions 1977-1979 (Figure 29). The predicted polypeptide precursor is 620 amino acids long (Figure 30). PR0227 is believed to have a transmemnbrane region. Clone UNQ201 (DNA33786-1 132) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209253.

Analysis of the amino acid sequence of the full-length PR0221 shows it has homology to member of the leucine rich repeat protein superfamily. including the platelet glycoprotein V precursor and the human glycoprotein

V.

EX1IEU Isolataon of cflNA Clones Encoding Humann PRO258 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA28746.

Based on the DNA28746 consensus sequence. oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO2S8.

PCR primers (forward and reverse) were synthesized: ford PCR primne 5'-GCTAGGAATI'CCACAGAAGCCC-3' (SEQ ID reverse PCR Dnrmer 5T-AACCrGGAATGTCACAGCTcrj3- (SEQ 11) NiO.86) :reverse PCR primer 5SCCTAGCACA*GTGACGAGGGACI1TyJ(-3' (SEQ ID NO:87) Additionally, synthetic oligonucleotdec hybridization probes were constructed from the consensus DNA28740 sequence which had the following nucoide sequence: C)GACACTCGC3(SEQ ID NO:88) 3 (SEQ ID NO:89) In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0258 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. DNAequitirg of the clones isolated as described above gave the full-length DNA sequence for PRO2S8 [herein designated as .UNQ225 (DNA35918-1174)J (SEQ ID NO:83) and the derived protein sequence for PR0258.

*25 The entire nucleodide sequence of UNQ225 (DNA3S918-1 174) is shown in Figure 31 (SEQ ID NO:83).

Clonie UJNQ225 (DNA35918-1 174) contains a single open reading frame with an apparent translational initiation site at nuclootidec positions 147-149 of SEQ ID NQ:83 and ending at the stop codon after nucleotide position 1340 of SEQ ID NO:83 (Figure 3 The predicted polypeptide prcusor is 398 amino acids long (Fgur 32). Clone UNQ225 (DNA35918-l 174) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209402.

Analysis of the amino acid sequence of the fuI-length PRO258 polypeptide suggests that portions of it possess significant homology to the CRTAM and the poliovirus receptor and have an Ig domain, thereby indicating that PRO258 is a new member of the Ig superfamily.

EXAMPLE 14: Isolation of cDNA Clones Encoding Human PRO266 An expressed sequence tag database was searched for ESTs having homology to SLIT, resulting in the identification of a single EST sequence designated herein as 173996. Based on the 173996 EST sequence, oligonucleotides were synthesized: 1) to idenify by PCR a eDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0266.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR prime S'-GTfGGATCMGOGCAACAATAAC-3- (SEQ ID NO:92) reverse PCR primer S-Afl fGTOCAGGCTGAGMAA-3' (SEQ MD NO:93) Additionally, a synthetic oligonuecotide hybridization probe was constructed which had the foMowing nucleotide sequence hybiizgaio Stob S'-GGTGGCTATACATGGATAGCAA1TACCTG;GACACGCGTCCCGG3 (SEQ ID NO:94) In order to screwn several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding die PR0266 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal brain tissue. DNAwcqmdrg of the clones isolated as described above gave the full-length DNA sequence for PR0266 [herein designated as *.UNQ233 (DNA37150-117S)J (SEQ ID NO:90) and the derived protein sequence for PR0266.

The entire nucleodde sequence of UNQ233 (DNA37150-1178) is shown in Figure 33 (SEQ MD 15 Clone UNQ233 (DNA37LSO-1178) contains a single open reading frame with an apparent translational initiation site at muceotide positions 167-169 and ending a the stop codon after nucleotide position 2254 of SEQ ID NO:90. The predficted polypeptide precursor is 696 amino acids long (Figure 34). Clone UNQ233 (DNA37150-1 178) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209401.

Analysis of the amino acid sequence of the full-length PR0266 polypeptide suggests that portions of it 20 possess significant homology to the SLIT protein, thereby indicating that PR0266 may be a novel leucine rich repeat protein.

~EXMP -F &M ISE.: Isolation of cDNA, Clones Encoding Human PRO269 A consensus DNA sequence was assembled relative to other EST sequences using pbrap as described in Example I above. This consensus sequence is herein designated DNA357OS. Based on the DNA35705 consensus sequence. oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a cdone of the full-length coding sequence for PR0269.

Forward and reverse PCR primers were synthesized: forward PCR primner (11) 5'-TGAAGGAGATGCGATGCCACCTG -3' (SEQ ID NO:97) forward PCR primier 5*-TGACCAGTGGGGAA3GACAG-3' (SEQ ID NO:98) forward PCR primer (M1) 5'-ACAGAGCAGAGGGTGCCTG-3' (SEQ ID NO:99) reverse PCR fnuipr 5*-TCAGGGACAAGTGGTGTCTCTCCC-3' (SEQ ID NO; 100) reverse PCR orinier (4r) 5'-TCAGGGAAGGAGTGTGCAGTTCTG3* (SEQ ID NO: 101) Additionally, a snthetic oligonucleodde hybridization probe was constructed from the consensus DNA35705 sequence which had the following nucleotide sequence: hybhidid =be -ACAGCTCCCGATCT'CAGUTAC7GCATCGCGGACGAAATCGGCGCTGCT3.(SEQ WD NO: 102) In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0269 gene using dhe probe oligonucleoide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence: for OR0269 (hertin designated as UNQ236 (DNA38260-i ISO)) (SEQ ID NO:95) and dhe derived protein sequence for PR0269.

The entire nucleotide sequence of UNQ236 (DNA3826O-1 190) is shown in Figure 35 (SEQ ID Clone UNQ236 (DNA38260-1180) contains a single open reading fram with an apparent translational initiation site at nucleotide positions 314-316 and ending at the stop codon at nucleotide positions 1784-1786 (Fig. 35; SEQ ED NO:95). lint predicted polypeptide precursor is 490 amino acids long (Fig. 36). Clone UNQ236 (DNA38260-1 180) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209397.

Analysis of the amino adid sequence of the full-length PR0269 suggests that portions of it possess significiint homology to the humn ixombomodulin proteins, thereby indicating that PR0269 may possess one or more thrornboxnodulin-like dotmains.

EXAMPLE 16: Isolation of eDNA Clones Encodiniy Human PRO297 20 A consensus DNA sequence encoding PRCY287 was assembled relative to the other identified EST sequences as described in Example 1 above, wherein the consensus sequence is designated herein as DNA2M78. Based on the DNA28M2 consensus sequence, oligonucleotides were syntheized to identify by PCR a eDNA library that contained :the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PR0287.

A pair of PCR primers (forward and reverse) were synthesized: forward PcR pd=me 5'-CCGAITCATAGACCrCGAGAGF.3- (SEQID NO:105) reerePCR vrimner S*-GTCAAGGAGTCCTCCACAATAC.3' (SEQ ID NO: 106) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA2W72 sequecem which had the following nucleotide sequence S*-GTGTACAATGGCCATGCCAATGGCCAGcGTGGCCGi~cGT.3' (SEQ ID NO:107) In order to screen several libraries for a source of a fullenagth clone, DNA from die libraries was screened by PCR amplification with the PCR primer pair identified. above. A positive library was then used to isolate clones encoding the PR0297 gene using the probe oligonucleofide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal iddney tissue.

DNA sequencing of thnt clones isolated as described above gave the ful-length DNA sequence for PR0287 (herein designated as UNiQ250 (DNA39969-1 195), SEQ ID NO: 1031 and the derived protein sequence for PROM.

The entire nucleotide sequence of UNQ25 (tDNA39969-1185) is shown in Figure 37 (SEQ ID NO: 103).

Clon UNQ2S (DNA39969-1 185) contains a single open reading frame with an apparent translational initiation site at nucleoade positions 307-309 and ending at the stop codon at nucleotide positions 1552-1554 (Fig. 37; SEQ ID NO:103). The predicted polyepide precursor is 415 amino ad~ds long (Fig. 38). Clone UNQ250 (DNA39969-1185) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209400.

Analysis of thc amin acid sequence of the full-length PR0287 suggests that it may possess one or more procollagen C-proteinase enhancer protein precursor or procollagen C-prteinase enhancer protein-like domains.

Based on a BLAST and FastA sequence alignment analysis oflthe full-length sequence. PR0287 shows nucleic acid sequence identity to procollagen C-proteinase enhanicer protein precursor and procoliagen C-proteinase enhancer protein, (47 and 54%, respectively).

£iXLIL 1: Isolation of cDNA Clones ncoding~ HMan PRO214 A consensus DNA sequence was assembled using phrap as described in Example I above. This consensus DNA sequence is designated herein as DNA28744. Based on this consensus sequence, oligonuecleotides were synthesized: 1)t dniyb C DAlbayta otie tesqec fitrsad2 o s spoe or 15 to isolate a clone of the full-length coding sequence.

In order to screen several libraries for a source of a full-length clone. DNA from the libraries was screened by PCR atmplification with the PCR primer pair identified below. A positive library was then used to isolate clones encoding the PR0214 gene using the probe oligonucleotide and one of the PCR primers RNA for construction of the cDNA libraries was isolated from human fetal lunig tissue. A cDNA clone .20 was sequenced in its entirety. The full length nrucleotide sequence of DNA32286I 191 is shown in Figure 39 (SEQ ID NO:lOS). DNA32286-1 191 conitains a single open reading frame with an apparent translational initiation site at nucleodde position 103 (Fig. 39; SEQ ID NO: 108). Thec predicted polypeptide precursor is 420 amino acids long :(SEQ ID NO: 109).

Based on a BLAST and FastA sequence aligzmrt analysis of the full-length sequence, PR0214 polypeptide 25 shows amino acid sequence identity to HT protein and/or Fibulin (49% and 38%. respectively).

The oligonucleotide sequences used in the above procedure were the following: 28744.p (0L1555) rGCACGAGGGrCATTTGTT ATATTA3(SEQ ID NO:1 28744.f (0L1556) 5'-ATT GCGTGAACACIYJAGyGC.3' (SEQ ID NO: 111t) 28744.r (011557) 5'-ATCTGCTrGTAGCCCTCGGCAC..3' (SEQ ID NO.:112) EAMEE,19: Isolation of cDNA Clones EcodinL Hman PR0317 A consensus DNA sequence was assembled using phrap as described in Example I above, wherein the consensus sequence is herein designated as DNA2872. Based on this consensus sequence, oigonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as Probes to isolate a clone of the ful-legth coding sequence. The forward and reverse PCR primers, respectively, synthesized for this purpose were: 5*-AGGACTGCCATAAC-1GCCTG (0U489) (SEQ ID NO: 115) and (0U490) (SEQ ID NO: 116).

The probe synthesized for this purpose was: 5'-TTGTGGACATAGACGAGTGCCGrAccGTAcJccAGcACC~ (01.488) (SEQ ID NO: 117) miRNA for construction of the cDNA libraries was isolated from human fetal idney tissue.

In order to scinen several libraries for a source of a full-length clone, DNA from thc libraries was screened by PCR amplification, as per Ausubel et al.. Clwrrent Protocols in Molecular Biology (1989). with the PCR primer pair identified above. A positive library was then used to isolate clones containing the PR0317 gemi using the probe oigonucleotide identified above and one of the PCR primers.

A eDNA clone was sequenced in its entirety. 1c cadire nucleotide sequence of DNA33461-1199 (encoding PR0317) is shown in Figure 41 (SEQ ID NO: 113). Clone DNA33461-1199 contains a single open reading frame with an appar-ent translational initiation site at nucleooide positions 68-70 (Fig. 41. SEQ ID NO: 113). The predicted polypeptide precursor is 366 amino acids long. The pralicted signal sequence is amino acids 1-18 of Figure 42 (SEQ ID NO:114). There is one predicted N-linked glycosylation site at amino acid residue 160. Clone DNA33461-1 199 has been deposited with ATCC and is assigned ATCC deposit no. ATCC 20967.

Based on BLAST~' and FastA:Tm sequence alignment analysis (using the AUIGNTN computer program) of the full-length PRO317sequence, PR0317 shows the most amino acid sequence identity to EBAF-I The results also demonstrate a signifixant homology between himan PR0317 and mouse LEFrY protein. The C-terminal :20 end of the PR0317 protein contains many conserved sequences consistent with the pattern expected of a member of TGF- superfamily.

In situ expression analysis in human tissues performed as described below evidences that there is distinctly :::strong expression of die PRO317 polypeptide in pancreatic tissue.

25 EXAME1LE.Jn: Isolation, of cDNA clones EncodinE Human PRO301 A consensus DNA sequence designated herein as DNA35936 was assembled using phrap as described in Example I above. Based on this consensus sequence, oligonuc-leotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence.

In order to scree several Libraries for a source of a full-length clone, DNA from the Libraries was screened by PCR amplification with the PCR primer pair identified below. A positive library was then used to isolate clones encoding the PRO301 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal kIdney.

A cDNA clone was sequenced in its entirety. The full lenigth nucleotide sequence of native sequence PRO30I is shown in Figure 43 (SEQ ID NO: 118). Clone DNA40628-1216 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 52-54 (Fig. 43; SEQ ID NO:118). The predicted polypeptide precursor is 299 amino acids long with a Predicted mnolecular weight of 32,583 daltons and pl of 8.29.

Clone DNA40628-1216 has been deposited with ATCC and is assigned ATCC deposit No. ATCC 209432.

Based on a BLAST and FastA sequence aligrnent analysis of the full-length sequence. PRO301 shows aid sequec idenity to A33 antigen precursor and coxsacuce and adenovirus receptor protein The oligonucleotide sequences used in the above procedure were the following: OLJ2162 (35936.fl) 5*-TCGCGGAGCTGTGirCTGrJTCCC3* (SEQ ID NO:120) 0L12163 3

S

936 .pl) 5'TACCAGGAAAGGAGTCAAGACGTTCT3 (SEQ MD NO: 121) 0L12164 (3593612) 5'-ACACCTGGITCAAAGATGGO-3- (SEQ ID NO:122) 0L1165 (3S936.rl) S5'TAGGAAGAGTGCTGAAGGCACGG-3- (SEQ ID NO:123) 0LI2166 (35936.3) 5*-7TCCTACTCAGGTOCTAC-3* (SEQ MD NO: 124) 01.12 167 (35936.r2) S'-ACTCAGCAGTGGTAGGAAAG-3y (SEQ ID NO: 125) EXAMPLE 20. Isolation of cDNA Clones Eneoin Human IPRO224 ExmpleA consensus DNA sequence assembled relative to dhe other identified EST sequences as described in Fim~c weren te cnsesussequenc is designated herein as DNA30845. Based on the DNA30845 consensus 1% 20 sequence. oligonucicotides were synthesized to identify by PCR a eDNA library that contained the sequence of interest and for wse as probes to isoate a clone of the full-length coding sequence for PR0274.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer S-AAGTCCAGTGCCGCACCAGTGGC-3' (SEQ ID 14O:128) :reverse PCR primer 5'-TGGTrCCACAGCCGAGCTCGTCG.3' (SEQ ID NO: 129) 25 Additionally, a synthetic oligonucleotdec hybridization probe was constructed from the consensus DNA30S45 sequence which had the following nucleotide sequence :hybridization robe 5SGAGGAGGAGTGCAGGATTGAGCCATGTACCAAJGGCATCCCACC-3*(SEQ ID) NO:130) In order to screen several libraries for a soure of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRI224 gene using the probc oigonucleotide n one of tePRpies RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. DNASrenckg of the clones isolated as described above gave die full-length DNA sequence for PR0224 [herein designated as UNQ198 (DNA33221-l 133)] and the derived protein sequence for PR0224.

The entire nucleotide sequence of UNQ198 (DNA33221-1 133) is shown in Figure 45 (SEQ ID NO: 126).

Clone UNQ198 (DNA33221-1133) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 96-98 and ending at the stop codon at nucleotide positions 942-944 (Figure 45; SEQ ID NO: 126). The start of a tratismembrane region begins at nu-ceotide position 777. The predficted polypeptide precuror is 282 amino adids long (Figure 46). Clone UNQ199 (DNA33221-1133) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 20963.

Analysis of the amino acid sequence of the ful-length PR0224 suggests that it has homology to very low-' density *ipprotein receptors. apolipoprotein E receptor and chicken oocyte receptors P95. Based on a BLAST and FastA sequence alignmient analysis of the full-length sequence. PR0224 has amino acid identity to portions of these proteins in the range from 28% to 45%. and overall identity with these proteins in the range from 33 to 39%.

EXAMPEB21: Isolation of eDNA Clones Enc:4ding Huma~n PRO222 A consensus DNA sequence was assembled relative to the other identified EST sequences as described in Example I above. wherein the consensus sequence is designated herein as DNA28771. Based on the DNA29771 consensus sequence, oligomucleonides were synthesized to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PR0222.

A pair of PCR primers (forward and reverse) were synthesized& forward PCR prime 5'-ATCTCCTATCGCTGCTFCCCG3* (SEQ ID NO:l33) reverse PCR pri=w 5'-AGCCAGGATCGCATAAACCC-.3' (SEQ ID NO: 134) Additionally. a synthetic oligonuicotide hybridization probe was constructed from the consensus DNA28771 sequence which had the following nucleotdec sequence: b~ridization groe s,-AT1TAAAcTGATGGGTcTGcGTATc r3AGTGrACAACTATT3' (SEQ ID NO:135) In order to sceen several libraries for a source of a ful-ength clone. DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO2 gene using the probe oigonucleotide and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0222 (herein designated as U1NQ196 (DNA33IO'7-1135)] and the derived protein sequtence for PR0222.

The entire nueceotide sequence of UNQ196 (DNA33107-1135) is shown in Figure 47 (SEQ ID NO:131).

Clone UNQ196 (DNA3310Y7-l 135) contains a single open reading frame with an apparent translational initiation site at nucleotode positions 159-161 and ending at the stop codon at nucleotide positions 1629-1631 (Fig. 47; SEQ ID NO:.131). The predictd polypeptde prcursor is490am acidslong (Fig. 48). Clone UNQ196 (DNA33107-1135) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209251.

Based on a BLAST and FastA sequence alignment analysis of the ful-ength sequence, PR0222 shows amino acid sequence identity to mouse complement factor h precursor complement receptor mouse complement C3b receptor type 2 long form precursor C25-47 and humian hypothetical protein liaaO247 ]EXAMPLE 22: Isolation of cDNA clones Encodine PR0234 A consensus DNA sequence was assembled (DNA30926) using phrap as described in Example I above.

Based on this consensus sequence. oligonucleotders were synthesized: 1) to identify by PCR a eDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence.

RNA for tie construction of the cDNA libraries was isolated using standard isolation protocols. e.g., Ausubel et at.. Cuirrent Prorocols In Molecular Biology, from tissue or cell line sources or it was purchased from commnercial sources Clontech). The cDNA libraries used to isolate the cDNA clones were constructed by standard methods Ausube at using commercially available reagents Invitrogen). This library was derived from 22 week old fetal brain tissue.

A cDNA dlone was sequenced in its entirety. The entire Micleotide scquence of PR0234 is shown in Figure 49 (SEQ M -NO: 136). The predicted polypeptide precursor is 382 amino acids long and has a calculated molecular weight of approximately 43.1 Wa.

The oligonueleotide sequenceswed in the above procedure were the following: 30926.p (0U8926) (SEQ WD NO: 138): GATGG7TGACrCGGATrGGGCTCA-3' 30926.f (0U827) (SEQ MD NO, 139): S*-AAGCCAMAGAAGCCT'GCAGGAGGG-3' 30926.r (01.828) (SEQ ID NO: 140): S*-CAGTCCMAGCATAAAGGTCCTGGC-31 EXAMPLE 23: Isolation of cDNA Clones Egco&=n Human PR,0231 A consensus DNA sequence was assembled relative to the other identified EST sequences as described in Example I above. wherein the consensus sequence was designated herein as DNA30933. Based on the DNA30933 consensus sequence, oligonucleotdecs were synthesized to identify by PCR a cONA library that contained the sequence of interest and for use as probes to isolate a clone: of the full-length coding sequence for PR023 1.

Three PCR primers (tw o forward and one reverse) were synthesized: forward PCR orimer I 5'-CCAACTACCAAAGCTGCTGGAGCC-3* (SEQ ID NO:143) forward PCR m2imer 2 S'-GCAGCTCTATTACCACGGGAAGGA-3* (SEQ ID NO:144) :20 reverse PCR Rrinier 5*-TCCTTCCCGTGGTAATAGAGCTGC-3* (SEQ ID N40:145) XAdditionally. a synthetic oligonucleotdec hybridization probe was constructed from the consensus DNA30933 sequence which had the following nuclcotdec sequence 5*-OGCAGAGAACCAGAGGCCGGAGGAGACTGCCTCnTACAGCCAGG-3' (SEQ ID NO: 146) In order to sa= cseveral libraries for asource of a full-length clone. DNA from the Libraries was screened by PCR amplification with the PCR primier pairs identified above. A positive library was then used to isolate clones encoding the PR0231 gene using the probe oligonudeotide and one of the PCR primers.

a'RNA for construction of the cDNA Libraries was isolated from human feta liver tissue. DNAsalILdzxi of the clones isolated as described above gave the full-length DNA sequecec for PR0231 [herein designated as UNQ205 (DNA34434-l 139)] and the derived protein sequence for PR0231.

The entire nucleotide sequence of UNQ205 (DNA34434-1 139) is shown in Figure 51 (SEQ ID) NO: 14 1).

Clone UNQ205 (DNA34434-1139) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 173-175 and ending at the stop codon a nucleotdec positions 1457-1459 (Fig. 51; SEQ ID NO: 141). The predicted polypeptide precuror is 428 amino acids long (FMg. 52). Clone UNQ205 (DNA34434-1 139) has been deposited with ATCC on September 16. 1997 and is assigned ATCC deposit no. ATCC 209252.

Analysis of the amino acid sequence of the full-length PR0231 suggests that it possesses 30% and 31 amino acid identity with the human and rat prostatic acid phosphatase precursor proteins, respectively.

EMAMPLE14A: Isolation of cDNA Clones Encodinp HuMan pR02 A consensus DNA sequence was assembled'relative to Other EST sequecesCC using phrap as described in Example 1 above. This consensus sequence is herein designated DNA28762. Based on the DNA28762 consensus sequence. oligoawuceotides were synthesized: 1) to identify by PCR a cDNA Library that contained the equene of Interest, and 2) for use as probes to isolate a clone ot the full-length coding sequence for pR0229.

A pair of PCR primers (forward and reverse) were synthesized: fowadPC jrm 5*-TCAGCTCATCACCTCACCTGCC..3. (SEQ ID NO: 149) reverse VCR primer S'-GGCTCATACATACCTAGGG-3' (SEQ ID Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28762 sequence which had the following nucleotide sequence hybridization probe GCCACG-GGAGCGGrGGTGAAGAGCAT3 (SEQ ID3 NO: 151) In order to screenseveral liries for a source of a full-length clone, DNA from the libraries was screened by PCR amplifiatinwit tho e PR~s primer pair identified above. A o itielbrr aste ue osltecoe encoding the PR0229 gene using the probe oigomiucleooide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. l2NAweqmia of the clones isolated as described above gave the fulfl-length DNA sequence for PR0229 Lhrein designated as UNQ203 (DNA3I0-1159)J (SEQ ID NO: 147) and the derived protein sequence for PR0229.

The entire nucleotide sequence of UJNQ203 (DNA331OD-1 159) is shown in Figure 53 (SEQ ID NO: 147).

20 Clone UNQ203 (DNA331IO- 159) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 98-100 and ending at the stop codon at nuclootide positions 1139-1141 (Figure 53). The predicted polypeptide precursor is 347 amino acids long (Figure 54). Clone UNQ203 (DNA33100-1 159) has been deposited with ATCC and is assigned ATCC deposit no.ATCC 209377 Analysis of the amino acid sequence of the full-length PR0229 polypeptide suggests that portions of it possess significant homology to antigen wcl.1, M130 antigen and CD6.

EXAMPLE 25: Isolation of cDNA Clones Enodina Human PROM3 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above in Example 1. This consensus sequence is herein designated DNA30908. Based on the DNA30908 consensus sequence, olig onacleotides were synthesized: 1) to identify by PCR a cDNA library tha contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PROM3.

PCR primers (forward and reverse) were synthesized: forward PCR 12rimer I 5'-GGTGCFrAAACTGGTG(FTCGC..3' (SEQ MD NO: 154) forward PCR uRdmer 7 5-CAGGGCAGATGACAITCC3' (SEQ ID NO: 155) reverse PCR prime 5*-TCATACGTrCATTCGGACGC- 3 1 (SEQ ID NO:156) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30908 sequence which had the following zmcloude sequence hybrikJdizio 2hob 5-AATGGTGGGGCCCTAGAAGAGCTCATCAGAGAACTCACCGCIrCTCATGC.3 (SEQ ID NO: 157) In order to screen several libraries for a soure of a full-length clone, DNA from the libraries was screened by PCR amp~lification with the PCR primer pair identified above. A positive library was then used to isolate clones ecding the PR0239 gene using the probe oligonucleodde and one of the PCR primers.

RN4A for construction of the cDNA libraries was isolated from human fetal liver tissue. DNAs~qmdng of the clone isolated as described above gave the full-length DNA sequence for PR0238 and the derived protein sequence for PROM3.

The entire nucleotide sequence of DNA35600-1162 is shown in Figure 55 (SEQ ID NO: 152). Clone DNA3S60071162 contains a single open reading framne with an apparent translational initiation site at nucleotide positions 134-136 and ending prior to the stop codon at rmcleotidec positions 1064-1066 (Figure 55). The predicted polypqptdec precursor is 3 10 amino acids long (Figure 56). clone DNA3560D-1 162 has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209370.

Analysis of the amino acid sequence of the full-length PR0238 polypeptide suggests that portions of it possess significant homology to reductase, particularly oxidoreductase. thereby indicating that PR0238 may be a novel reductase.

EXAMEI.E.26: Isolation of cDNA Clones Encodin, Human PRO233 The extracdlular domain (ECD) sequences (icuding the secretion signal. if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases. The EST dalabases included public EST databases GenBank) and a proprietary EST DNA database (UIFESEQT. Incyte Pharmaceuticals, Palo Alto. CA). The search was performed using the computer program *.:BLAST or BL.AS72 (Altshul et al.. Mehd nF~aoy2(&6460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequence. Those comparisons; resulting in a BLAST score o f 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program 4phrap* (Phil Green, University of Washington. Seattle. Washington.

'http://bman.mt.washington.ed/h ap.dosprap.hd).

An expressed seqenc tag MESM was identified by the EST database search and a consensus DNA sequence was assembled relative to other EST sequences using phrap. This consensus sequence is herein designated DNA30945. Based on the DNA30945 consensus sequence, oligotrueleotdes were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0233.

Forward and reverse PCR primers were synthesized: forward PCR primer S'-GGTOAAGGCAGAAA7T3GAGAT.3' (SEQ ID NO: 160) reverse PCR lirimer 5'-ATCCCATUCATCAGCCTGTrTACC-3' (SEQ ID NO: 161) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30945 sequence which had the following micleotide sequence S'-GCTGGTGTAGTCTATACATCA

A'JIGCACACAAATCCTCAG-

3 (SEQ ID NO: 162) In order to screen several libraries for a source of a ful-lengtht clone, DNA from the libraries was screened by PCR amplification w~ith the PCR primer pair identified above. A positive library was then used to isolate lonies encoding the PR0233 gene using the p'robc oligornicleodde.

RNA for construction of the cDNA libraries was isolated from human fetal brain tissue. DNAsqmiqg of the clones isolated as described above gave the MIl-length DNA sequence for PR0233 (hercin designated as UNQ207 (DNA34436-1238)J (SEQ ID) NO:l158) and! the derived protein sequence for PR0233.

-The entire micleotide sequence of UNQ207 (DNA34436-l238) is shown in Figure 57 (SEQ ID NO: 158).

Clone UNQ207 (DNA34436-128) cntains a singe open reading framec with an apparent tramilational initiation site .at nucleotide positions 101-103 and ending at the stop codon at nucleotide positions 1001-1003 (Figur 57). The *.predicted polypeptide precursor is 300 amino acids long (Figure 58). The fuI-length PR0233 protein shown in Figure 58 has an estimated molecular weight of about 32.964 daltons and a p1 of about 9.52. Clone UNQ207 (DNA34436-1238) has been deposited with ATCC arnd is assigned ATCC deposit no. ATCC 209523.

Analysis of the amino acid sequence of the Mlength PR0233 polypeptide suggests that portions of it possess significant homology to reductase proteins, thereby indicating that PR0233 may be a novel reductase.

EXAMPLE 27: Isolation of cDNA Clones Encdin Human PR223 20 A consensus DNA sequence was assem~bled relative to other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA30836. Based on the DNA30836 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of :interest, and 2) for -use as probes to isolate a clone of~ MI fl-length coding sequence for PR0223.

primer pairs (one forward and two reverse) were synthesized: forward PCR primer 5*-TrCCATGCCACCTAAGGGAGACTC.3* (SEQ ID NO:165) reverse PCR grimer 1 5*-TGGATGAGGTGTGCAATGGCTGCcJ(3* (SEQ ID NO: 166) reverse PCR primer 2 S'-AGCTCTCAGAGGCTGGTCATA(JcJ(.3' (SEQ ID NO: 167) Additionally, a synthetic oligonuclotdec hybridization probe was constructed from the consensus DNA30836 sequence which had the following nucleotide sequence M OAGCGAAGAG rTCG~rCCC3(SEQ ID NO: 168) In order to screen several libraries for a source of a ful-lngth clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate lonies encoding the PR0223 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated ftrm human fetal liver tissue. DNAseming of the clones isolated as described above gave the full-length DNA sequence for PR0223 therein designated as UNQ197 (DNA33206-l 165)] (SEQ ID NO: 163) and the derived protein sequence for PR0223.

The entire nuclotide sequence of UNQI 97 (DNA33206-1 165) is shown in Figure 59 (SEQ ID NO: 163).

Clone UNQ197 (DNA33206-l 165) contains a Aingle open reading frame with an apparent tanslational initiation site at nucleotide positions 97-99 and ending at the stop codon at micleotide positions 1525-1527 (Figure 59). The predicted polypeptide precursor is 476 amino acids long (Figure 60). Clone UNQ197 (DNA33206.1 165) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209372.

Analysis of the amino acid sequence of the full-length PR0223 polypeptide suggests that it possesses significant homology to various samine carboxypeptidase proteins, thereby indicating that PR0223 may be a novel serine carboxypeptidase.

EXAMPLE.2R: Isolation of cT)NA Clone Encoftn Human PRO235 A consensus DNA sequence was as sembled relative to other EST sequences using pbrap as described in' Example 1 above. Ibis consensus sequence is herein designated *DNA30927*. Based on the DNA30927 consensus sequence. oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as. probes to isolate a done of the full-length coding sequence for PROM3.

A pair of PCR primers (forward and reverse) Were synthesized: forward PCR primer S'-TGGAATACCGCCrCCTGCAG-3' (SEQ ID NO: 171) reverse PCR grimer 5*-CTTCTGCCC1TIGGAGAAGATGGC.3' (SEQ ID NO: 172) Additionally, a synthetic oligomiceotide hybridization probe was constructed from the consensus DNA30927 sequence which had the following nucleotide sequence hbbridization probe 5'-GGACTCACTGGCCCAGGCCTrCAATATCACCAGCCAGGACGAT-3 (SEQ 11) NO: 173) In order to screii several libraries for a source of a ful-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones :encoding the PR0235 gene. using the probe oligonuclctide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. DNAnwqxeimg of the clone isolated as described above gave the full-length DNA sequence for PR0235 [herein designated as UNQ209 (DNA35558-1167)J (SEQ ID NO: 169) and the derived protein sequence for PROM3.

The entire nucleotide sequence of UNQ209 (DNA35558-1167) is shown in Figure 61 (SEQ ID NO. 169).

Clone UNQ209 (DNA355S-1167) Contains a single open reading frame with an apparent translational initiation site at nucleotide positions 667-669 and ending at the stop codon at nucleotide positions 2323-2325 (Figure 6 The predicted polypetide precursor is 552 amino acids long (Figure 62). Clone UNQ209 (DNA35558-1167) has been deposited with ATCC and is assigned ATCC deposit no. 209374.

Analysis of the amino acid sequence of the full-length PR0235 polypeptide suggests that portions of it possess significan homology to the human, mouse and Xciwpus plexin protein, thereby indicating that PRO235 may be a novel plexin protein.

EXAMI!.&Z29: Isolation of eDNA Cognes Enenodina Humn PRO236 MWd Human PRO262 Consensus DNA sequences were assembled relative to other EST sequences using phrap as described in Example 1 above. These consensus sequences are herein designated DNA30901 and DNA30847. Based on the DNA30901 and DNA30847 consensus sequences. oligoamcleoides were synthesized: 1) to idenify, by PCR a cDNA library that-contained dhe sequence of interest, and 2) for use as probes to isolate a clone of dhe full-length coding sequence for PR0236 and PR0262. respectively.

Based upon the DNA3090 consensus sequence. a pair of PCR primers (forward and reverse) were synthesized: forward PCR primier 5'-TGGCTACTCCA ACCGCATG-3- (SQ IDNO:178) reverse PCR nrmr 5'-TGGACAAATCCCCrTGCTCAGCCC..3' (SEQ ID NO: 179) Additionally. a synthetic oligonucleotide hybridization probe was constructed from die consensus DNA30901 sequence which had the following nuclcode sequence lhbidimtion jmhe 3 (SEQ ID, NO:180) Based upon the DNA30847 consensus sequence. a pair of PCR primers (forward and reverse) were orwarsd: C rimier S*-CCAGCTATGACTATGATGCACC-3' (SEQ ID NO:18l) .reverse PCR urime 5'-TGGCACCCAGAATGGTGTGGCTC.3 (SEQ ID NO: 192) Additionally, a synthetic oligonucleolide hybridization probe was constructed from the consensus DNA30847 sequence which had the following nucleotide sequence hyrdzto rb T rCAGGA GGAAC I I AC--AC~rC.3'(SEQ ID NO:183) In order to screen several libraries for a source of fuil-length clones. DNA from the libraries was screened ~9:by PCR amplification with the PCR prim pairs identified above. Positive libraries were then used to isolate clones encoding the PR0236 and PR0262 genes using the probe oligonucleotides and one of the PCR primers.

25 RNA for construction of the cDNA libraries was isolated from human fetal lung tissue for PR0236 and human fetal liver tissue for PR0262.

DNA sequencing of the clones isolated as descibed above gave the ful-length DNA sequence for PR0236 therein designated as UNQ210 (DNA3SS99-I 168)] (SEQ ID NO: 174). the derived protein sequence for PROM3, the full-length DNA sequence for PRO262 [herein designated as UNQ229 (DNA36992-1 168)] (SEQ ID NO: 176) and the derived protein sequence for PR0262.

The entire nucleotide sequence of UNQ210 (DNA35599-1 168) is shown in Figure 63 (SEQ ID NO:174).

Clone UNQ210 (DNA35599-l 168) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 69-71 and ending at the sto codon at nucleotide positions 1977-1979 (Figure 63). Ile predicted polypeptide precursor is 636 amino acids long (Figure 64). Clone UNQ210 (DNA3SS99-i 168) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209373.

The entire tasclcotide sequence of UNQ229 (DNA36992-1 168) is shown in Figure 65 (SEQ ID NO: 176).

Clone UNQ229 (DNA36992-1168) contains a single open reading framne with an apparent translational initiation site at nucide positions 240-242 and ending at the stop codon at nucicotide positions 2202-2204 (Figure 65). The Predicted polypeptide precursor is 654 amino ads long (Figure 66). Clone UNQ229 (DNA36992.1168) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209382.

Analysis of the amino acid sequence of the full-length PR0236 and PRO262 polypeptides suggests that Portions of dios polypepidcs possess significant homology to P-galactosidase proteins derived from various sources, thereby indicating that PR0236 and PR0262 may be novel P-galactosidas'e homologs.

EXAMPE..I Isolation of eD)NA Clones Encodine Human PRO239 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA30909. Based on the DNA30909 consensu sequence, oligonucleoddes were synthesized: 1) to idenif by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PROM3.

A pair of PCR primers (forward and reverse) were synthesized.

.*forward PCR primrer 5'-CCTCCCTCTATTACCCATGTC..3' (SEQ ID NO:186) reverse PCR primner S'-GACCAAC7nCTCGGGAGTGAGGJ.3* (SEQ ID NO: 187) Additionally, a synthetic oligonudceotdec hybridization probe was constructed from the consensus 'DNA30909 sequence which had the following macleotide sequence hybridizationprobe (SEQ ID NO: 18S) In order to screen several libraries for a source of a full-length clone. DNA from th e libraries was screened by PCR ampIification with the PCR primer pair identified above. A positive libray was then used to isolate clones encoding the PR0239 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. DNAwmin of the clones isolated as described above gave the ful-lergth DNA sequence for PR0239 therein designated as UNQ213 (DNA34407-1169)] (SEQ ID NO:184) and the derived protein sequence for PROM3.

The entire nucleotide sequence of UNQ213 (DNA34407-1 169) is shown in Figure 67 (SEQ ID NO: 184.

Clo UN21 (DN~A0711 ontin a single opnrveadn t with an apparent translational initiation site at nucleotide positions 72-74 and ending at the stop codon at micleodde positions 1575-1577 (Figure 67). The predicted polypeptide precursor is 501 amin acids long (Figure 68). Clone UNQ213 (DNA34407-1 169) has been deposited with ATCC and is assigned ATCC deposit no.ATCC 209383.

Analysis of the amino acid sequence of the full-lngt PR0239 polypeptide suggests that portions of it possess significant homology to the densin protein. thereby indicating that PR0239 nay be a novel molcule inf the dcnsin family.

EXAELE 31: Isolation of cDNA Clones ncodirut Huma pR025 A consensus DNA sequence was assembled relative to other EST scquences using phrap as described in Eixamplel1above. This consensus sequence is herein designated DNA28731. Based on the DNA28731 consensus sequence. oligonwcleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for usc as probes to isolate a clone of the full-length coding sequence for PR0257.

A pair of PCR primers (forward and reverse) were synthesized: foward PCR Vdril S-TCTCTATrCCAACTGTGGCG-3 (SEQ ID NO: 191) reverse POR primer 5'-MTGATGACGATCGAAG(TGG-3- (SEQ ID NO: 192) Additionally. a synthetic oigonucleotide hybridization probe was constructed from the consensus DNA28731 sequence which had t following nucleotide sequence (SEQ ID NO: 193) In order to screen several libraies for a soumce of a fudll-ength clone. DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO2S7 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from humran fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PRO2S7 thercin designated as UNQ224 (DNA35841-1173) (SEQ IM NO:l189) and the derived protein sequence for PR0257.

*The entire nucleotide sequence of UNQ224 (DNA35841-1173) is shown in Figure 69 (SEQ ID N6:189).

Clone UNQ224 (DNA3SS41-l 173) contains a single open readinig frame with an apparent tanslational initiation site at nucleotidc positions 964-966 and ending at the stop codon at nucleotide positions 2785-2787 (Figure 69). The predicted polypeptide precursor is 607 ammno acids long (Figure 70). Clone UNQ224 (DNA3584 1-1 173) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209403.

20L Analysis of the amino acid sequence of the fulfl-length PR0257 polypeptide suggests that portions of it ***possess significant homoology to the ebnrcrn protein. thereby indicating that PRO2S7 may be a novel protein member related to the ebnerin protein.

EXAM-PLE 3: Isolation of cDNA Clones Encoding Human PR0260 025 A consenss DNA sequence was assembled relative to other EST sequences using phrap as described in Examiple 1 above. This consensus sequence is herein designated 0NA30834. Based on the DNA30834 consensus sequenice, oligonuecotides were synthesized: 1) to identify by FCR a cDNA library that contained the sequence of interest. and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0260.

PCR primers (forward and two reverse) were synthesized: forward PcR nruce 5'-TGGTGMACCAGGCCAAG-ryJ(.3' (SEQ ID NO: 196); reverse ECR primer A: 5'-GAITCATCCTCAAGGAAAGCJG-3* (SEQ ID NO:197); and reversce CR urimerl~ B AACffGCAGCATCAGCCA~CT~rGC3' (SEQ IDNO:198) Additionally. a synthetic oligonucleotide hybridization probe was constructed from t consensus DNA30834 sequence which had the following miecotide sequence: Wvridization yrobe: ID NO:199) In order to sal several libraries for a source of a full-ength clone. DNA from the libraries was screened by PCR amplification with die PCR prir pair identifled above. A positive library was then used to isolate clones encoding the PR0260 gene using the probe oligonucleotide andi one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue.

DNA seqmenirg of the cloe isolated as described above gave the fll-length DNA sequence for PR0260 (berein designated as UNQ227 (DNA33470-1175)J (SEQ ID NO: 194) and the derived protein sequece for PR0260.

The entire nuclentide sequence of UNQ227 (DNA33470-l 175) is shown in Figure 71 (SEQ ID NO: 194).

Clone UNQ227 (D5NA33470-1 175) contains a single open reading framc with an apparent translational initiation site at nucleotide positions 67-69 and ending at the stop codon 1468-1470 (see Figure 71). The predicted polypeptide precusor is 467 amino acids long (Figure 72). Clone UNQ227 (DNA33470-l 175) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209398.

Analysis of die amino acid sequence of the full-length PR0260 polypeptide suggests that portions- of it possess significant homology to the alpha-l-fucosidase precursor, thereby indicating that PR0260 may be a novel 9 9fucosidase.

LEXML 3: Isalaiion of cDNA Clones Encodinar Human PRO263 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA30914. Based on the DNA30914 consensus sequence, oligonuicleotides were synthesized: 1) to identify by PCR a eDNA library that contained thc sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PROM6.

.:20 PCR primers (tow firward and one reverse) were synthesized: forward PCR 2rimer 1: 5'-GAGC1TCCATCCAGGTGTCATGC-3* (SEQ ID NO:202); forward PCRurimer 2- S'-GTCAGTGACAGTACCTACTCGG-3' (SEQID NO:203); rCesePCR nrUMer: .:5'-TGGAGCAGGAGGAGTAGTAGTAGG-3- (SEQ ID NO:204) 9999 Additionally, a synthetic oligonucleotide hybridization probe was constructcd from the consensus DNA30914 sequence which had the following nucleotide sequence: hbridztin roe 5'-AGGAGGCCTGTAGGCrGCTGGGACrAAGTrrUGCCGGCAAGACCAAYT-3' (SEQ ID NO:205) order to screen several libraries for a source of a I-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0263 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated from human fetal liver tissue. DNtAseiaudzin of the clones isolated as described above gave the MIl-length DNA sequence for PR0263 [herein designated as UNQ230 (DNA34431-1177)I (SEQ ID NO:200) and the derived protein sequence for PROM6.

The entire raicleotide sequence of UNQ230 (DNA34431-i 177) is shown in Figure 73 (SEQ ID NO:200).

Cow UNQ230 (DNA34431-1177) contains a single open reading frame with an apparent tanslational initiation site at nucleotde positions 16D-162 of SEQ IDI N02W uund ending at the stop codon after the nucleotide at position 1126- 1128 of SEQ MD NO:200 (Figure 73). The Predicted polypeptide precursor is 322 amino acids long (Figure 74).

Clone UNQ230 (D)NA34431-1177) has been deposited with ATCC mid is assigned ATCC deposit no. ATCC 209399.

Analysis of the amino acid sequence of the full-lengt pR0263 polypeptide suggests that portions of it Possess significan homology to CD44 antigen, thereby indicating that pR0263 may be a novel cell surface adhesion molecule.

EXAMPLE34: Isolationi of cDNA Clones Enicoding Hurn2n PR07 A consnsu DNA sequence was assembled relative to the other identified EST sequences as described in EMa*g 1 above. wherein the consensus sequence was designated herein as DNA35712. Based on the DNA35712 consensus sequence. oligonrucleodides were synthesized: 1) to identify by PCR a eDNA library that contained the sequence of interest, and 2) for use as probes-to isolate a clori of the full-length coding sequence for PR0270.

Forward and reverse PCR primers were synthesized: f~oW PR V 5'-GCJ'TGATATrCGCATGGGCCTAC..3, (SEQ ID NO:208) :Oseo orwrdPCRRrier(1ffl S'-TGGAGACAATATCCCTGAJG-3, (SJ D NO:209) reverse PCR primer (.rll 5'-AACAGTTGGCCACAGCATGGCAGG..3. (SEQ ID NO:210) Additionally. a synthetic oigoncleotide hybridization probe was constructed from the consensus DNA35712 sequence which had the following nucleotide sequence *to* LS...ztonRrb S SCCATTGATGAGAACTAGACGGGACAAGAGGGGATGTGAG- 3 (SEQ ID NO:21 1) Inorder to screen several libraries for a source of a full-length clone, DNA from the libraries was screened 20 by PCR amnplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0270 gene using the probe oligoruicleotde and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated from human fetal lung tissue. DNAwpcix 5 of the clones isolated as described above gave the ful-length DNA sequence for PR0270 (herein designated as UNQ237, DNA395 10-1181] (SEQ ID NO:206) and the derived protein sequence for PROM7.

The entire muclotide sequence of UNQ237. DNA39510-1181 is shown in Figure 75 (SEQ ID N0:206).

Clon UNQ237 (D)NA39510-1 181) contains a single open reading fram with an apparent translational initiation site at nucleotide positions 3-5 and ending at the stop codon at nucleotide positions 891-893 (Fig. 75; SEQ ID NO:206).

see.The predicted polypeptide precursor is 296 amino acids long (Fig. 76). Clone UNQ237 (DNA3951O-1l8l) has been depo sited with ATCC and is assigned ATCC deposit no.. ATCC 209392.

Analysis of the amino adid sequence of the full-length PRO270 suggests that portions of it possess significant homology to the tioredoxin-protein, thereby indicating that the PR0270 protein may be a novel member of the thioredoxin family.

EXAMPE 35:~ Isolation of cDNA Clones Encodiag Humn PR271 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designiated DNA3S737. Based on die DNA35737 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a eDNA library that contained the sequence of interest. and 2) for use as probes to isolate a clone Of the full-length coding sequence for PR0271.

Forward and reverse PCR primers were synthesizecd: fOrward PCR nrimer I S'-TGCrFGCTACTOCCC'rC-3* (SIEQ ID NO:214) forward PCR primer 2 S*-1TCC CTTGTGGGTrGGAG-3' (SEQ ID NO:21S).

forward PCR nrimr 3 5S-AGGGCTGM0CCAUMrC3' (SEQ ID N0:216) reverse PCR primer I S'-AGCCAGTGAGGAAATGCG.3. (SEQ ID NO:217) 7 reverse PCR Drimer 2 5'-TGTCCAAAGTACACACACCTGAGG.3- (SEQ ID NO:218) Additionally. a synthetic oligomicleodde hybridization probe was constructed from die consensus DNA35737 sequence which had tbe following aucleotide sequence s,-GAToccAcGATcGccAAGGTGGAcAcc~rC CTGAA-3, (SEQ iD N0.219) In order to screen, several libraries for a source of a full-length clone, DNA from die libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones ce encoding the PR0271 gene using the probe oligonucleotide and one of the PCR primers.

:**ooRNA for construction of the eDNA libraries was isolatd from human fetal brain tissue. DNAseqrmting of tie clones isolated as described above gave the full-length DNA sequence for PR0271 [herein designated as UNQ238 (DNA39423-l 182)] (SEQ ID NO:212) and the derived protein sequence for PR0271.

The entire nucleotide sequence of UNQ238 (DNA39423-1182) is shown in Figure 77 (SEQ ID NO:212).

Clone UNQ238 (DNA39423-1 182) contains a single open reading firame with an apparent translational initiation site at nucleotide positions 101-103 and ending at the stop codon at nucleotide positions 1181-1183 (Figure 77). The predicted polypeptide precursor is 360 amino acids long (Figure 78). Clone UNQ238 (DNA39423-1l82) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209387.

Analysis of the amino acid sequence of the full-length PR0271 polypeptide suggests that it possess ~significant homology to the proteoglycan link protein. thereby indicating that PRO271 may be a link protein homolog.

EXAMPLE 36: Italatinn of eDNA Cones Eciag HmnP272 A consensus DNA sequence was assembled relative to other EST sequences using phrap, as described in Example 1 above. This consensus sequence is herein designated DNA36460. Based on the DNA36460 consensus seqenc, oligonucleotides were synthesized: 1) to identifyr by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PROM7.

Forward and reverse PCR primers were synthesized: fowadPC imr(.fl) 5'CGCAGGCCCTCAT G0C(AG3- (SEQ ID NO:222) forward IPCR primne 5'-GAAATCCtGGGTAAl(3.3..3' (SEQ ID NO:223) reverse PCR ptrimer 5S-GTGCGCGGTGCTCACAGarCATC.3. (SEQ ID NO.224) Additionally, a synthetic oigonucleotide hybridization probe was constructed from the consensus DNA36460 sequence which had the following nucleotide sequence 5'CCCTACAGTCCAGTAGCAGGA~C3 (SEQ MD NO:225)

I-

In order to seme several libraies for a source of a full-length clone. DNA from the libraries was screened by PCR amplification with the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0272 gene using the probe oligonuicleetide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from humian fetal -lung tissue. DNAequening of the clones isolated as described above gave the full-lenigth DNA sequence for PR0272 [herein designated as UNQ239 (DNA4O620-1183)] (SEQ MD NO:220) and the derived protein sequencie for PROM7.

The entire nucleotide sequence of UINQ239 (DNA40620-1 183) is shown in Figure 79 (SEQ ID NO:22D).

Gone UNQ239 (DNA40620-l1893) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 35-37 and ending at the stop codon at micleotide positions 1019-1021 (Figure 79). The predicted polypetidc precursor is 328 amino acids long (Figure 80). Cone UNQ239 (DNA40620-1183) has been deposited with ATCC and is assigned ATCC deposit tn. ATCC 209388.

Analysis of the ammo acid sequence of the full-length PR0272 polypetide suggests that portions of it possess significant homology to the human and mouse reticulocalbin proteins, respectively. thereby indicating that PR0272 may be a novel reticulocalbin protein.

*.15 EXAMPLE 37: Isolation of eDNA Clones Encniag Human PRO294 A consensus DNA sequecwe was assembled relative to other EST sequences using phrap as described in Example I above. This consensus sequence is herecin designated DNA35731. Based on the DNA35731 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest. and 2) for use as probes to isolate a clone: of the full-length coding sequence for PR0294.

Forward and reverse PCR primers were synthesized: forward PCR primer nf) 5'-TGGTCTCGCACACCCIATC-3' (SEQ ID N0:228) fovodPC R=(12) 5*-CTGCTGTCCACAGOGGAG-3' (SEQ U) N'O:229) foinvard..E PCR msL( 13) 5'CTGACTCGr-' (SEQ ID N0:230) forward PCR Drimer (14) 5'-GAGATAGCAAflTCCGCC.3' (SEQ ID NO:23 1) revrsePCRg 5-TTCCTCAAGAGGGCAGCC-3' (SEQ ID NO:232) ree C rmr S'-C'rrGGCACCAATGTCCGAGATMr-3* (SEQ ID NO:233) Additionally, a synthetic oligonueceotidc hybridization probe was constructed from the consensus DNA35731 sequence which had the following nucleotde sequence hykdizaton probe 5'TCTAGAAGTGACGCGCGGGGCCTCCGAACCMGCraCc1lGo3' (SEQ ID NO:234) In order to screen several libraries for a source of a full-length clone. DNA from the libraries was screened by PCR arnplificatior with t PCR pd=ie pairs identified above. A positive library was then used~ to isolate clone encoding the PR0294 gene Using the probe oligonucleodde and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated from huiman fetal brain tisse. DN~eqmdng of the clones isolated as described above gave the full-lengdh DNA seque=c for PR0294 (herein designatied as 144 UNQ257 (I440604-1187) (SED ID NO:226) and the derive protein sequence for PR0294.

The entire nucleotide sequence of UNQ257 (1R40604-1187) is shown in Figure 81 (SEQ ID NO:226). Clone UN257 (DA40604-1187) contains a single open reading franre with an apparent translational initiation site at nucleotide positions 396-398 and ending at the stop codonat nucleotide positions 2046-2048 (Figure 81). The predicted polypeptide precursor is 550 amino acids long (Figure 82). Clone NQ257 (E1M40604-1187) has been deposited with ATCC and is assigned A'IC deposit no. 209394.

Analysis of the amino acid sequence of the full-length PRO294 polypeptide suggests that portions of it possess significant harology to portions of various collagen proteins, thereby indicating that PR0294 may be collagen-like nlecule.

EXAMP L 38: Isolation of cl Clones Encoding thman PR0295 A consensus IU. sequence was asserbled relative to other EST sequences using phrap as described in Ecanple 1 above. This consensus sequence is herein designated SIE35814. Based on the 1NA35814 consensus sequence, oliganucleotides were synthesized: 1) to identify by PCR a ct1rM 20 library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0295.

Forward and reverse PCR primers were synthesized: :forward PCR priner(.fl) 5' -CAL 'IOCA GOMCPTI-3' (SEQ ID N3:237) forward PCR priner(.f2) 5'-CCCAIGCATGACTfCCAG-3' (SED ID 10:238) forward PCR priner(.f3) 5'-TIGGCAGrICAIMGG-3' (SD2 ID NO:239) forward PCR priner(.f4) 5' -CCI3GGCAAAA3ZAAGC (SDQ ID NO:240) *reverse PCR primer(.rl) 5' -CCAGCXO3T-3' (SEB ID NO:241) Additionally, a synthetic oligonucleotide hybridization probe was constructed fran the consensus U0A35814 sequence which had the following nucleotide sequence.

hybridization probe 51 3I (SEQ ID ND:242) In order to screen several libraries for a source of a fulllength clone, MA frcan the libraries was screened by ICR anplification with the PCR priner pairs identified above. A positive library was then used to isolate clones encoding the PR0295 gene using the probe oligonucleotide and one of the PCR priners.

RNA for construction of the clNA libraries was isolated fran hunan fetal lung tissue. EA sequencing of the clones isolated as 145 described above gave the full-length aDa sequence for PRO295 (herein designated as t1N2258 (I2NA38268-1188) (SEQ ID NO:235) and the derived protein sequence for PI)2 The entire nucleotide sequence of LNM258 (ENA38268-l188) is shown in Figure 83 (SE)Q ID NO: 235). Clone UNQ258 (EN38268-1l88) contains a single open reading fraire with an apparent,-translational initiation site at nucleotide positions 153-155 and ending at the stop codon at nucleotide positions 1202-1204 (Figure 83). The predicted polypeptide precursor is 350 amino acids long (Figure 84). Clone UN1QR258 (EA38268-1188) has been deposited with A1TCC and is assigned AIVC deposit no. 209421.

Analysis of the amino acid sequence of the full-length PR0295 polypeptide suggests that portions of it possess significant hcxmology to the integrin proteins, thereby' indicating that PR0295 way be a novel integrin.

Ioai f~C~n~odn ~ma ~9 EC7InWL 39: The extracellular dcznain (BCD) sequences (including the secretion signal, if any) of frarn about 950 known secreted proteins from the Swiss-Prot public protein dat-abase were used to search expressed :20 sequence tag (ET) databases. The EST databases included public EST' databases GenBank) and a proprietary EST U-Zk database (LIFES~gm, Incyte Pharamceuticals, Pal Alto, Ch). The search was performed using the :caipuiter programn BLAST or BIAST2 (Altshul et al., Methods in Enizymology 266:460-480 (1996)) as a cczrparison of the ECD protein sequences to a 6 frame translation of the EST sequence. -Those carparisons resulIting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and asseabled into consensus EMA sequxences *with the progre-un phrap, (Phil Green, University of Vbashington, Seattle, Washingiton; http: //bozeman.rbt .washirigton ecl/phrap. docs /Phrap .htmnl).

Based on an expression tag sequence designated herein as T08294 identified in the above analysis, oligonucleotides were synthesized: 1) to identify b~y PRa crIsM library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0293.

A pair of PCR primrers (forward and reverse) were synthesized: forward PCRir 5'-AACAAGrAAGO~XCC= (SE72 ID NO:245) reverse pCRiz 5 '-AAACIG1ICAAGACCA'r=t-3' (SEQ ID O: 246) Additionally, a synthetic oligonucleotide hybridization probe was constructed frun the expr-ession sequence tag %ftich had the following 146 nucleotide sequence.

hybridization probe QAICCAGC-3' (SEBQ ID ND:247) In order to screen several libraries for a source of a fulllength clone, E M from the libraries was screened by PCR.anplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO293 gene using the probe oligonucleotide and one of the PCR priners.

RNA for construction of the cDNA libraries was isolated from human fetal brain tissue. EA sequencing of the clones isolated as described above gave the full-length MA sequence for PR0293 (herein designated as UNQ256 (ENA37151-1193)] (SEQ ID N0:243) and the derived protein sequence for PR0293.

The entire nucleotide sequence of UND256 (INA37151-1193) is 15 shown in Figures 85A-B (SEQ ID NO:243) Clone UNQ256 (ENA37151-1193) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 881-883 and ending at the stop codcn after nucleotide position 3019 of SB ID ND0:243, Figures 85A-B). The predicted polypeptide precursor is 713 amino acids long (Figure 86). Clone 20 UNQ256 (ENA37151-1193) has been deposited with AICC and is assigned AT'CC deposit no. ATICC 209393.

Analysis of the amino acid sequence of the full-length PRO293 polypeptide suggests that portions of it possess significant hamology to the NLRR proteins, thereby indicating that PR0293 may be a novel NLRR 25 protein.

EXAMPLE 40: Isolation of cCa Clones Encoding Haian PRO247 A consensus INA sequence was assembled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus 3 0 sequence is herein designated ENA33480. Based on the EIA33480 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cIEA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0247.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer 5'-CAACAAWIGAGnCCCAAGC-3' (SEQ ID NO:250) reverse PCR primer 5'-GA TAG GAGCG3' (SEBQ ID N0:251) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the ENA33480 expression sequence tag which had the following nucleotide sequence.

147 hybridization probe -CAACC1rGCOC0-3' (SE3 ID O3:252) In order to screen several libraries for a source of a fulllength clone, I fran the libraries was screened by PCR anplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0247 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the ca1A libraries was isolated froman human fetal brain tissue. IA sequencing of the clones isolated as described above gave the full-length IMA sequence for PRO247 [herein designated as UND221 (E1'A35673-1201) (SEQ ID N:248) and the derived protein sequence for PRO247.

The entire nucleotide sequence of 1NQ221 (EIN35673-1201) is shown in Figures 89A-B (SEQ D M:248). Clone UNQ221 (INA35673-1201) contains a single open reading frane with an apparent translational initiation site at nucleotide positions 80-82 of SEQ ID NO:248 and ending at the stop codon after nucleotide position 1717 of SEQ ID N3:249 (Figures 89A-B). The predicted polypeptide precursor is 546 amino acids long (Figure 88). Clone UNQ221 (INA35673-1201) has been deposited with AC and 20 is assigned A'TC deposit no. 209418.

Analysis of the amino acid sequence of the full-length PR0247 polypeptide suggests that portions of it possess significant hamlogy to the densin molecule and KIAAO231, thereby indicating that PR)247 may be a novel leucine rich repeat protein.

EXAMPIE 41: Isolation of c1lt Clones Encriing Hmnan PR0302, PRO303, PRO304, PRO307 and PRO343 Consensus EIA sequences were assebled relative to other EST sequences using phrap as described in Exanple 1 above. These consensus sequences are herein designated EItA35953, EIA35955, INA35958, EWA37160 and IlA30895. Based on the INA35953 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cIIA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO302.

PCR primers (forward and reverse) were synthesized: forward PCR primer 1 5' -GI-3'CAGGATrTA' (SEQ ID D0:263) forward PCR primer 2 5' -3CAGAM ITAAGGI'IG-3' (SEM ID 10:264) reverse PC primer 5' -AGIrAOCAACCOC I-3' (SE2 ID ND:265) Also, a synthetic oliganucleotide hybridization probe was constructed fran 148 the consensus INA35953 sequence which had the following nucleotide sequence hybridization probe 3' (SEQ ID NO:266) In order to screen several libraries for a source of a fulllength clone, DA from the libraries was screened by PCR anplification with the PCR priner pairs identified above. A positive library was then used to isolate clones encoding the PR0302 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cENA libraries was isolated fron human fetal kidney tissue (LIB228).

IA sequencing of the clones isolated as described above gave the full-length INA sequence for PRO302 [herein designated as UNQ265 (INA40370-1217)] (SEQ ID N0:253) and the derived protein sequence for PRO302.

15 The entire nucleotide sequence of UNQ265 (EMA40370-1217) is shown in Figure 89 (SEQ ID N3:253). Clone UNM265 (ENA40370-1217) contains a single open reading frae with an apparent translational initiation site at nucleotide positions 34-36 and ending at the stop codon at nucleotide positions 1390-1392 (Figure 89). The predicted polypeptide precursor is 452 amino acids long (Figure 90). Various unique aspects of the PRO302 protein are shown in Figure 90. Clone UNQ265 (IEA40370-1217) has been deposited with the ACC on November 21, 1997 and is assigned ATOC deposit no. ATC 209485.

Based on the ENA35955 consensus sequence, oligonucleotides were 25 synthesized: 1) to identify by PCR a cI'A library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO303.

pair of PCR priners (forward and reverse) were synthesized: forward PCR primer 5'-GCAWTICA'IGACATIGCC-3' (SEQ ID N1:267) reverse PCR priner 5' -GAA C CA AAC (SEQ ID NO:268) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus IiA35955 sequence which had the following nucleotide sequence: hybridization probe 5' (SEQ ID NO:269) In order to screen several libraries for a source of a fulllength clone, ENA from the libraries was screened by PCR anplification with the PCR priner pairs identified above. A positive library was then used to isolate clones encoding the PRO303 gene using the probe oligonucleotide and 149 one of the PCR priners.

RNA for construction of the cIMA libraries was isolated frcn hurrman fetal lung tissue UA sequencing of the clones isolated as described above gave the full-length NA sequence for PR0303 [herein designated as UQ266 (MA42551-1217)] (SEQ ID ND:255) and the derived protein sequence for PR0303.

The entire nucleotide sequence of U2266 (EA42551-1217) is shown in Figure 91 (SED ID NO:255). Clone UL266 (ENA42551-1217) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 20-22 and ending at the stop codon at nucleotide positions 962-964 (Figure 91). The predicted polypeptide precursor is 314 amino acids long (Figure 92). Various unique aspects of the PRO303 protein are shown in Figure 92. Clone UNQ266 (DN42551-1217) has been deposited on 15 Novenmber 21, 1997 with the =tC and is assigned =MC deposit no. ATCC *Ci" 209483.

Based on the DAN35958 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the 20 full-length coding sequence for PR0304.

Pairs of PCR priners (forward and reverse) were synthesized: forward PCR primer 1 -GOGAAGOCAGAAIGGGACIA G3 (SEQ ID NO:270) S. forward PCR priner 2 5' -CAGCICc(CACtATGTIC-3' (SEQ ID NO:271) forward PCR priner 3 5'-TAC1GGGIGGCAGCAAC-3' (SEQ ID NO:272) reverse PCR pri5er 5' GG AGCG'IM3' (SEQ ID NO:273) Additionally, a synthetic olignucleotide hybridization probe was constructed fran the consensus rNA35958 sequence which had the following nucleotide sequence hybridizationprobe S'GooTcICA AAw m maw o 3 (SEQ ID NO:274) In order to screen several libraries for a source of a fulllength clone, CA fraom the libraries was screened by PCR anplification with the CR prinrer pairs identified above. A positive library was then used to isolate clones encoding the PRO304 gene using the probe olignucleotide and one of the PCR prinrers.

RA for construction of the cEIA libraries was isolated fran 22 week human fetal brain tissue (LIB153).

EM sequencing of the clones isolated as described above gave the full-length CNA sequence for PR03-4 [herein designated as U=267 150 (IA39520-1217) (SE) ID NO:257) and the derived protein sequence for PR0304.

The entire nucleotide sequence of MNQ267 (ENA39520-1217) is shown in Figure 93 (SEQ ID N:257). Clone tNQ267 (MA39520-1217) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 34-36 and ending at the stop codon at nucleotide positions 1702-1704 (Figure 93). The predicted polypeptide precursor is 556 amino acids long (Figure 94. Various unique aspects of the PR0304 protein are shown in Figure 94. Clone UNQ267 (I1A39520-1217) has been deposited with ACC on Noverber 21, 1997 and is assigned AO deposit no.

ATC 209482.

Based on the IEA37160 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cEA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0307.

*Pairs of PCR priners (forward and reverse) were synthesized: ****!forwnard FPCR priser 1 5 '-GGCAGGGAICAGGCIOC-3' (SEQ ID N:275) forward PCR priner 2 5' -GGCIGACAGCAGTIC-3' (SEQ ID NO:276) forward PCR.priner 3 5' -IGACAAACGA CCAGG3-3' (SEQ ID NO:277) 20 reverse PCR priner 5' -AGCA'ICATIGCIrGAGAGCAAG-3 (SEQ ID NO:278) Additionally, a synthetic olignucleotide hybridization probe was constructed fran the consensus I[A37160 sequence ihich had the following nucleotide sequence hybridization probe -25 (SEQ ID ND:279) In order to screen several libraries for a source of a fulllength clone, ENA frn the libraries was screened by PCR anplification with the PCR pairs identified above. A positive library was then used to isolate clones encoding the PRO)307 gene using the probe olignucleotide and one of the PCR priners.

RNA for construction of the clA libraries was isolated from human fetal liver tissue (LIB229).

IA sequencing of the clones isolated as described above gave the full-length OHA sequence for PRO307 (herein designated as UN,270 (IEA41225-1217)) (SED ID NO:259) and the derived protein sequence for PR0307.

The entire nucleotide sequence of UNQ270 (IEA41225-1217) is shon in Figure 95 (SEQ ID NO:259). Clone UQI270 (EmA41225-1217) contains a single open reading frane with an apparent translational initiation site 151 at nucleotide positions 92-94 and ending at the stop codon at nucleotide positions 1241-1243 (Figure 95). The predicted polypeptide precursor is 383 amino acids long (Figure 96). Various unique aspects of the PRO307 protein are shown in Figure 96. Clone UtQ270 (ENA41225-1217) has been deposited with ATC on November 21, 1997 and is assigned Al'CC deposit no.

ATCC 209491.

Based on the IENA30895 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cENA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0343.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer 5' -O IGAG ATAA' (SEQ ID N0:280) reverse PCR prir 5'-G

G

CICAIr1CAGTA-3' (SEQ ID N: 281) 15 Additionally, a synthetic olignucleotide hybridization probe was constructed from the consensus IINA30895 sequence which had the following nucleotide sequence hybridization probe

T

2 0 GAAG GC GIGOCOCrACT-3' (SEQ ID NO:282) In order to screen several libraries for a source of a fulllength clone, EA from the libraries was screened by PCR amplification with the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PRO343 gene using the probe olignucleotide and one of the PCR primers.

:.RNA for construction of the clA libraries was isolated frcmn human fetal lung tissue (LIB26).

NA sequencing of the clones isolated as described above gave 3 0 the full-length DA sequence for FR0343 (herein designated as UQ302 (IENA43318-1217) is shown in Figure 97 (SEQ ID N0:261) and derived protein sequence for PR0343.

The entire nucleotide sequence of UNQ302 (ENA43318-1217) is shown in Figure 97 (SEQ ID N0:261). Clone UNV302 (DNA43318-1217) contains a single open reading, frame with an apparent translational "initiation site at nucleotide positions 53-55 and ending at the stop codon at nucleotide positions 1004-1006 (Figure 97). The predicted polypeptide precursor is 317 amino acids long (Figure 98). Various unique aspects of the PRO343 protein are shown in Figure 98. Clone UNQ302 (EMA43318-1217) has been 152 deposited with ATCC on November 21, 1997 and is assigned AIC deposit no.

ATIC 209481.

EXMPLE 42: Isolation of cEM Clones Encoding DHman PR0328 A consensus MlA sequence was assembled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus sequence is herein designated ENA35615. Based on the ENA 35615 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cENA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO328.

Forward and reverse PCR primers were synthesized: forward PCR primer 5' -TCCAGT TIGA'IGC-3' (SEQ ID N0:285) reverse PCR primer 5'-CTCAT ATIO ACCAC ATIOG-3' (SEBQ ID N0:286) Additionally, a synthetic olignucleotide hybridization probe was 15 constructed from the consensus UNA35615 sequence which had the following nucleotide sequence.

hybridization probe GGAA-3 (SEQ ID NO:287) In order to screen several libraries for a source of a full- 20 length clone, INA fran the libraries was screened by PCR anplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO328 gene using the probe olignucleotide and one of the PCR priners.

RNA for construction of the cENA libraries was isolated from 25 human fetal kidney tissue.

IEA sequencing of the clones isolated as described above gave *the full-length ENA sequence for PRO328 (herein designated as UNQ289 (EMA40587-12131) (SEQ ID N0:284) and the derived protein sequence for PRO328.

The entire nucleotide sequence of UNQ289 (ENA40587-1231) is shown in Figure 99 (SEB ID N0:283). Clone UNQ289 (IA40587-1231) contains a single open reading frane with an apparent translational initiation site at nucleotide positions 15-17 and ending at the stop codon at nucleotide positions 1404-1406 (Figure 99). The predicted polypeptide precursor is 3 5 463 amino acids long (Figure 100). Clone UNQ289 (ENA40587-1231) has been deposited with ATCC and is assigned AICC deposit no. ATOC 209438.

Analysis of the amino acid sequence of the full-length PRO328 polypeptide suggests that portions of it possess significantly homology to the human glioblastana protein and to the cysteine rich secretory protein 153 thereby indicating that PR0328 my be a novel gliablastara protein or cysteine rich secretory protein.

EXAHPLE 43: Isolation of cEVA Clones EFoding Eki PRM35, PRO331 or PROD326 A consensus E1; sequence was assernbled relative.to other EST sequences using phrap as described in Etaxrple 1 above. This consensus sequence is herein designated E1M36685. Based on the EM'6685 consensus sequence, and Incyte ESr sequence no. 2228990, oligonucleotides were synthesized: 1) to identify by PCR a crZ4 library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0335, PRO331 or PR0326.

Forward and reverse PCR primers were synthesized for the determination of PR0335: forward FCR prin r 5' -GGAACAAtICAi'A-3' (SB2 ID NO:294) foriard PC rier 5' -CCTAAAC"1GAACTGGACEA-3' (SE ID ND):295) forward P primer 5' -GIZGAGC AC7 NX-3' (SEQ ID N):296) forward P primer 5' -AIC2CvAGCICA(=AA-AAG1=-3' (SEp ID NTh 297) reverse FOR primer 5' -C-TI3CA1r~rAAAA flTt-3' (SD2 ID N:2 98) I :20 reverse PC praner 5' -GOiCrii1GAhALA-3' (SE2 ID N1S:299) reverse PM primer 5' -G1,CCI('1(XfTAC-3' (SEQ ID NX1:300) Additionally, a synthetic oligonucleotide hybridization probe was constructed for the determination of PR0335 which had the following nucleotide sequence.

hybridization probe 51 AGAAACAGOCG=AC-3 (SBQ ID ND:301) Forward and reverse PCR priners were synthesized for the determination of PR0331: forward primer 5' 3O'ITI1AACX.'P11ACINA1;It)3-3' (SEe ID N:302) reverse C rprier 5' -Ofl C1tIA't-3' (SEYQ ID ND:303) Additionally, a synthetic oligonucleotide hybrization probe was constructed for the deterination of PRO33l which had the following nucleotide sequence hybridization probe 51 3 (SE) ID NO:304) Forward and reverse PCR prizers were synthesiied for the determination of PR0326: forward FGR prier 5' -AcrocAAGGAAArcGAr c (SE2 ID 143:305) reverse IC primer 5' -TAOCAGOIGAGGAYOGOOAC (SEY MI 14:306) Additionally, a synthetic olignucleotide hybrization probe was constructed 154 for the determination of PRO331 which had the following nucleotide sequence hybridization probe 'IiGC-3''ICTAG (SEQ ID NO:307) In order to screen several libraries for a source of a fulllength clone, 1NA from the libraries was screened by PCR anplification with the PCR priner pairs identified above. A positive library was then used to isolate clones encoding the PR0335, PRO331 or PR0326 gene using the probe olignucleotide and one of the PCR priners.

RNA for construction of the cMA libraries was isolated fromn human fetal kidney tissue (PR0335 and PRO326) and human fetal brain (PRO331).

ssA sequencing of the clones isolated as described above gave the full-length NA sequence for PR0335, PRO331 or PRO326 (herein designated as SEQ ID NOS:288, 290 and 292, respectively; see Figure 103A-B, 15 105 and 107, respectively], and the derived protein sequence for PRO335, .6 PRO331 or PR0326 (see figures 104, 106 and 108, respectively; and the derived protein sequence for PRO335, PRO331 or PR0326 (see Figures 104, 106 and 108, respectively; SEQ ID NOS:289, 291 and 293, respectively).

The entire nucleotide sequences are shown in Figures 103A-B, 20 105 and 107, deposited with the ATTC on June 2, 1998, November 7, 1997 and November 21, 1997, respectively.

Analysis of the amino acid sequence of the full-length PR0335, PR0331 or PR0326 polypeptide suggests that portions of it possess significant homology to the LIG-1 protein, thereby indicating that PR0335, 25 PR0331 and PR0326 may be a novel LIG-1-related protein.

EXAMPLE 44: Isolation of cEaL Clones Encoding Hmann PR332 Based upon an BCD homology search performed as described in SExample 1 above, a consensus EMA sequence designated herein as ENA36688 was assembled. Based on the E1A36688 consensus sequence, oligonucleotides were synthesized to identify by PCR a cENA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PRO332.

A pair of PCR priners (forward and reverse) were synthesized: 5' -GCA3T OGX3AGACITOC-3' (SBQ ID NO:310) -GCGGCCA3CnlOTIGAAAN3-3' (SEBQ ID ND:311) A probe was also synthesized: 3CA (SEQ ID NO:312) In order to screen several libraries for a source of a full- .155 length clcne, CN fran the libraries was screened by CRamplification with the PCR pruner pair identified above. A positive library was then used to isolate clones encoding the PR0332 gene using the probe olignucleotide and one of the PCR prixters.

RNA for construction of the cENA libraries was isolated from a human fetal liver library (L1B2 29).

I14A sequencing of the clones isolated as described above gave the full-length I1'R sequence for CM440982-1235 and the derived protein sequence for PR0332.

The entire nucleotide sequence of r.NA40982-1235 is showin in Figures 109A-B (SEQ ID NO1:308). Clone MJ440982-1235 contains a single open reading frame (with an apparent -translational initiation site at nucleotide positions 342-344, as indicated in Figures 109A-B). The predicted polypeptide precursor is 642 amino acids long, and. has a calculated mnolecular weight of 72,067 (pI:6.

6 Clone UI40982-1235 has been deposited with AltCC and is assigned A=I' deposit no. A= 209433.

Based on a BLASr and FastA sequence aligimnt. analysis of the full-length sequence, PRO332 shows about 30-40% amino acid sequence identity with a series of know~ proteoglycan sequces, including, for example, fibramodulin and fibrcimodulin precursor sequence of various species (FrL)BOVIN, F'DCHIICK, FVM lDRAT, FT'fDJW F-DJU1'AN, PJR36773), ostearmedulin sequences (AB010l4j1, AB0078481), decorin :sequences (CFU83141.1, (rU0339 1 pR42266, FPjR42267, P R42260, P R89439), keratan sulfate proteoglycans (B1114836CL_1, AF02289..LI), corneal proteoglycan (AF0222561j), anid bone/cartilage proteoglycans; and proteoglycane precursors (PGSl.BOVIN, PS2-PMSE, EGS2JIUMiN).

EXAMP~LE 45: Isolatici of cIZa Clonea Ikncodizag Ilmu PRO334 A consensus ENA sequence %as assembled relative to other EST sequences using phrap as described in Excanple 1 above. Based on the consensus sequence, oligonucleotides ware synthesized: 1) to identify by PCR a cM library that contained the sequence of interest, and 2) for use as probes isolate a clone of the full-length coding sequence for PR0334.

Forward and reverse PCR prizvers were synthesized for the determination of PIR0334: forwvard pCRir 5' -3 (SEp ID NO: 315) reverse JPCRi~ 5.1 -TiucAI'IrAGCAACCk=UIAO-3' (SE)2 ID NO: 316) Additionally, a synthetic olignucleotide hybridization probe was constructed for the determination of PRO334 which had the following 156 nucleotide sequence.

hybridization probe (SEQ ID NO:317) In order to screen several libraries for a source of a fulllength clone, MA franom the libraries was screened by PCR anplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0334 gene using the probe oligonucleotide and one of the PCR primers.

Human fetal kidney cINA libraries used to isolate the cENA S 10 clones were constructed by standard methods using cnamercially available reagents such as those from Invitrogen, San Diego, CA.

EM sequencing of the clones isolated as described above gave **the full-length DNA sequence for PR0334 (herein designated as DA41379- 1236] (SEQ ID N3:313) and the derived protein sequence for PO334.

15 The entire nucleotide sequence of ENA41379-1236 (also referred to as UNQ295) is shown in Figure 109 (SEQ ID N0:313). Clone EA41379-1236 0: contains a single open reading framre with an apparent translational initiation site at nucleotide positions 203-205 and ending at the stop codon at nucleotide positions 1730-1732 (Figure 109). The predicted 20 polypeptide precursor is 509 amino acids long (Figure 110). Clone ".ENA41379-1236 has been deposited with AMTC and is assigned ACC deposit no.

ATCC 209488.

Analysis of the amino acid sequence of the full-length PR0334 polypeptide suggests that portions of it possess significant homology to the fibulin and fibrillin proteins, thereby indicating that PR0334 may be a novel member of the BGF protein family.

EXAMPLE 46: Isolation of c2a Clones Eooding HRan PRO346 A consensus DA sequence was identified using phrap as described in Example 1 above. Specifically, this consensus sequence is herein designated DMA38240. Based on the IMA38240 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cIEA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length PR0346 coding sequence.

RNA for construction of the ca libraries was isolated from human fetal liver. The cNA libraries used to isolate the cDMA clones were constructed by standard methods using cummercially available reagents Invirtogen, San Diego, CA; Clontech, etc). The cNA was primed with oligo dTr containing a NotI site, linked with blunt to Sail hemnikinased 157 adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRK5D that does not contain the SfiI site; see, Holnes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

A cMA clone was sequenced in entirety. The entire nucleotide sequence of ENA44167-1243 is shown in Figure 111 (SEQ ID NO:318). Clone MA44167-1243 contains a single open reading frame with an apparent translational initiation site-at nucleotide positions 64-66 (Fig. 113; SEQ ID JND:318). The predicted polypeptide precursor is 450 amino acids long.

Clone EIA44167-1243 has been deposited with AT and is assigned AICC deposit no. AICC 209434 (designation IEA44167-1243).

~Based an a BLAST, BALST-2 and FastA sequence alignent analysis (using the ALIGNJ carputer program) of the full-length sequence, PR0346 shows amino acid sequence identity to carcinoembryonic antigen The oligonucleotide sequences used in the above procedure were the following: OLI2691 (38240.f1) -GA-I3CIICAAGC (SEQ ID NO:320) OLI2693 (38240.rl) 5' -C 3- 3' (SEQ ID NO:321) OLI2692 (38240.p1) I-3' (SEQ ID NJ:322) EIMUE 47: Isolation of dcAI Clones Ecoding Imnan P1RO268 A consensus IIJA sequence was asserbled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus sequence is herein designated 1EA35698. Based on the DIA35698 consensus sequence, olignucleotides ware synthesized: 1) to identify by PCR a cEA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO268.

Forward and reverse PCR printers were synthesized: forward PCR priner 1 5' -'-A'OAAGdGAAA (SEQ ID ND:325) forward PCR prirmer 2 5' -TAMIGKAMAMGGAOIGOC-3' (SEp ID NO3:326) forward PCR printer 3 5' -'I33CAGGGICAGLITTAGATIG-3' (SBE ID NO: 327) reverse PCR priner -T CA AAAC'-3' (SED ID NO:328) Additionally, a synthetic olignucleotide hybridization probe was constructed fromn the consensus lW35698 sequence which had the following nucleotide sequence 157a hybridization probe 3' (SEQ ID M :329) In order to screen several libraries for a source of a fulllength clone, IWiA froman the libraries was screened by PCR anplification with the PCR priner pair identified above. -A positive library was then used to isolate clones encoding the PR268 gene using the probe olignucleotide and one of the PCR priners.

RNA for construction of the clIA libraries was isolated from human fetal lung tissue. IA sequencing of the clones isolated as described above gave the full-length M2 sequence for PR0268 [herein designated as UIN)235 (IUA39427-1179)] (SEQ ID ND:323) and the derived protein sequence for PRO268.

The entire nucleotide sequence of IUN235 (I39427-1179) is shown in Figure 113 (SDEQ ID NO: 323). Clone U12235 (IW39427-1179) contains 15 a single open reading frame with an apparent translational initiation site at nucleotide positions 13-15 and ending at the stop codon at nucleotide positions 853-855 (Figure 113). The predicted polypeptide precursor is 280 amino acids long (Figure 114). Clone UNQ235 (I39427-1179) has been deposited with ALtCC and is assigned AtCC deposit no. AICC 209395.

20 Analysis of the amino acid sequence of the full-length PRO268 **polypeptide suggests that it possess significant hamology to protein 9disulfide isacerase, thereby indicating that PRO268 may be a novel protein disulfide isarerase.

EKAMP2E 48: Isolation of CM& Clones Encoding Han PRO330 A consensus U1 sequence was assembled relative to other EST *sequences using phrap as described in Eanple 1 above. This consensus ae nteIA53 osnu sequence is herein designated ITA35730. Based on the E135730 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cIWA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO330.

Forward and reverse ECR priners were synthesized: forward PCR priner 1 5' CC'CACAATFIrCCAGA-3' (SED ID ND:332) forward PCR priner 2 O CI UIG-ICCAG-3' (SEQ ID N:333) forward PCR priner 3 5'-GUlrIC AAcAAciIW'CfII-3' (SEQ ID NO:334) reverse PCR priner 5' -ACACItACAI'GOC IGX3TI'I-3' (SE2 ID NO:335) Additionally, a synthetic olignucleotide hybridization probe was constructed from the consensus which had the following nucleotide sequence 157b hybridization probe GCACAACIATITA (SEQ ID ND:336) In order to screen several libraries for a source of a fulllength clone, IA fran the libraries was screened by PCR anplification with the PCR priner pair identified above. A positive library was then used to isolate clones encoding the PRO330 gene using the probe olignucleotide and one of the PCR priners.

RNA for construction of the cWA libraries was isolated from human fetal lung tissue. IUA sequencing of the clones isolated as described above gave the full-length UA sequence for PRO330 (herein designated as UNQ290 (IM40603-1232)] (SEQ ID N1:330) and the derived protein sequence for PR0330.

The entire nucleotide sequence of .Q290 (IUA40603-1232) is shown in Figure 115 (SE2 ID NO:330). Clone UN290 (EA40603-1232) contains 15 a single open reading frare with an apparent translational initiation site at nucleotide positions 167-169 and ending at the stop codon at nucleotide positions 1766-1768 (Figure 115). The predicted polypeptide precursor is 533 amino acids long (Figure 116). Clone UNQ290 (EA40603-1232) has been deposited with MICC and is assigned A'TCC deposit no. A=C 209486 on November 21, 1997.

Analysis of the amino acid sequence of the full-length PR0330 polypeptide suggests that portions of it possess significant homnlogy to nouse prolyl 4-hydroxylase alpha subunit protein, thereby indicating that PR0330 may be a novel prolyl 4-hydroxylase alpha subunit polypeptide.

EAMPLE 49:. Isolation of c1NA Clones Enhoding uman PRD310 ~A consensus IIA sequence was asseSrbled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus sequence is herein designated M40553. Based on the [1A40553 consensus sequence, olignucleotides ware sythesized: 1) to identify by PCR a cENA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence.for PRO310.

Forward and reverse PCR priners were synthesized: forward FPCR prinmer 1 5' -00CCCAAGN0CGT7=AAa0G-3' (SEQ ID NO: 341) forward PCR prinmer 2 5' -CIIGGPITIOCTIGCA (SE3Q ID i):342) reverse PCR priner 5' AAM tCAA TACCCC-3' (SEQ ID EN:343) Additionally, a synthetic olignucleotide hybridization probe was constructed fran the consensus wihich had the following nucleotide sequence 157c hybridization probe -3'I3X GTIGACA1IGCTI T (SEQ ID Nb:344) In order to screen several libraries for a source of a fulllength clone, DlA from the libraries was screened by PCR anplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO310 gene using the probe olignucleotide and one of the PCR priners.

IR for construction of the cZNA libraries was isolated fromn human fetal liver tissue. EIA sequencing of the clones isolated as described above gave the full-length IA sequence for PRO310 [herein designated as (ElA43046-1225) (SEQ ID NO:339) and the derived protein sequence for PRO310 (SE2 ID 17:340).

The entire nucleotide sequence of (IEI43046-1225) is shown in Figure 119 (SEQ ID ND:339). Clone (IM43046-1225) contains a single open 15 reading frame with an apparent translational initiation site at nucleotide positions 81-83 and ending at .the stop codon at nucleotide positions 1035- 1037 (Figure 119). The predicted polypeptide precursor is 318 amino acids long (Figure 120) and has a calculated mrolecular weight of approximately 36,382 daltons. Clone (IN43046-1225) has been deposited with ATCC and is 20 assigned AIC deposit no. A'IC 209484.

Analysis of the amino acid sequence of the full-length PRO310 polypeptide suggests that portions of it possess hrology to C. elegans proteins and to fringe, thereby indicating that PRO310 may be involved in developnent.

EXAMPIE 50: Isolation of CESI. clones EncodLiwg an PR0339 An expressed sequence tag (ESr) INA database (LIFESE2' 5 Incyte Pharnmaceuticals, Palo Alto, CA) was searched and Emrs were identified. An assembly of Incyte clones and a consensus sequence was forned using phrap as described in Exanple 1 above.

Forward and revers PCR priners were synthesized based upon the assaenbly-created consensus sequence: forward PCR prinrer 1 5' -GCaAII( IIG3-3' (SED ID NO:345) forward PCR priner 2 5' -CC'ICNIGPAACCGIt-3' (SEQ ID 1NO:346) forward PCR priner 3 5'-GlGIGACACAOMIGWC-3' (SEQ ID N:43) forward PCR primer 4 5' -GAC0CAfCTIC 'IGO-3' (SEQ ID NO:44) reverse PCR prinmer 1 5' 3' (SEQ ID reverse PCR prirer 2 5' -CGC3IG7G O-3' (SED ID NO:46) Additionally, a synthetic olignucleotide hybridization probe was N:1Akrot\Keeptspecilg3178-98*2.doc 10108/00 157d constructed fran the consensus sequence which had the following nucleotide sequence.

hybridization probe ACXCA A AGCCA-3' (SEQ ID NO: 47) In order to screen several libraries for a source of a fulllength clone, E[A fran the libraries was screened by PCR anplification with the PCR priner pairs identified above. A positive library was then used to isolate clones encoding the PCR339 gene using the probe oligonucleotide and one of the PCR prinmers.

RNA for constructing of the cl:A libraries was isolated frnom human fetal liver tissue. A cINA clone was sequenced in entirety. The entire nucleotide sequence of DNA43466-1225 is shown in Figure 117 (SEQ ID 10:337). Clone ENA43466-1225 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 333-335 and 15 ending at the stop codon found at nucleotide positions 2649-2651 (Figure 117; SEQ ID N0:337). The predicted polypeptide precursor is 772 amino acids long and has a calculated nolecular weight of approximately 86,226 daltons. Clone ENA43466-1225 has been deposited with ATC and is assigned ATCC deposit no. ATCC 209490.

20 Based on a BLAST and FastA sequence alignment analysis (using the ALIGN computer program) of the full-length sequence, PRO339 has hamology to C. elegans proteins and collagen-like polymer sequences as well as to fringe, thereby indicating that PRO339 nay be involved in development or tissue growth.

EXAMPLE 51: Isolation of eNra Clones Encoding Hman PRO244 A consensus INA sequence was assembled relative to other EST sequences using phrap as described in Exanple 1 above. Based on this consensus sequence, oligonucleotides were synthesized to identify by PCR a cENA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PRO244.

A pair of PCR priners (forward and reverse) ware synthesized: 5'-TICAGCTICTGGGATGrAGGG-3' (30923.fl) (SEQ ID NO:377) 5'-TAT'ICCACCATrrCACAAACO-3' (30923.rl) (SEQ ID NO:378) A probe was also synthesized: TIGG-3' (30923.pl) (SEQ ID NO:379) In order to screen several libraries for a source of a full-length clone, mNA fran the libraries was screened by PCR anplification with the PCR 157e primer pair identified above. A positive library was then used to isolate clones encoding the PRO244 gene using the probe olignucleotide and one of the PCR primers.

RNA for construction of the cENA libraries was isolated from human fetal kidney library. ENA sequencing of the clones isolated as described above gave the full-length RA sequence and the derived protein sequence for PRO244.

The entire nucleotide sequence of PRO244 is shown in Figure 121 (SBQ ID NO:375). Clone 1NA35668-1171 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 106- 108 (Fig. 121). The predicted polypeptide precursor is 219 amino acids long. Clone ENA35668-1171 has been deposited with AT C (designated as [I A35663-1171) and is assigned ATCC deposit no. ATC 209371. The protein has a cytoplasmic dcmain (aa 1-20), a transmembrane domain (aa 21-46), and 15 an extracellular dcuain (aa 47-219), with a C-lectin domain at aa 55-206.

Based on a BLAST and FastA sequence alignment analysis of the full-length sequence, PR0244 shows noitable ami- add sequence identity to hepatic lectini gallus gallus (43 HIC hp120-binding C-tYPe lectin (42%) macrophage lectiri 2 (HUMHML2-1, 41 and sequenc-e PR32199 (44%) 10CAMPLE 52: Use of PRO Poypetide-Encoftn Nucleic Acid as Hybridization Probes The following method describes use of a nucleotide sequence encoding a PRO polypeptide as a hybridization probe.

DNA comprising the coding sequence of of a PRO polypeptide of interest as disclosed herein may be employed as a probe or used as a basis from which to prepare probes to screen for homologous DNAs; (such as those encoding naturally-occurring variants of the PRO polypeptide) in human tissue cDNA libraries or human tissue genomic libraries.

Hybridization andi washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled. PRO polypeptide-encoding nucleic acid-derived probe to the filters is performed in a solution of 50 fonnamide. 5x. SSC. 0. 1% SDS. 0. 1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42 0 C for 20 hours. Washing of the filters is performed in an aqueous solution of Ohl SSC and 0.1l% SDS at 42C.

o DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO polypeptide can then be identified using standard techniques known in the art.

EAML.53- Expression of PRO Polvoentides in E. coli This example illustrates preparation of an unglycosyland form of a desired PRO polypeptide by recombinant expression in E. ccli.

The DNA sequence encoding the desired PRO polypeptide is initially amplified using selected PCR primers.

TIbrprimiers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar er aL.. fkn. 2:95 (1977)) which contains genes for anipicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector wUi preferably include sequences which encode for an antibiotic resistance gene, a tip promoter, a polyhis leader (including the first six ST1l codons, polyhis sequence, and enteroldnase cleavage site). the specific PRO polypeptide coding region, lamibda transcriptional terminator. and an argU gene.

The ligation mixture is then used to tiansform, a selected E. coli strain using the methods described in Sambrook et al., Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plassnid DNA can be isolatd and confirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid cultur medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to Jinoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.

After culturing fte cells for several more hours, the cells can be harvested by Centrifugation. The cell pellet obtained by the centrifugation can be solubjijzed using various agents known in thc art, and the sohibilized PRO polypeptide can then be purified using a metal chelating colum under conditions that allow tight binding of the protein.

PRO]IS7, PR0317, PR0301, PR0224 and PR0238 were successfully expressed in E. co/i in a poly-His tagged form, using the following procedure. The DNA encoding PROWS, PR0317, ORO30I, PR6224 or PR0238' was initially amiplified using seetdPCR primers. The primers contained restriction enzme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelaton column. and proteolytic removal with eniterokinase. Thec PCRZ-anipliflcd. poly-His tagged sequences were then ligated into an expression vector, whichwas used to transform an E. coil host based on strain 52 (W31 10 fuihA(tonA) Ion galE rpoHzs~brRts;) clpP(laclq).

Transformants were first grown in LB containing 50 ng/ml carbenicillin at 30@C with shakig until an O.D.600 of 3-S was reacbed. Cultures were then diluted 50-100 (old into CRAP media (prepared by mixing 3.57 g (NHASO 4 0 0.71 g sodium citrate-2H20, 1.07 g KCI, 536 g Difco yeast extract 5.36 g Sheffield hycase SF in 500 miL water, V 0 as well as 110mrM MPOS. pH 7.3, 0.55 (wtv) glucose and 7 mM MgSO4) and grown for approximately 20-30 hours at 30*C with shaking. Samples were removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cWls. Cell pellets were frozen until purification and refolding.

E. coli paste from 0.5 to I L fermentations (6-10 g pellets) was resuspended in 10 volumes (wlv) in 7 M guanidinr. 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium teathionate is added to make final concentrations of 0.lM and 0.02 M. respectively, and the solution was stirred overnight at 40C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution was centrifuged at 40,000 e rpm in a Bockman Ukracentige for 30 min. The supernatant was diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine. 20 mM Tris, pH 7.4) and filtcred through 0.22 micron filters to clarify. Depending the 0* clarified extract was loaded onto aS5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochemn, Utrol grade), pH 7.4. Thec protein was eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at VC. Protein concentration was estimated by its absorbance at 280 am using th calculated extinction coefficient based on its amino acid sequence.

The proteins were refolded by diluting sample slowly into freshly prepared refolding buffer consisting of:, mM. Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine. 20 mM glycine and I mM EDTA. Refolding volums were chosen so that the final protein concentration was between 50 to 100 micrograms/mI. The refolding solution was stirred gently at VC for 12-36 hours. The refolding reaction was quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately Before further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to 2-10% final concentration. T7he refolded protein was chruawgraphed on a Pores RI/H reversed phase colum using a mobile buffer of 0. 1% TFA-with elution with a gradient of acetonitrilc from 10 to 80%. Aliquots of fractions with A280 absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were pooled. Generally, the properly refolded species of most proteins are cluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form. the reversed phase step also removes endotoxin from the samples.

Fractions containing the desired folded PR0187, PRO317, PRO301, PRO224 and PR0238 proteins, respectively, were pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution.

Pteins were formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.

EXAMPLE 54: Exnression of PRO Polvpeotides in Mammalian Cells This example illustrates preparation of a glycosylated form of a desired PRO polypeptide.by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published March 15. 1989). is employed as the expression vector.

S" Optionally, the PRO polypcptide-ncoding DNA is ligated into pRKS with selected restriction enzymes to allow insertion of the PRO polypeptide DNA using ligation methods such as described in Sambrook et al., spa. The resulting vector is called pRK5-PRO polypeptide.

15 In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) arc grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 pg pRKS-PRO polypeptide DNA is mixed with about 1 pg DNA encoding the VA RNA gen [rhimnappaya et al., £Cll. 21:543 (1982)] and dissolved in 500 pl of 1 mM Tris-HCI. 0.1 mM EDTA. 0.227 M CaCI. To this nxture is added, dropwise, 500 pl of 50 mM HEPES (pH 7.35), 20 280 mM NaCI, 1.5 mM NaPO,, and a precipitate is allowed to form for 10 minutes at 25'C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.

Approximately 24 hours after the transfections the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 pCi/ml "S-cysteine and 200 pCi/ml "S-methionine. After a 12 Shour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15 SDS gel.

The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.

In an alternative technique, PRO polypeptide may be introduced into 293 cells transicntly using the dextran sulfate method described by Sompasyrac ct al., Proc Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 pg pRKS-PRO polypeptide DNA is added. The cells are first concentrated from the spinner flask by ccntrifugation and washed with PBS. The DNA-dcxtran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 pg/ml bovine insulin and 0.1 ptg/ml bovine transferrin. After about four days, the conditioned media is ccntrifuged and filtered to remove cells and debris. The sample containing expressed PRO polypeptide can then be concentrated and purified by any selected method, such as dialysis andor column chromatography.

In another embodiment, PRO polypeptides can be expressed in CHO cells. The pRKS-PRO polypepdde can be transfacted into CHO cells using known reagents such as CaPO, or DEAE-deXtuin. As described above, the cell culmmrs can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as "S-methionine. After determining the presence of PRO polypeptide, the culture medium may be replaced with se=u free medium. Preferably, the culturs arn incubated for about 6 days, and thenthie conditioned medium is harvested. The medium containing the expressed PRO polypeptide can then be concentrted anid purified by any selected method.

Epitope-tagged PRO polypeptide may also be expressed in host CH0 cells. The PRO polypeptide may be subcloncd out of the pRKS vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-bis tag into a Baculovinis; expression vector. The poly-bis tagged PRO polypeptide insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones.

Finally, the CHO cells can be transfeeted (as described above) with the SV40 driven vector. Labeling may be performd, as described above, to verify expression. The culture mneditum containing the expressed poly-His tagged PRO polypeptide can then be concentrated and Purified by any selected method, such as by Ni 2 +-chlatc affinity chromatograhy.

PRO21 1, PR0217, PROM.0 PROM1. PROM4. PRO2. PRO258, PR0301. PR0224, PRom2, PR0234.

PR0229, PR0223, PR0328 and PR0332 were successfully expressed in CHO cells by both a transient and a stable expWsion procedure. In addition, PROM3, PROM6, PROM,6 PR0228, PR0227, PR0220, PR0266, PR0269, 20 PROM8, PR0214. PR0231, PR0233. PROM,8 PR0244, PROM3. PR0236, PR0262. PROM3. PR0257, 2 PROM6, PROM6, PROM7, PR0271. PROM7, PR0294, PROM9, PR0293. PR0247. PR0303 and PR0268 were successfully transiently expressed in CHO cells.

Stable expression in CHO cells was performed using the following procedure. The proteins were expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms extraceliular domains) of the respective proteins were fused to an IgG I constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged formt.

Following PCR amplification. the respective DNAs were subeloned in a CHO expression vector using standard techniques as described in Ausubel ct al., OUrrent Protocols of Molecular Biology, Unit 3.16, John Wiley woo* and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used expression in CHO cells is as describedin Lucas er al.. Nuct. Acdsf Res. 9 (1774-1779 (1996). and uses the SV40 early promotertenhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.

Twelve micrograms of the desired plasmid DNA were introduced into approximately 10 million CHO cells using commercially available transfectIon regent Superftci (Quiagen). Dosper or Fuge~e* (Boebringer Mannheimi).

The cells were grown and described in Lucas et at. supra. Approximately 3 x 1O'7 cells are frozen in an ampule for further growth and production as described below.

The ampules, containing the plasmid DNA were thawed by placement into water bath and mixed by vortexirg. Ile contents were pipced into a cenatriug tube containing 10 mILs of media and cetrfifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the Cells Were resuspended in 10 niL of selective media (0.2 pm filtered PS20 with 5 S 0.2 pm diafiltered fetal bovine serumn). The cells were then aliquoted into a 100 niL spinner containing 90 niL of selective media. After 1-2 days. dhe cells were transferred into a 250 niL spinner filled with 150 niL selective growth medium and incubated at 37*C. After another 2-3 days. a 250 mil, 500 niL and 2000 mL spinners were seeded with 3 x 10 cells/mL. The cell media was exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in US Patent No. 5,122,469. issued June 16. 1992 was actually used. 3L production spinner is seeded at 1.2 x 10' celia/niL. On day 0. the cell numnber pH- were determined. On day 1. she spinucr was sampled and sparging with filtered air was comnedl. On day2. t spinner was sampled, the temperature shifted to 33VC, and mL of500 glL glucose and 0.6 nL of 10% antifoam 35 %polydimethysiloxnne emulsion, Dow Corming 365 Medical Grade Emulsion). Thiroughout the production. pH1 was adjusted as necessary to keep at around 7.2.

d: .After 10 days, or until viability dropped below 70%. the cell culture was harvested by eetrfifugtion and filtering through a 0.22 pin filter. The filtrate was either stored at VC or imdalyloaded onto columns for purification.

For the poly-His tagged constructs, the proteins were purified using a Ni-NTA column (Qiagen). Before S purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 mI Ni-NTA colmin equilibrated in 20mM Hepes. pH 7.4, buffer containing 0.3 MNaCI and izaidazole at a flow rate of 4-5 mI/mmn. at 40C. After loading, the columm was washed with additional equilibration buffer and the protein ehared with equilibration buffer containing 0.25 M imidazole. The highly purified protein was .20 subsequently desaitcd into a storage buffer containing 10 mM Hepes. 0.14 M NaCI and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharinacia) column and stored at Immunoadhesin (Fe containing) constructs of were purified from the conditioned media as follows. The conditioned meiutm was pumped onto aS5 ml Protein A column (Phannacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elsilon with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 mi fractions "too: into tubes containing 275 puL of 1 M Tris buffer, PH 9. The highly purified protein was subsequently deialted into storage buffer as described abovc for the poly-His tagged proteins. The homogeneity was assessed by SDS see* polyacrylamide gels and by N-teminal amino acid sequencing by Ed-an degradation.

PRO211, PR0217. PROM,0 PR0232* PROIS7. PROW6, PRO219, PROM4, PR0228, PROM,3 PROW4, PR0221, PR0227, PR0220. PR0258. PR0266, PR0269. PROW9. PR0214, PROM1, PRO301, PR0224, PR0222. PR0234, PR0231. PRO229, PR0233, PROM3, PM0223. PROM.5 PROM3, PROM6, PR0239.

PR0257. PR0260, PROM6, PROM7, PR0271, PROM7, PROM9, PR0295, PR0293, PROM4, PR0304, PR0302, PR0307. PR0303. PROM.3 PR0328. PR0326, PR0331. PR0332, PR0334, PR0346, PROMS8, PR0330, PRO310 and PR0339 were also succssfully transiently expressed in COS cells.

EXAMELLU: Expessowi of PRO Polvpnides in Yeast The following method describes recombinant expression of a desired PRO polypeptide in yeast.

First, Yeast expression vectors are consttuctnd for intracellular production or secretion of PRO polypeptides from the ADH2IGAPDH pro moter. DNA encoding a desired PRO potypeptide. a&selected signal peptide and the promoter isinserted into suitable restrction enzyme sites in the selected plasmid to direct intracelluiar expression of tie PRO polypepude. For secretion, DNA encoding dhe PRO polypeptidd can be cloned into the ilected plasmid;* together with DNA encoding the ADH2/GAPDH promoter, the yeast alpha-factor secretory signal/leader sequence.

and Lincr sequences (if needed) for expression of the PRO polypeptide.

Yeast cells, such as yeast strain ABI 10. can then be tranisformed with the expression plasids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10 trlchioroacetic acid and separation by SDS-PAGE, followed by staining -of the gels with Coomassie Blue stain.

Recombinant PRO polypeptide can subsequently be isolated and purified by removing the yeast cells from *.the fermenation medium by centrifugation and theni concentrating the medium using selected cartridge filters. The .Concenrate containing the PRO polypeptide may further be purified using selected column chromatography resins.

EAMPLE.56: Exnrssion of PRO Polvpeptdes in Baculovis-lnfected Insect Celts The following method describes recombinant expression of PRO polypeptides in Baculovirus-infected insect cells.

The desired PRO polypeptide is fused upstream of an epitope tag contained with a baculovirus expression vector. Such epitope tags include poly-his tags and immunioglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived fr-om. commercially available plasmids such as pVL1393 ***.(Novagen). Briefly, the PRO polypeptide or the desired portion of the PRO polypeptide (such as the sequence encoding ie extracellu domain of a transnienirane protein) is amplified by PCR with primers complementary to the 5 and3 region~s. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subeloned into the expression vector.

Recombinant baculovirus is generated by co-ranfecting the above plasmid and BaculoGoldrm virus DNA 0 0(Phanningen) into Spodopterafrugiperda (*S19) cells (ATCC CR1 1711) using lipofiectin (commercially available 0 ~from GIBCO-BRL). After 4-5 days of incubation at 28*C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression is performed as described by O'Reilley et aL. Baculovirus expression vectors: A laboratory Manual. Oxford: Oxford University Press (1994).

Expressed poly-bis tagged PRO polypeptide can then be purified, for example, by Nilchelate affinity chrmatography as follows. Extracts are Prepared from recomnbinnt viruis-infected S(9 cells as described by Rupert cc Nature, 32: 175-179 (1993). Briefly. Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, PH 7.9; 12.5 mM MgCI 2 0. 1 mM EDTA; 10% Glycerol; 0. 1% NP-40; 0.4 M KCI), and sonic~ted twice for seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer mM phosphate, 300 mM NaCI. 10% Glycerol, PH 7.8) and filtered through a 0.45 pum filter. A Ni*-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL.. washed with 25 mL.

of water and equilibrated with 25 niL of loading buffer. 'Me filtered cell extract is loaded onto the column at 0.5 niL per miun. he columri is washed to baseline Aio with loading buffer, at which point fraction collection is started.

Next. the column is washed with a secondary wash buffer (50 mM phosphate; 300 mnM NaCI, 10% Glycerol, pH which elutes nionspecifically bound protein. After reaching AjObaseline again, the column is developed with a 0 to 500mM Imidazolc gradient-in the secondary wash buffer. One niL fractions are collected and analyzed by SDS-PAOE and silver staining or western blot with Ni 1 -NTA-conjugatcd to alkaline phosphaiase (Qiagen).

Fractions containing the eluted Hs,6-tagged PRO polypeptide are pooled and dialyzed against loading buffer.

Alteruanvely. pwiflcaiion of the IgG tagged (or Fe tagged) PRO polypeptide can be performed using known chromatography techniques, including for instance. Protein A or protein G column chromatography.

PRO21I1. PR0217. PROM3. PRO1S7. PROM6, PROM4, PR0228, PROM3. PROM4. PR0221. PR0220, PRO258, PR0266. PR0269. PR0287. PROM14 PRO30I, PR0224. PR0222, PR0234, PR023 1. PR0229.

PROM3. PR0257. PROM7. PROM.4 PR0295. P1-0328. PR0326. PROM3, PR0334. PR0346 and PRO3 10 were successfully expressed in baculovurus infected SM or higbSisec cells. While the expression was V0000* actually performed in a 0.5-2 L scale, it can be readily scaled up for larger 8 L) preparations. The proteins were expressed as anlIgG constrct (inmuicadhesint). in which dhe proteint extracellular region was fused to an IgGl constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms.

Following PCR amoplilication, die respective coding sequences were subcloned into a baculoviuus expression vector (pb.PH.IgG for lgG fusions and pb.PH.His.c for poly-His tagged proteins), and the vector and BaculogoldO baculovirus DNA (Phariningen) were co-transfected into 105 Spodoprerafnugiperda C*Sf9') cells (ATCC CRL 1711). using Upofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are rmdifications of the commtrercially available baculovirus expression vector pVL1393 (Pharaningcn). with modified polylinker regions so include the His or Fc tag **sequences. The cells were grown in Rink's TNM-FHi medium supplemented with 10 FBS (Hyclone). Cells were inaibamd for S days at2S 9 C. The supernatant was harvested and subsequently used for the first viral amplification by infecting SM cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells were incubated for 3 days at 28*C. The supernatant was harvested and t expression 25 of the constructs in the baculovimus expression vector was determined by batch binding of 1 ml of supernatant to niL of Ni-NTA beads (QL4GEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard 999 by Coomassie blue staining.

Thie first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf cells grown in ESF-921 medium (Expression System LW) at an approximate MOI of 0.1. Cells were incubated for 3 days at 28 9 C. The supernatant was harvested and filtered. Batch biding and SDS-PAGE analysis was repeated, as necessary. until expression of the spinner culture was confirmed.

The conditioned medium from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For dft poly-His tagged constructs. the protein construct were purified using a INi-NTA columrn (Qiagen). Before purification, isuidazole was added to the conditioned media to a conicentration of 5 mM. The c :onditioned media were puimped onto a 6 ml, Ni-NTA column equilibrated in 20 mM Hepies, pH 7.4, buffer containing 0.3 M NaCI and 5 mM imidazole at a flow rate of 4-5 mI/mmn. at 4*C. After loaft.~ the column was washed with additional equilibration buffer and the protein ehited with equilibration buffer containig 0.25 M imidazoic. The highly purified protein was subsequently desalted into a storage buffer containing mM Hcpes, 0.14 M NaCI andI 4% mannitol, pH 6.8, with a 25 ad G25 Superfine (Phannacia) column and stored at -800C.

Innunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows.

lT conditioned media were pumped onto a 5.1l Protein A column (Pharniacia) which had been ciiiilibrated in mM Na phosphate buffer, pH 6.8. After Woading, the column was washed extensively with equilibration buffer before elusion with 100 mM citric acid, pH 3.5. Met ehited prin was immediately neutralized by collecting 1ad fractions into tubes containing 275 mL of I M Tris buffer. pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the proteins vas verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.

EXML 7 Prenaration of Antibodies that Bind to PRO Polvpeptids This example Illustrates preparation of monoclonal antibodies which can specifically bind to a PRO polypeptide.

.15 Techniques for producing the monoclonal antibodies are known in the art and am described. for instance.

in Goding. w=pz. Imnunogens that may be employed include purified PRO polypeptide, fusion proteins- containing the PRO polypeptide, and cells expressing recmbinant PRO polypeptide on the cell surface. Selection of the inmmogen can be made by the skilled artisan without undue experimentation.

.Mice, such as Dalh~c, ame immunized with the PRO polypeptide imimmogen emulsified in complete Freund's 20 adjuvant and injected subcutaneously or intraperiwoncally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in PL-TDM adjuvant (Ribi Inmunochemnical Research. Hamilton. MI) and-injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for .25 testing in ELISA assays to detec anti-PRO polypeptide antibodies.

After a suitable antibody titer has been detected, the amals 'Positive* for antibodies can be injected with a final.inravnous injecton of PRO polypepfide. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells ame then fused (using 35 polyethylene glycol) to a selected murine mycloma cell linec such as P3X63AgU. 1. available from ATCC. No. CRL 1597. The fusions generate hybridorna cells which can then be plated in 96 well tissue Culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) meditn to inhibit proliferation of non-fused cells, mycloma hybrids, wan spleen cell hybrids.

The bybridoma cells will be screened in an EUSA for reactivity against the PRO polypeptide.

Determnination of 'positive" hyWdoma cells secreting the desired monoclonal antibodies against the PRO polypeptide is within the sill in the art.

Ile positive hybridagna cells can be injected intraperitoneally into syngcneic Balb/c mice to produce ascites conaiin the ansi-PRO polypeptide mnonoclonal antibodies. Alternatively, the haybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation. followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.

EAMPT ltj: Chineric PRO Polvnentides PRO polypptdes may be eVxpesd as chimeric proteins with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domazins include. but are not limited to, metal checlating peptides such as histidine-Arptophan modules that allow purification on immobilized nmetals. protein A domains that allow purification on immobilized iinmunoglobulin. and the domain utilized in the FLAGSyh ceson/affinity purification system (l[miunex. Corp.. Seattle Wash.). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (rinvitrogen, San Diego Calif.) between the purification domain and the PRO polypepde sequence may be useful to facilitate expression of DNA encoding the PRO polypeptide.

EXAMPLP!19: Purification of PRO Povoetides Usine SneCific Antibodies Native or recombinant PRO polypeptides may be purified by a variety of standard techniques in the art of protein purificauion. For example, pro-PRO polypeptide. mature PRO polypeptide, or pre-PRO polypeptide is purilled by immunoalfnity chreenatography using antibodies specific for the PRO polypeptide of interest. In general, an immunoaffinity colurrn is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin.

Polyclonal iminmogilobulins ame prepared from immune sera either by precipitation with amomoniumn sulfate or by purification on immobilized Protein A (Pharruacia LKB Biotechnology, Piscataway, Likwise, mnomclonal antibodies art prepared from moause ascites fluid by ammoniumn sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE'' (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to dhe manufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subciular firaction obtained via differential centrifugation by the addition of detergent or by other methodis wenl known in the art. Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.

A soluble PRO polypeptide-containing preparation is passed over the iminunoafinity column, and the column is washed under conditions that allow the preferential absorbanee of PRO polypeptide high ionic stregth buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PRO poblyrde bindling a low pH buffe~r such as apmxinnsly pH 2-3. or a high concentration of a chaotrope such as urea or thiocyaziate ion), and PRO polypeptide is collected.

EXAMPLEI 6: Dru&-crening This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drug screening techniques. The PRO polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intraccliluarly.

One method of drug screening utilizes cukaryotic or prokayotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form. can be used for standard binding assays. One.may measure, for exampl, the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively one can examine the diminution in complex formation between the PRO polypeptide and its target cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying for the presence of a complex between the agent and the PRO polypeptide or fragment, or for the presence of a complex between the PRO polypeptide or fragment and the cellc by methods well known in the at. In such competitive binding assays, the PRO polypeptide or fragment is typically labeled. Afer suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, o *and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypcptide/cll complex.

15 Another technique for drug screening provides high throughput screening for compounds having suitable :binding affinity to a polypeptide and is described in detail in WO 84/03564. published on September 13, 1984.

Briefly staed, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PRO polypeptide, the peptide test compounds are reacted with PRO polypcptide and washed. Bound PRO polypeptide is detected by methods well known in the an. Purified PRO polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding PRO polypcptide specifically compete with a test compound for binding to PRO polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide S 25 which shares one or more antigenic determinants with PRO polypeptide.

:EXAMPLE 61: Rational Dug Design The goal of rational drug design is to produce structual analogs of biologically active polypcptide of interest a PRO polypcptide) or of small molecules with which they interact, agonists, antagonists, or inhibitors.

Any of these examples can be used to fashion drugs which are more active or stable forms of the PRO polypeptide or which enhance or interfere with the function of the PRO polypeptidc in vive Hodgson, Bio/rcchnologv. 2: 19-21 (1991)).

In one approach, the three-dimensional strcture of tie PRO polypeptide, or of an PRO polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the PRO polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant strnjal infortnation is used to design analogous PRO polyeptite-like molecules or to identify efficient inhibitors.

Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biocg~Mil U1:7796-7801 (199) or which act as inhibitors. agonists, or antagonists of native pqptides; as shown by Atbauda et at. LBlohem-, 11.3742-746 (199).

Is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal stucture. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional. pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the antxwids would be expected to be an analog of die original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.

By virtue of the present invention, sufficient amounts of the PRO polypptde may be rade available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the PRO polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.

EXAMPLEh: D~iagnostic Test Usting PRO317 PovEd ntideSneific Antibodies Particular anti-PR0317 polypeptide antibodies are usefil for the diagnosis of prepathologic conditions, and chronic or acute diseases such as gyncological diseases or ischernic diseases which amc characterized by differences in the amount or distribution of PROM 1. PR0317 has been found to be expressed in human kidney and is thus likely to be associated with abnormalities or pathologies which affect this organ. Further, since it is so closely related to EBAF-l, it is likely to affect the endomerrium and other genital tissues. Further, due to library sources of certain ESTs. it appears that PR0317 may be involved as well in forming blood vessels and hence to be a modulator of angiogencsis.

:Diagnostic tests for PR0317 include methods utilizing the antibody and a label to detect PR0317 in human body fluids, tissues, or extracts of such tissues. The polypeptide and antibodies of the present invention may be used with or without modification. Frequently, the polypepside and antibodies will be labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature.

Suitable labels include radionuctides, enzymes, substrates. cofactors, Inhibitors, fluorescent agents. chemihiminescent agents, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos. .3.817.837; 3,850,752; 3,939.350; 3,996,345; 4.277,437; 4,275,149; and 4,366,241. Also, recombinant imniunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567.

A variety of protocols for measuring soluble or membranc-bound PRo3 17, using either polyclonal or monoclonal antibodies specific for that PROM 1, are kown in the art. Examples include enzyme-linked innanosorbent assay (flSA). radioirnmunoassay (RIA), radioreceptor assay (RRA), and fluorescent activated cell sorting (FACS). A two-site monoclonal-based immiunoassay utilizing monoclonal antibodies reactive to two n-interfering epitopes an PR0317 is preferred, but a competitive binding assay may be employed. These assays are described, among other places. in Maddox er al. l Ez. Med.. -1U:1211 (1983).

EXAMPLE 63: Identification of PR0317 Receptors Purified PR0317 is useful for characterization and purification of specific cell surface receptors and other binding molecules. Cells which respond to PR0317 by metabolic changes or other specific responses are likely to express a receptor for PR0317. Such receptors include, but ar not limited to. receptors assdciated with and activated by tyrosine and serine/threonine kinases. See Kolodziejczyk and Hall, supra, for a review on known receptors for the TGF- superfamily. Candidate receptors for this superfamily fall into two primary groups, termed type I ad type II receptors. Both types are scrin/hreonine inases. Upon activation by the appropriate ligand, type I and type 1 receptors physically interact to form hetero-oligomers and subsequently activate intracellular signaling cascades, ultimaely regulating gene transcription and expression. In addition. TGF- binds to a third receptor class, type III, a membrane-anchored protcoglycan lacking the kinase activity typical of signal transducing molecules.

PRO317 receptors or other PRO317-binding molecules may be identified by interaction with radiolabeled S PRO317. Radioactive labels may be incorporated into PR0317 by various methods known in the art. A preferred embodiment is the labeling of primary amino groups in PR0317 with '2I Bolton-Hunter reagent (Bolton and Hunter.

15 Biochem., 1-529 (1973)), which has been used to label other polypeptides without concomitant loss of biological activity (Hebert a. J. Biol. Chem.. 2:18989 (1991); McColl et al.. Immunol.. 15:4550-4555 (1993)).

Receptor-bearing cells arc incubated with labeled PRO317. The cells are then washed to removed unbound PR0317.

and receptor-bound PR0317 is quantified. The data obtained using different concentrations of PRO317 are used to calculate values for the niumber and affinity of receptors.

Labeled PRO317 is useful as a reagent for purification of its specific receptor. In one embodiment of affinity Spurification, PRO317 is covalently coupled to a chromatography column. Receptor-bearing cells arc extractd, and the extract is passed over the column. The receptor binds to the column by virtue of its biological affinity for PRO317. The receptor is recovered from the column and subjected to N-terminal protein sequencing. This amino acid sequence is then used to design degenerate oligonucleotide probes for cloning the receptor gene.

In an alternative method, mRNA is obtained from receptor-bearing cells and made into a cDNA library. The library is uansfcted into a population of cells, and those cells expressing the receptor are selected using fluorescently Slabeled PRO317. The receptor is identified by recovering and sequencing recombinant DNA from highly labeled cells.

In another alternative method, antibodies are raised against the surface of receptor bearing cells, specifically monoclonal antibodies. The monoclonal antbodies are screened to identify those which inhibit the binding of labeled PRO317. These monoclonal antibodies are then used in affinity purification or expression cloning of the receptor.

Soluble receptors or other soluble binding molecules are identified in a similar manner. Labeled PR0317 is incubated with extracts or other appropriate materials derived from the uterus. After incubation, PR0317 complexes larger than the size of purified PR0317 are identified by a sizing technique such as size-exclusion chromatography or density gradient centrifugation and are purified by methods known in the art. The soluble receptors or binding protein(s) are subjected to N-terminal sequencing to obtain information sufficient for database identification, if the soluble protein is known, or for cloning, if the soluble protein is unknown.

EXAMPLE 6: Determination of PR0317-lnduceii Cellular Respons The biological activity of PR0317 is measured. for example. by binding of an PRO3 17 of the invention to an PR0317 eceptor. A test conqhound is screened as an antagonist for its ability to block binding of PR0317 to dhe receptor. A test compound is screened as an agonist of the PR0317 for its ability to bind an PR0317 receptor and influence thetam physiological events as PR0317 using, for example, the KIRA-EUSA assay described by Sadick er at.. Anajycal Biocbemistrv 2U:207-214 (1996) in which activation of a receptor tyrosine kinase is monitored by immuno-capture of the activated receptor and quantitation of the evel of ligand-induced phosphorylation. The assay may be adapted to nmtitr PRO3l7-inducod receptor activation through the use of an PR0317 receptor-specific antibody to capture tde activated receptor. These techniques are also applicable to other PRO polypeptides described -herein.

EXAMPIE 65: Use of PR022A- fr Screeine Comnounds PR0224 is expressed in a cell stripped of membrane proteins and capable of expressing PR0224. Low density lipoproteins having a detectable label are added to the cells and incubated for a sufficient time for endocytosis.

The cells am washed. The ofll are then analysed for label bourl to the membrane and within the cell after cell lysis.

Detection of the low density lipoproteins withi the celideeminesthat PRO224is within the fanmilyof low density lipoprotei receptor proteins. Memabers fond within this family are then used for screening compounds which affect these receptors. and particularly the uptake of cholesterol via these receptors.

EXAMPLE 6: Ability of PRO oyetdst nibit Vascular Fxdothelial Growth Factor VEGF Stimulated- Proliferation of Endothelial Cell Growh The ability of various PRO polypeptides to inhibit VEGF stimulated proliferation of endothelial cells was tested. Specifically, bovine adrenal cortical capillary endothelial (ACE) cells (from primary culture, maximum 12-14 passages) were plated on 96-well microtiter plates (Amersham Life Science) at a density of 500 cellslweil per 100 ~*uL in low glucose DMEM. 10% calf serum, 2 mM glutamine x. pen/strept and fungizone, supplemented with 3 ng/mL VEGF. Controls were plated the same waiy but some did not include VEGF. A test sample of the PRO polypeptide of interest was added in a 100 $d volume for a 200 ILI final volume. Cells were incubated for 6-7 days at 37*C. The media was aspirated and the cells washed lx with PBS. An acid phosphatase reaction mixture (100 1 pl, 0.lIM sodium acetate, pH 5.5. 0.1 Triton-100, 10 mM p-nirophenyl phosphate) was added. After incubation for 2hours at 37C, the reaction was stopped by addition of 10jpd iN NaOH. OD was measured on microtiter plate reader at405 tm. Contonls were no cells, cell aone, cells +FGF (5 n/mL).cells+ VEGF (3 ng/mL), cells VEOF (3 nj/mI) TGF-P (1 ng/mI), and cells +1 VEGF (3ng/mL) UF (S ng/mL). (TGF-3 at a I ng/rnl concentration is known to block 70-90% of VEGF stimuilated cell proliferation.) The results were assessed by calculating the percentage inhibition of VEOF (3 nglml) stimulated cells proliferation, determined by measuring acid phospharase activity at 0D405 mu. relative to cells without stimuilation, and relative to the reference TGF-ft inhibition of VEGF stimulated activity. The results, as shown in Table 2 below, are indcative of t utility of the PRO polypeptides in cancer therapy and specifically in inhibiting humor angiogenesis. The numerical values (relative inhibition) shown in Table 2 are determined by calculating the percent inhibtion of VEGF stimnlated proliferation by the PRO polypeptide relative to cells without stimulation and then dividing that percentage into the percent inhibition obtained by TGF.-3 at I ng/ml which is known to block of VEGF stimulated cell proliferation.

Iabil2 PRO Name PRO211 PRO211 PRO211 PRO211 PRO211 PR0211 PRO217 PRO217 PRO217 PRO217 PRO217 PRO217 PRO187 PRO187 20 PRo1S7 PRO219 PRO219 PRO219 PRO246 PRO246 PRO246 PRO228 PRO228 PRO228 30 PRO228 PRO228 PRO228 PRO228 PRO228 35 PRO228 PRO245 PRO245 PRO245 PRO245 PRO245 PRO245 PRO221 PRO221 PRO221 PRO221 PRO221 PRO221 PRO258 PRO258 PRO258 PRO258 PRO258 PRO258 PRO Concentration 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% 0.01% 0.1% 1.0% 2.5 pM 25 nM 250 nM 0.01% 0.1% 1.0% 5.7 pM 57 nM 570 nM 0.01% 0.1% 1.0% 0.01% 0.1% 1.0% 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% 0.01% 0.1% 1.0% 0.48 nM 4.8 nM 48 nM 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% Relative hiion 99.0 1.09 0.95 67.0 0.27 20.0 1.06 0.84 .0.39 0.2 0.88 0.58 0.91 0.82 0.44 0.61 1.09 0.97 1.04 0.49 0.99 0.93 0.57 0.95 0.98 0.77 0.88 0.16 0.48 0.76 0.35 0.11 1.03 0.95 0.49 1.03 1.06 0.82 0.93 0.31 0.43 0.98 1.06 0.95 1.02 0.6 0.69 Tabe cnt PRO3Nam PRO301 PRO301 PRO301 PRO301 PRO301 PR0224 PR0224 PRO224 PR0272 PR0272 PR0272 PR0328 PR0328 PR0328 PR0331 PR0331 PR0331 RR onenrion puM 70 jiM 700,uM 0.01% 0.1% 1.0% 0.01% 0.1% 1.0% 0.01% 0.1% 1.0% 0.01% 0.1% 1.0% 0.01% 0.1% 1.0% Relative Inhibiion 1.02 0.8 0.44 0.92 0.85 0.68 101.0 65.0 23.0 0.95 0.57 0.18 0.98 0.96 0.6 0.88 0.82 0.56 a a. a a EXAMPLE 67: Retinal Neurn Survival This example demonstrates that PR0220 popeptides have efficay in enc ng the survival of retinal neuron cells.

25 Sprague Dawley rat pups at postnatal day 7 (mixed population: glia and retinal neuronal types) arc killed by decapitation followin CO. anesthesia and the eyes are removed undcr sterlc conditions. The neural retina is dissected away from the pigment epithelium and other ocular tissue and then dissociated into a single cell suspension using 0.25 trypsin in Ca M g2-free PBS. The retina are incubated at 37C for 7-10 minutes after which the trypsin is inactivated by adding 1 ml soybean trypsin inhibitor. The cells arc plated at 100,000 cells per well in 96 well planes in DMEM/F12 supplemented with N2 and with or without the specific test PRO polypeptide. Cells for all expenrunents are grown at 370C in a water saturated atmosphere of 5% CO, 1 After 2-3 days in culture, cells are stained with calcein AM then fixed using 4% paraforinaldehyde And stained with DAPI for determination of total cell count. The total cells (fluorescent) are quantified at 20X objective magnification using CCD camera and NIHimg software for Macintosh. Fields in the well are chosen at random.

The effect of various concentration of PRO220 polypeptdes are reported in Table 3 below where percent survival is calculated by dividing the total number of calcein AM positive cells at 2-3 days in culture by the total number of DAPI-labeled cells at 2-3 days in culture. Anything above 30% survival is considered positive.

Tab1ig PRO Name PR0220 PR0220 PRO220 PR0220 PR0220 PRO220 0R.Cncntato 0.01% 0.1% 0.1% 010% 1.0% Perce-nrival 2.4% 4.1% 3.0 7 3.1% 72.4% 42.1% EXAMPLO:Ro htec asri ibis example demconstrates tat PR0220 polypcptidcs have efficacy in enhancing the survival of rod photoreceptor cells.

Sprague Dawley rat pups at 7 day postnatal (nixed population. glia and retinal neuronal Cell types) am illed by decapitation following CO% anesthesis And the eyes arc removed wider sterile conditions. ThIe neural retina is dissected away form the PMa epielium and other ocular tissue and then dissociated into a single cell suspension using 0.25% trypsin in Can*. mg 14 -ftee PBaS. The retinas are incubated at 37*C for 7-10 minutes after wh ich the tl~sin is inactivated by adding 1 MI soYbean "rpin inhibitor. The cells are plated at 100,000) cells per well in 96 Well plates in DMEMIFl2 supplemented with N2 and with or without the specific test PRO polypeptide. Cells for alexcerimenis ar grown at 37C in awater saturated atmosphere of 5% C02. After 2-3 days in culture. cells are fixed Using 4% paraforinaldehyde. and then stained using Celrrracker Green CMFDA. Rho 4D2 (ascites or IgG; 1: 100). a mono~clonal antibody directed towards the visual pigmntt rhodopsin is used to detect rod photoreceptor cells by indirect Immunofluorescn. The results are reported as survival: total cumber of calcein/Cclrrracker rhodopsmn positive cells at 2-3 days in culture. divided by the total number of rhodopsin positive cells at time 2-3 days in cultirm. The total cells (fluorescent) arc quantified at 20x objective magnification using a CCD camera and NIH image software for Macitosh. Fields in the well amc chosen at random.

Thei effect of various concentration of PRO220 poluvvepe amrepre in Table4beo.Ayhnaov 1%survival is cons idered positive..

Table 4 20 ERo Nam PRO Concentration Prcnt Suial *PR0220 0.1% 0.0% PR0220 0.1% 0.0% PRO220 1.0% 0.0% *25 POz2on 20% 66.9% PRO220 1.0% 56.9% EXAMPLE 69: Induction of Endothelial Cell Aontosis Th~e ability of PR0228 polypeptides; to induce apoptosis in eindothelial cells was tested in Imman venous umbilical veinendothelial cells (HuvEC. Cell systems), using a 96-well format, in 0% serum media supplemented with 100 og/mI VEGF. (As HUVEC cells are easily dislodged from the plating surface, all pipetting in the wells must be done as gently as practicable.) The n-dis was aspired and the ceis washed once with PBS. 5 ml of Ix typsin was addedto the cells in a T..175 flask, and the cells were allowed so stand until they were released from the plate (about 5-10 minutes).

Tsysinization was stopped by adding 5 ml of growth media. The cells were spun at 1000 rpm for S minutes at VC.

The media was aspirated and the cells were resuspended in 10 ml of 10% serum complemented medium (Cell Systems), 1 x penn/strep.

The cells were plated on 96-well microtiter plates (Amershan Wfe Science. cytostar-T scintillating snicroplate. RPNQI6O, sterile, tissue-culture treated, individually wrapped), in 10% serum (CSG-medium, Cell Systems). a adensity of 2x l0'clls per well ina total volume of 100 The PRO228 polypeptide was added in trilicate atdiltions of 1 0.33 andO0.l11%. Wells withut cellsweeuse asa blank and wells with cells only as a negative control. As a positive control 1:3 serial dilutions of 50jul of a 3x stock of staurosporine were used.

Tlie ability of the PR0228 polypeptide to in&=c apoptosis was determined using Anmexin V. a member of the calcium and phospholipid binding proteins, to detect apoptosis.

0.2ml Annexin V itnsokslto 10u/l)wr iue n46m 0 binding buffer and BSA (1.:25 dilution). 50 ph of the diluted Annexin V Biotin solution were added to each well (except controls) to a final concentration of 1.0 #g/mI. 'The samples were incubated for 10-15 minutes with Annexiin-Biotin prior to direct addition of 'S-Streptavidin. 5 8-Streptavidin was diluted in 2x Ca2* binding buffer. 2.5% BSA and was adde to all wells at a final concezltrationof 3 x 10'cptn/weil. The plates were then sealed, centrifuged at 1000 rpm for 15 mnutnes and placed on orbital shaker for 2 hours. The analysis was performed on 1450 Microbeta Trilux (Waflac). The results am shown in Table 5 below where percent above background represents the percentage amount of counts per minute above the negative controls. Percents greater than or equal to 30% above background are considered positive.

Table *PRO Name PROCoQcnraim Percent Above flack~round *PR0228 0.11% 0.7% *PR0228 0.11% 47.6% PR0228 0.33% 92.2% PR0228 0.33% 123.77o PR0228 1.0% 51.4% PR0228 1.0% 95.3% EAMPLE2Q FDB12 Cell Inhibition This example demonstrates that various PRO polypeptides have efficacy in inhibiting protein production by :PDB12 pancreatic ductal cells.

PDB12 pancreatic ductal cells are plated on fibronecdn coated 96 well plates at 1.5xl0' cells per well in 100 pL of growth media. 100 1 &L of growth media with the PRO polypeptide test sample or negative control lackting the PRO polypeptide is then added to well. for a final volume of 200 1& Controls contain growth medium cotiigapoensont eiadei hsasy elsaeicbtdfr4dy t3*.2 uo lmrBu Dye (ABD) is then added to each well and the flourescent reading is measured at 4hours post addition of AB. on a microtiter plate reader at 530 run excitation and 590 mm emission. The standard employed is cells without Bovinc Pituitary Extract (BPE) and with various concentrations of BPE. Buffer or CM controls from unknowns are run 2 times on each 96 well pla te.

The results from these assays are shown in Table 6 below whierein percent decrease in protein production is calculated by comparing the Ala=na Blue Dye calculated protein concentration produced by the PRO polypeptide.

treated cells with the Alum Blue Dye calculated protein concentration produced by the negative control cells. A percent decrease in protein production of greater than or equal to 25 as compared to the negative control cells is considered positive.

Tabl PRO21 1 PRO211I PR0211 PR0287 PR0297 PR0287 PR0297 PR0297 PR0287 PR0301 PR0301 PRO301 PR0293 PRO293 PRO293 PRO Concentration 0.1% 0.01% 1.0% 2.0% 10% 50% 2.0% 10% 50% 2.0% 10% 50% 2.0% 10% 50% Percent Decreas in Protein Produckin 0.0% 0.6% 59.7% 22.3% 18.2% 67.5% 45.53% 57.3% 52.24% 0.0% 59.8% 65.6% 0.0% 40.4% 56.7% *5

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5555 EXAMPLE 71: Stimulation of Adult Heart Hvyertrophy This assay is designed to measur the ability of various PRO polypeptides to stimulate hypertrophy of adult 20 heart.

Ventricular myocytes freshly isolated from adult 9250g) Sprague Dawley rats are plated at 2000 cell/well in 180ju1 volume. Cells are isolated and plated on day 1. t PRO polypeptide-containing test samples or growth medium only (negative control) (20 p1 volume) is added on day 2 and the cells are then fixed and stained on day Aftr staining, cell size is visualized wherein cells showing cc growth enhancement as compared to control cells are givecn a value of 0.0. cells showing small to moderate growth enhancement as compared to control cells are given a value of 1.0 and cells showing large growth enhancement as compared to control cells are given a value of Any degree of growth enhancement as compared to the negative control cells is considered positive for the assay.

TIe results are shown in Table 7 below.

30 PRO Na PR0287 PR0301 35 PR0301 PRO293 PR0293 PR0303 PR0303 Tale PRO Concentration 20% 20% 20% 20% 20% 20% 20% 20% Growth Enhancement Score EXAMEJ2: PDB12 Cell Proliferation This example demnxtrAICs tha various PRO polypeptides have efficacy in inducing proliferation of PDBl2 pancreatic ductal cells.

PDB12 paricrearic ductal cells are plated on fibronoctin coated 96 well plates at I .5xl0' cells per wdll in 100 uUIJ80 pl. of growth media. 100 pzL of growth media with the PRO polypeptide test sample or negative contol lacking the PRO polypeptide is then added to well. for a final volume of 200 Controls contain growth medium containing a protein shown to be inactive in this assay. Cells arm incubated for 4 days at 37C. 20 pL of Alamar Blue Dye (AB) is then added to each well and the flourescent reading is measured at 4 hours post addition of AB. on a microtiter plate reader at 530 an excitation and 590 em emission. The standard employed is cells without Bovine Pituitary Extract (BPE) and with various concentrations of DPE. Buffer or growth medium only controls from unknowns amre nn 2 times on each 96 well plate.

The results from these assays are shown in Table 8 below wherein percent increase in protein production is calculated by comparing the Alamar Blue Dye calculated protein concentration produced by the PRO polypeptidetreated cells with the Alakmr Blue Dye calculated protein concentration produced by the negative control cells. A percent increase in protein production of greater than or equal to 25 as compared to the negative control cells is considered positive.

PR aePRO Concentration Percent Increase in Protein Production .PRO301 2.0% 44.0% PRO301 10% 67.4% PRO301 *50% 185.8% PR0303 2.0% 27.9% PR0303 10% 174.9% PR0303 50% 193.1% EXAMPL~F7: Enhancem~ent of Heart Neonta Hvnetrnhy Induced by PROM2 Thiis assay is desigred to measure the ability of PR0224 polypeptides to stintulate hypertrophy of neonatal heart.

Cardiac myocytes from 1Way old Harlan Sprague Dawley rats were obtained. Cells (180 jul at 7-5 x 104/ml, serum fitshly isolated) ame added on day 1Ito 96-well plates previously coated with DMEMIF12 4% FCS.

C: Test samnples containing the test PRCY224 polyetide or growth medim only (hegative control) (20 p1/well) are added directly to the wells on day 1. PGF (20 p/well) is then added on day 2 at final concentration of W0 M. The cells are then stained on day 4 and visually scored on day 5, wherein cells showing no increase in size as compared to negative controls are scored 0.0 cells showing a small to moderate increase in size as compared to negative controls are scored 1.0 and cells sliewinga large increase in size as compared to negative controls are scored 2.0. The results are shown in Table 9 below.

Table9 PRO Name PRO Concentration rth Eman cmenSore PR0224 0.01% 0.0 PR0224 0.1% 0.0 PR0224 1.0% EXMPE 7: In situ Hybridization In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tisse preparations. It may be useful, for example, to identify sites of' geme expression.

zilyne the tissue distrbution of transcription. identify and localize viral infection, follow changes im specific mRI4A synthesis and aid in chromosome mapping.

In siz* hybridization was performed following an optimized version of the protocol by Lu and Gillett, W YXaim 1:169-176 (1994), using PCR-gencrated "P-labeled riboprobes. Briefly, forinalin-fixed. paraffin-embedded human tsiues were sectioned. deparaffinized, deproteinated in protcinasc K (20 guml) for 15 mimues at 37*C. and.

further processed for in situ hybridization as described by Lu and Gillett, supra. A UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55'C overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

"YPRlbonrobe synthesis p1 (125 mai) of "P-uTp (Amersham BF 1002, SA <2000 Ci/minol) were speed vac dried. To each tube containing dried the following ingredients were added: p1l 5x transcription buffer Id DTF (100 mM) 152.0 IdNT mix (2.5 mM it-,each of 10mM GTP. CTP &A7 10 idHIO) 1.0 pl Rnasin p1 DNA template (lgg) 11 H0 ;d RNA polymerase (for PCR products T3 AS. T7 usually) The tubes were incubated at 37*C for one hour. 1.0 JAI RQ1 DNase wcre added, followed by incubation :20 at 37*C for 15 minutes. 90 Idl TE (10 mM Tris pH 7.6/1mM EDTA pH 8.0) were added, and the mixture Was pipetted oat DE81 paper. 'eremainingt solution was loaded in aMicrocon-50 ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes).

After the final recovery spin. 100 Otl TE were added. I id of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II.

.25 Thieprobe was rnon a TBFurca gel. 1-3 Idof the probe or5pl of RNA MrkcIII were added to 3P1 of loading buffer. After beating on a 95*C heat block for three minutes, the gel was immedtiaitely placed on ice. The wells of gel were flushed, the sample loaded, and nun at 180-250 volts for 45 minutes. 'Me gel was wriappe in saran wrap and exposed to XAR film with'an intensifying screen in -70*C freezer one hour to overnight.

HEIybridization A. Pretreatment of frozen sections The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for nmites. The trays were placed in 55*C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC 975 ml SQ H2,0). After deproteination in 0.5 #&g/mI proteinass K for 10 minues at 37*C (12.5 Id of 10 mg/mI stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in x SSC for 10 minutes at room temperature. The sctions were dehydrated in 70%. 95%, 100% ethanol, 2 minues each.

178

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B. Pretreatment of paraffin-ebedded sections The slides were deparaffinized, place in SQ H 2 0, and rinsed twice in 2 x SSC at roon temperature, for 5 minutes each time. The sections were deproteinated in 20 14/ml proteinase K (500 V1 of 10 ng/ml in 250 ml RNase-free RNase buffer; 37 0 C, 15 minutes) human embryo, or 8 x proteinase K (100 pl in 250 ml RNase buffer, 37 0 C, 30 minutes) formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were perforred as described above.

C. Prebybridization The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) saturated filter paper. The tissue was covered with 50 p1 of hybridization buffer (3.75g Dextran Sulfate 6 ml SQ H 2 0), vortexed and heated in the microwave for 2 minutes with the cap loosened.

After cooling on ice, 18.75 ml formamide, 3.75 ml 20 x SSC and 9 ml SQ H 2 0 15 were added, the tissue was vortexed well, and incubated at 42 0 C for 1-4 hours.

D. Itybridization x 106 cpn probe and 1.0 ,ul tRNA (50 ng/ml stock) per slide were heated at 95 0 C for 3 minutes. The slides were cooled on ice, and 48 pl hybridization buffer were added per slide. After vortexing, 50p1 3 1 "P mix were added to 50 pl prehybridization on slide. The slides were incubated overnight at 55 0

C.

E. Washes Washing was done 2 x 10 minutes with 2 x SSC, EDIA at roan 25 tarperature (400 ml 20 x SSC 16 ml 0.25M EDTA, Vf=4L), followed by RNaseA treatment at 37C for 30 minutes (500 pl of 10 mg/ml in 250 ml RNase buffer 20 pj/ml). The slides were washed 2 x 10 minutes with 2 x SSC, EDIA at roan terperature. The stringency wash conditions were as follows: 2 hours at 55C, 0.1 x SSC, EDrA (20 ml 20-x SSC 16 ml EDIA, Vf=4L).

F. oliganucleotides In situ analysis was performed on a variety of IA sequences disclosed herein. The oligonucleotides earployed for these analyses are as follows: IMA33094-1131 (PRO217) pi 5' 3ATIAATAAAA- '(SEQ2 ID NO: 347) p2 CAGAAA TAA*AG AI AC (SE2 ID NO:348) I3M3223-1136 (PED230) pi 5'-0ATIE3ATAOGCItATCW (SE3 ID N0:349)

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9cS0 179 p2 5' LAAAaQOA1X-3' (SEQ2 ID NO: 350) (3) P1 p 2 (4) P1 p2 ENA34435-3140 (PRO232) 5' '-T~ATL~ArTX~CCQQC~~ -3 (SEQ 5' 3 (SEQ ID NO: 351) ID NO: 352) ID NO:353) ID 13: 354) 0

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15 (6) P1 p2 (7) p1 p2 (8) p1 p2 ENA35639-1172 (PRO246) 5' -3 5' 3' ENM.9435-1219 (PIM533) -CM3~~A~cA~oActAn1,IArA3' ENA35638-1141 (PW245) 5' -ATtrTmArA'A.-u:. CGflO-31 5' -3 IXM33O89-M12 (PR0221) 5' TIrAAA'crAh GITt1xCr-31 5' 31 EM35918-174 (PR258) 5' -3 5' 3f

(SEQ

(SEQ

(SEC?

(SEQ

ID NO: 355) ID NO:356) (SE)Q ID (SEQ ID (SEQ ID (SEQ ID NO: 357) NO: 358) NO: 359) NO: 360) 00 4, 0 050 00S

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(SEQ ID NO:361) (SEQ ID NO:362) 1Rt32286-1191 (PRO214) p1 5' -TIr TA ALIcAT rtcIrrtRC-3' P2 3CGATAMtI -3' p1 50 ;AfGGA-C3ACAT p2 5' 3' (SEQ ID NO:363) (SEQ ID NO:364) (SEQ ID NO:365) (SEQ ID NO:366) (U1) p1 p2 EIM35557-1137 (PF4234) 5' -3 5' 3'

(SEQ

(SEQ

ID NO:367) ID ND:368) EM331OO-M19 (P1V229) 180 p1 5' -OGATITAATACGACITACTAQ CITG1A (SED ID NO:369) p2 5' -CrAIWGAAATrAACTACrAAD ACACACT TAC-3' (SEX ID N3:370) (13) E34431-1177 (PRO263) pl 5' -GATAATAACICACTATAAAAA'I (SED ID NO:371) p2 5' -CrAAAMAGCIACATAGAG3AGACGXTA-3' (SED ID NO:372) (14) IN38268-1188 (PRO295) pl 5' -3ATICTACOA G G' (SEX ID 13:373) p2 5' -CAIGAAA3TAA CICATAA t AICCTGALT'AA' (SEQ ID NO:374) G. Results In situ analysis uas performed on a variety of UEA sequences disclosed herein. The results frcm these analyses are as follows.

A,33094-1131 (P=a217) Highly distinctive expression pattern, that does not indicate an obvious biological function. In the human eatryo it was expressed in outer smooth nuscle layer of the GI tract, respiratory cartilage, branching respiratory epithelium, osteoblasts, tendons, gonad, in the optic nerve head and developing dermis. In the adult expression was observed in the .i epidernal pegs of the chip tongue, the basal epithelial/myoepithelial cells of the prostate and urinary bladder. Also expressed in the alveolar lining cells of the adult lung, nesenchynal cells juxtaposed to erectile tissue in the penis and the cerebral cortex (probably glial cells). In the 25 kidney, expression was only seen in disease, in cells surrounding thyroidized renal tubules.

Itanan fetal tissues eamined (E12-E16 weeks) include: Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus, stcrach, snall intestine, spleen, thynus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower lirb.

Adult Ifnn tissues examined: Kidney (nornmal and end-stage), adrenal, myocardiumn, aorta, spleen, lynph node, gall bladder, pancreas, lung, skin, eye (inc. retina), prostate, bladder, liver (normal, cirrhotic, acute failure) NRm-anan priate tissues exmined: Chinp Tissues: Salivary gland, stcuach, thyroid, parathyroid, skin, thynus, ovary, lynph node Rhesus monkey Tissues: Cerebral cortex, hippocanpus, cerebellum, penis.

180a EtA33223-MG3 (f2O23O') Sections show an intense signal associated with arterial anid venous vessels in the fetus. In arteries the signal appeared to be confined to snnooth-nscle/pericytic cells. The signal is also seen in capillary vessels and in gicireruli. It is not clear vether or not endothelial cells are expressing this rMR. Exession is also observed in epithelial cells in the fetal lens. Strong expression was also seen in cells within placental trophoblastic villi, these cells lie between the trophoblast and the fibroblast-like cells that express HGF uncertain histogenes is. In the aduslt, there was no evidence of expression and the wall of the aorta and nest, vessels appear to be negative. However, expression was seen over vascular channels in the normal prostate and in the epitheliu lining the gallbladder. Insurers expression was seen in the vessels of the soft-tissue sarcana and a renal cell carcinomia. in summary, this is a mlecule that shows relatively specific vascular expression in the fetus as well as in scime adult organs. Expression was also 0:.

Observed in the fetal lens and the adult gallbladder.

In a secondary screen. iascular expression Was observed. similar to that observed above, seen in fetal blocks. Expression is on vascular smooth muscle. rather than endotheium. Expression also seen in smooth muscle of the developing oesphagus. so as reported previously, this molecule is not vascular Specific. Expresion was examinod i&4 lung and 4 breast carcinomas. Substantial expression was seen in vascular smooth muscle of at least 314 hung cana andl 2/4 breast cancers. In addition, inone breast carcinoma, expression was observed in peritumoral stromnal edhi of unermtain histogemis (possibly myofibroblasts). No endotlial cell expression was observed in this study.

DNMA4435:1140 (P110232) Strong expression in prostaric epithelium and bladder epithelium, lower level of expression in bronchial epitlelum. High background low Level expression seen in a nussuber of sites, including amotng others, bone. blood.

chondrosarcoina. adult heart AMd fetal liver. It is felt that this level of signal represents background. partly bccause signal at this level was s9e over die blood. All other tissues negative.

Humn fetal tissues examined (El 2ME6 weeks) inlude: Placenta umbilical cord, Dier, kidney. adrenals, -thyroid.

lungs, heart great vessels. oesophagus, stomach, smu intestine.

spleen, thymuis. pancreas, brain, eye, spinal cord, body wall, pelvis, testis and lower limb.

Adut hlull tssus cimid:Kidney (normal and end-stage), adrenal, spleen, lymph node, pancreas, lung, eye (inc.

retina), bladder, liver (normal, cirrhotic. acute failure).

Non-human rimate tissues examin d: Chimp Tissues: adrenal Rhesus Mnkey Tissues: Cerebral cortex, bippocampuis In a secondary screen, expression was observed in the epithelium of the prostate, the superficial layers of *:the uretheium of the urinary bladder, the urethelium liming the renal pelvis and the ureclium of the ureter (I out of 2 experimients). The urethra of a rhesus monkey was negative; it is unclear whether this represents a true lack of exession byticethra,or if itisthe result of afail=reof the probe to cross react with rhesus tissue. Ile findings in the prostate and bladder are similar to those previously described using an isotopic detection technique. Expression of the naRNA for this antigen is NOT prostate epitheliul specific. The antigen may serve as a useful marker for uretlilal derived tissues. Exression in the superficial, post-mitotic cells, of the urinary tract epitheliunm also suggest that kt is unlikldy to represent a specific stem cell marker, as this would be expected to be expressed specifically in basal epitheum.

DNA35639-1172 (PRO246) Strongly expressed in fetal vascular endotheium, including tissues of the CNS. Lower level of expression in adult vaseulature, inicluding the CNS. Not obviously expressed at higher levels in tmor vasular endotheium.

Signal also seen over bone miatrix and adult spleen, not obviously cell associated, probably related to non-specific background at these sites.

Human fetal tissues eamined 12-1316 weeks) include: Placenta, umbilical cord, liver, kidney. adrenals. thyroid, kings, heart, great vessels. oesophagus. stomach, small intestine. spleen, thymus. pancreas. brain. eye, spinal cord, body *wall, pelvis, testis and lower limb.

Aduk b=Mdsn= cx Ki d: ny (normal and eril-stage), adrenal, spleen. lymph node, pancreas, lung, eyc (inc.

retina), bladder, liver (normal, cirrhotic, acute failure).

Non-human primate tissues examined: Chimp Timea: adrenal 7 Rhesus Monkey-Tissues: Cerebral cortex, hippocampus DlNA-49435-1219 (PR0533) Moderate expression over cortical neurones in dhe fetal brain. Expression over the inner aspect of the fetal retina possible expression in the developing lens. Expression over fetal skin. cartilage, small intestine, placental villi and umbilical cord. In adult tissues there is an extemely high level of expression over die gallbladder epitheliumn.

Moderate expression over the adult kidney, gastric and! colonic epithl Low-level expression was observed over many cell types in many tissues, this may be related to stickines of the probe, these data should therefore be interpreted with a degree of caution.

Human fetal tissues examined (El2E6wes)icue Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart great vessels, oesophagus. stomach, small intestine, spleen, thymurs, pancreas, brain, eye, spinal cord, body wall, pelvis, testis and lower limb.

Adut hmantisuesc~ffind:Kidney (normal and end-stag), adrenal, spleen, lymph node, pancreas, hing, eye (inc.

retina), bladder, liver (normal, cirrhotic, acute failure).

Non-human prigrate tissues examined-, Chininissurt: adreiial Rhesus Monkey Tissues: Cerebral cortex, hippocAmpus, cerebellum.

DNA3563R-1 141 PRO245) Expression observecd in the enidothelium lining a subset of fetal and placental vessels. Endothelial expression was coadined to these tissie blocks. Expression also observed over intermediate trophoblast cells of placenta. All other tissues negative.

Fetal tissues exmined (E12-E]6 weeks) include. Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels. oesophagus. stomach, small intestine, spleezi, thymus. pancreas, brain; eye, spinal cord, body wall. pelvis and lower limb.

Adult tissues examined: Liver, kidney, adrenal, myocardium, aorta. spleen, lymph node, pancreas, lung, skin, cerebral cortex hippocanpus(rm). cecebelhm(rm), penis, eye, bladder, stomach, gastric carcinomai, colon, colonic carcinoma, thyroid (chimp), parathyroid (chimp) ovary (chimp) and chondrosarcoma.

Acetorninophen induced liver injury and hepatic cirrhosis DNA33089-1132 (PRO22fl Specific express ion over fetal cerebral white and grey mat ter, as well as over neurones in the spinal cord.

Probe appear to cross react with rat.. Low level of expression over Ccbllar neurones in adult rhesus brain. All other tissues negative.

Fetal tissuex examined (E12-E1 6 weeksl include: Placenla, umbilical cord, liver, kidney, adrenals, thyroid. hang, huat, great vesscls. oeSOPhagus. stomach. small intestine, spleen. thymus,pjancreas. brain eye, spinal cord, body wall, pelvis and lower limb.

Adult tissues exaimined: liver, kidney, adrenal, myocardium, aorta, spleen, lymph node.

pancreas. lure, skin, cerebral cortex hippocampus(rin). cerebellum(rm). penis. eye, bladder, stomch, gastric canaxuna colon, colonic =acim Arid chondrosarcoim. Acetoninophen induced liver injury and hepatic cirrhosis DNA3591 &1174 (PRO258) Strong expression in the, nervous system. In the rheus monkey brain expression is observed in cortical, hippocainpal and cerla r a neuroneCs. Exprsson over spinal neurones in the fetal spinal cord, te developing brain and die inner aspects of the fetal retina. Expression over developing dorsal root and autonomic ganglia as well as enterlc nerves. Expression observed over ganglion cells in the adult prostate. In die rat, there is strong expression over the developing bind brain antd spinal cord, Strong expresion over interstitial cells in the placenta vill All other tissues were negative.

FeWa tissues examined (E12-E16 weekcs) include: Placena,. umbilical cord, liver, kidney. adrenals, thyroid, lungs.

20 heart, great vessels, oesophagus, stomach, small intesine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult tissues examined: Liver, kidney, renal cell carcinoma, adrenal. aorta, spleen lymph node, pancreas, lung, myocardiuin, skin, cerebral cortex (tnn), hippocampus(m), cerebelzn(rm), bladdcr. prostate, stomach, gastric carcinoma, colon, colonic carcinoma, thyroid (chimp), parathyroid (chimp) ovary' (chimp) and chondrosarcoma. Acetorwnophen induced liver injury and hepatic cirrhosis.

DNA32296-1191 (PR0214) Fetal tissue: Low level throughout mesenehyme. Moderate expression in placental stromal cells in miembranous tissues and in thyroid. Low level expression in cortical neurones. Adult tissue: all negative.

Fetal tissues examined (E12-EI6 w~eeks)I include: Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels, oesopbagus, stomach. small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adut tssus cmind ichxe:Liver, kidney, adrenal, mnyocardium, aorta, spleen, lymph nodc, pancreas, lung and skin.

(10) DNA3322min11 (PRO224 Expression limited to vascular endotheiumn in fetal spleen, adult spleen, fetal liver, adult thyroid and adult lymph node (chimp). Additional site of expression is the developing spinal ganglia. All other tissue negative.

Human fetal tluques examined (El 2-E16 weeks) include: Placenta, umbilical cord, liver, Iddney, adrenals. thyroid, lungs, heart, great vessel, oesophagus, stomach, small'intestine, spleen, thymus. pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult humn tissues examinetl: Kidney (normal and end-stage), adrenal, mnyocardium, aorta, spleen, lymph node, pancreas, lung, skin, eye (inc. retina), bladder, liver (normal, cirrhotic, acute failure).

Non-human primate tissues examined: ChjuxlTisuest: Salivary glan, stomach, thyroid. parathyroid, sin, thymnus, ovary, lymph node.

Rhesus Monkey Tissues: Cerebral cortex, hippocanmpus, cerebellum, penis.

(11) QNA35557-1 137 (PRO234) Specific expression over developing motor nourones in ventral aspect of the fetal spinal cord (will develop into ventral horns of spinal cord). All other tissues negative. Possible role in growth, differentiation and/or development of spinal motor neurons.

Fetal tissues examined (E2-R16 weekcs) include: Placenta, umbilical cord, liver, kIdney, adrenals,-thyroid, lungs, heart, great vessels, ocsophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult tissues examined: Liver, kidney, adrenal, myocardiumn, aorta, spleen, lymph node, pancreas, lung, sin, cerebral cortex OWn. hippocampus(rm), cerebellum(rmn). penis, eye, bladder, stomach, gastric carcinoma. colon colonic carcinomna and chondrosarcosna. Acetominiophen induced liver injury and hepatic cirrhosis (12) DNA,33100-1 159 (PRO229) Strikin expression in mononuclear phagocytes (mnacrophages) of fetal and adult spleesi liver, lymph node ard adultI tyvms in tingible bod~y marohge). The hgetexpression is in the spleen. All other tissues negative.

Localisation and homology are entirely consistent with a role as a scavenger receptor for cells of dhe reticuloendothelial system. Expression also observed in placental mononuclear cells.

Hu=a fetal tissues examined M1l2-1116 weeks) include: Placenta, umbilical cord, liver, idney, adrenals, thyroid, lungs, heart, great vessels, oesophagus, stomach, small intestine, spleen, thymus. pancreas, brain. eye, spinal cord, body wall, pelvis and lower limb.

Adult human tissues examined: Kidney (normal and end-stage), adcrenal, myocardiuma, aorta, spleen, lymph node, gall bladder. pancreas, lung, skin, eye (inc. retina), prostate, bladder, liver (normal, cirrhotic, acute failure).

Non-human pirimate tissues examined: Cbimp..Thisse: Salivary gland, stomach, thyroid, parathyroid, skin, thymnus. ovary, lymph node.

Rhesus Monke Tissues, Cerebral cortex, hippocanipus, cerebellum, penis.

(13) DNA34431-1 127 (PRO263) Widepread expression in human fetal tissues and placenta over mononuclear cells, probably macrophages +-lymphocytes. The cellular distribtion follows a perivascular pattern in many tissues. Strong expression also seen in epithelial cells of the fetal adrenal cortex. All adult tissues were negative.

Fetal tissues examined (E12-EI6 weeks) include: Placenta, umbilical cord, liver. kidney, adrenals. thyroid, lungs.

heart. great vessels, oesophagus. stomach, small intestine, spleen, thynms. pancreas. brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult tissues examined: Liver, kidney, adrenal. splcen. lymnph node. pancreas, lung. skin, cerebral cortex (rin).

hippocampus(rm), cerebellum(rmn), bladder, stomach, colon and colonic carcinoma. Acetomninophen induced liver injury and hepatic cirrhosis.

A secondaty screen evidenced expression over strornal mononuclear cells probably histiocytes.

(14) DNA3 8269-118 I (PR0295i High expression over ganglion cells in human fetal spinal ganglia and over large neurones in the anterior horns of the developing spinal cord. In die adult there is expression in the chimp adrenal mieduilla (neural), neurones of the di~tesus mney brain (hippocanipus ++)and cerebral cortex) and neutrones in ganglia in the niormal adult human prostate (the only section that contains ganglion cells. ie expression in this cell typ is presumed NOT to be confined to the prostate). All other tissues negative.

Hiimnin fetal fisuneq examinedt (E12-E16 weeks) ene-de: Placenta, umbilical cord, liver, kidney, adrenals. thyroid.

lungs, great vessls stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis, testis and lower limb.

Adult himian tissues e-xamtwii: Kidney (normal and end-stage), adrenal, spleen, lymph node, pancreas, lung, eye (inc.

retina), bladder, liver (normal, cirrhotic, acute failure).

Non-humnan priMate tissues examined: *~Ci Tbz.issues: adrenal Rhesus Monkey Tissues: Cerebral cortex, bippocampus, cerebellum.

Densit of Material Tbe follwing materials have been deposited with the American Type Culture Collection, 12301 Parlawn Drive, Rockville, MD. USA (ATCC): Material ACC Del.o M tDe DNA32292-1131 ATCC 209258 September 16, 1997 DNA33094-1131 ATCC 209256 September 16, 1997 DNA33223-1 136 ATCC 209264 September 16, 1997 DNA34435-1 140 ATCC 209250 September 16, 19917 DNA27864-1155 ATCC 209375 October 16, 1997 DNA36350-1 158 ATCC 209378 October 16, 1997 DNA32290-1 164 ATCC 209384 October 16, 1997 DNA35639-1 172 ATCC 20996 October 17, 1997 DNA33092-1202 ATCC 209420 October 28, 1997 DNA,49435-1219 ATCC 209480 November 21, 1997 DNA35638-1 141 ATCC 209265 September 16, 1997 DNA32298-1132 ATCC 209257 September 16, 1997 DNA33089-1 132 ATCC 209262 September 16, 1997 DNA33786-1 132 ATCC 209253 September 16, 1997 DNA35918-1 174 ATCC 209402 October 17. 1997 186 DNA3 7150-1178 DNA382 60-1180 DNA3 9969- 1185 DNA3 2286-1191 DNA3 3461-1199 DNA4 0628-1216 DNA3 3221-1133 DNA3 3107-1135 DNA3 5557-1137 DNA3 4434-1139 DNA3 3100-1159 DNA3 5600-1162 DNA3 4436-1238 DNA3 3206-1165 DNA3 5558-1167 DNA3 5599-1168 DNA3 6992-1168 DNA3 4407-1169 DNA3 5841-1173 DNA3 3470-1175 DNA3 4431-1177 DNA39510-1181 DNA3 9423-1182 DNA4 0620-1183 DNA40604-1187 DNA3 8268-1188 DNA3 7151-1193 DNA3 5673-1201 DNA40370-1217 DNA42551-1217 DNA39520-1217 DNA41225-1217 DNA43318'-1217 DNA40587-1231 DNA41338-1234 ATCC209401 ATCC2 09397 ATCC2 094 00 ATCC2 09385 ATCC2093 67 ATCC209432 ATCC2 092 63 ATCC209251 ATCC2 092 55 ATCC2 09252 ATCC2093 77 ATCC209370 ATCC209523 ATCC2093 72 ATCC209374 ATCC2093 73 ATCC2 09382 ATCC209383 ATCC209403 ATCC209398 ATCC2093 99 ATCC2 09392.

ATCC2093 87 ATCC2093 88 ATCC2093 94 ATCC2 09421 ATCC209393 ATCC2 094 18 ATCC209485 ATCC2O94 83.

ATCC209482 ATCC209491 ATCC2 094 81 ATCC209438 ATCC209 927 October 17, 1997 October 17, 1997 October 17, 1997 October 16, 1997 October 15, 1997 November 7, 1997 September 16, 1997 September 16, 1997 September 16, 1997 September 16, 1997 October 16, 1997 October 16, 1997.

December 10, 1997 October 16, 1997 October 16, 1997 October 16, 1997 October 16, 1997 October 16, 1997 October 17. 1997 October 17, 1997 October 17, 1997 October 17, 1997 October 17, 1997 October 17, 1997 October 17, 1997 October 28, 1997 October 17, 1997 October 28. 1997 November 21, 1997 November 21, 1997 November 21, 1997 November 21, 1997 Novem~ber 21, 1997 November 7, 1997 June 2, 1998 5,

S

186a DNA40981-1234 DNA37140-1234 DNA40982-1235 DNA41379-1236 DNA44167-1243 DNA39427-1179 DNA40603-1232 DNA43466-1225 DNA43046-1225 DNA35668-1171 ATCC209439 ATCC209489 ATCC209433 ATCC209488 ATCC209434 ATCC209395 ATCC209486 ATCC209490 ATCC209484 ATCC209371 November 7, 1997 November 21, 1997 November 7, 1997 November 21, 1997 November 7, 1997 October 17, 1997 November 21, 1997 November 21, 1997 November 21, 1997 October 16, 1997 r r r These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty).

This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposits will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc.

10 and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined r by the U.S. Comissioner of Patents and Trademarks to be entitled thereto according to 35 Usc 122 and the Conunissioner's nibs pursuant thereto (incluin 37 CFR 1. 14 with particular reference to 88600G638).

Thw assignee of tde present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when ailtiared uider suitable conditions, the materials will be promptly replaced on notification with another of the same. Xvailability of the deposited material is not to be coninid as a li.cense to practice the invention in comsravention of the rights granted under the authority of any government in accordance with its patent laws.

The foregoing written specificationus considered to be sufficient to enable one skiled in the ant to practice the invention. The present invention is not to be limited in scope by the coustruct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and ay constructs that are fuinctioally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, includieg t best mode thereof, nor is it to be construed as limiting the scope of the claim to the specific Mlustations that it represents. Ineed. various znodificatim of the invention in addition to those shown and described herein will become apparen~t to t=s skiled in the art from the foregoing description and fall within the scope of the appended claims.

Claims (21)

1. An isolated nucleic acid molecule having at least sequence identity to a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence which has the amino acid sequence shown in Figure 86 (SEQ ID N0:245).

2. A nucleic acid molecule according to Claim 1, wherein said nucleotide sequence comprises a nucleotide sequence as shown in Figure 85A-B (SEQ ID NO:244), or the complement thereof.

3. A nucleic acid molecule according to Claim 1, wherein said nucleotide sequence comprises the full-length coding sequence of the sequence shown in Figure 85A-B (SEQ ID NO:244), or the complement thereof.

4. An isolated nucleic acid molecule which comprises the full-length coding sequence of the DNA deposited under accession 20 number ATCC 209393.

5. A vector comprising a nucleic acid according to any one of Claims 1 to 4. eo,,r e6 *e 1 4

6. control sec vector. A vector according to Claim 5, operably linked to quences recognized by a host cell transformed with the

7. A host cell comprising a vector according to Claim or Claim 6.

8. A host cell according to Claim 7, wherein said cell is a CHO cell.

9. A host is an E. coli.

A host is a yeast cell. cell according to Claim 7, wherein said cell cell according to Claim 7, wherein said cell 189

11. A process for producing a PRO polypeptide, comprising the step of culturing a host cell according to any one of Claims 7 to 10 under conditions suitable for expression of said PRO polypeptide, and recovering said PRO polypeptide from the cell culture.

12. Isolated native sequence PRO polypeptide having at least 80% sequence identity to the amino acid sequence shown in Figure 86 (SEQ ID NO:245).

13. Isolated PRO polypeptide having at least 80% sequence identity to the amino acid sequence encoded by the nucleotide deposited under accession number ATCC 209393.

14. A chimeric molecule comprising a polypeptide according to Claim 12 or Claim 13, fused to a heterologous amino acid sequence.

15. A chimeric molecule according to Claim 14, wherein said heterologous amino acid sequence is an epitope tag sequence.

16. A chimeric molecule according to Claim 14, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin. e

17. An antibody which specifically binds to a PRO polypeptide according to Claim 12 or Claim 13.

18. An antibody according to Claim 17, wherein said antibody is a monoclonal antibody.

19. A composition comprising a) a nucleic acid molecule according to any one of Claims 1 to 4; b) a polypeptide according to Claim 12 or Claim 13; c) a chimeric molecule according to any one of 190 Claims 14 to 16; or d) an antibody according to Claim 17 or Claim 18, together with a pharmaceutically-acceptable carrier.

A nucleic acid according to Claim 1 substantially as hereinbefore described with reference to any one of the examples.

21. A polypeptide according to Claim 12 or Claim 13, substantially as hereinbefore described with reference to any one of the examples. Dated this 1st day of February 2002 15 GENENTECH, INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia 4