AU772723B2 - 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 references states what their authors assert, and the applicants 15 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 0 herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in 20 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 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 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. PR0211 and PRO217 Ep)iderma growth fhto (I3GF) is a convcnional mktogenic: factor that stimulates the proliferation of various types of cells includirV epithelial cells and fibroblasts. EGF binds to and activates the EGF receptor (EGFR). which initiates intracellular signaling and subsequnt effects. Thec EGFR is expressed in neurons of the cerebral cortex.

certbellum, Wn 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 postiiotic neurons. In fact. many studies have indicated tat EGF has neurotrophic 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 septat eholinergic and mesencephalic dopaminergic neurons, indirctly 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 signalin -in initotc cells is bettr uderstood than in postmnitotic neurons. Studies fcldoned pheochromocytoma PC12 cells and cultured cerebral cortical neurons have suggested that the EGF-induccd neurotrophic actions are mediated sustained activation of the EGFR and mitogen-activated protein kinase (MApK) in response to EGF. The sustained intracellular signialing correlates with the decreased rate of EGFR down-regulation, which might determine the response of neuronial cels to EGF. It is liely that EGF is a multi-potent growth factor that acts upon various types of cells including mitotic cells and postruitotic neurons.

EUF is produced by the salivary and Brunner'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.

crebrospinal fluid (CSP), urine, amniotic fluid, prostatic fluid, pancreatic juice, and breast milk, Plata-Salamnan, 20 Peptides .12: 653-663 (1991).

EGF is mediated by its membrane specific receptor, which contains an intrinsic tyrosine kinase. Stoscheck el Cell Biodzem. Ut: 135-152 (1986). EGF is believed to function by binding to the,extracellular portion of :its receptor which induces a transmembrane 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 thre peptide loops, Savage et J. Biol. COwm. 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 7

CX

4

,CX

1 CXCXGXC4. where X represents any non-cystcine amio acid, and n is a variable repeat zmer. Non isolated peptides; having this motif include TGF-cs, amphiregulin, schwairmoma-derived growth factor (SDGr-), beparin-bindiag EGF-Iike growth factors and certain virally encoded peptidcs Vaceinia virus, Reisner, Nature Mi: 801-803 (1985). Shope fibroma virus, Chang et al., Mol Cell Biol. 7: 535-540 (1987).

Molluscunneontagiostim, Porter and Archard, J. Gen. Virol. ff 673-68 (1987), and Myxoma virus, Upton et a1..

J. Virol. fil: 1271-1275 (1987), Prigent and Lemioine. Prog. Growth Factor Res. 1: 1-24 (1992).

EGF-Iikec domains are not confined to growth factrs but have been observed in a variety of cell-surface and extracelhrular proteins which have interesting properties in cell adhesion, protein-protein interaction and development, LUrzence and Gusterson, Tumor Biol. 11: 229-261 (1990). These proteins include blood coagulation factors (factors VI. Ex. X. XlI. protein C, protein S. protein Z, tissue plasminogen activator, urokinase), extracellular matrix componenits (laminin, cytotactin, entactin), cell surface receptors (LDL receptor, thrombomodulin reeptor) and immunity-relaed proteins (complement CMr. uromodulin).

Even more interesting. the general structure pattern of EUF-like Precursors is Preserved through lower organisms as well as in mammnalian cells. A number of genms with dcvelopmcntal significance have been identified in invertebrates with EGF-I-c repeats. For example, the notch gene of Drosophila encodes 36 tandemtly arranged amino aid repeats which show homology to EGF. Wharton er aL. cell 557- '581 (1985). Hydropathyplots Sindicate a putative membrane spanning domain. with the EGF-rehated sequences being located on tie exatacetfular side of the membrane. Other homeotic genes with EGF-lice repeats include Delta, 95F and SZD which were identified using probes based on Notch. arnd the nematode gene Lin-12 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 nairosa and! the repair of acute and chmonic mucosal lesions. Kontuek et al.. Eur. J. Gartroenterol Hiepatol. 2 933-37 (1995), including dhe treatment of necrotizing enterocolids, Zollinger-Ellison syndrome, gastrointestinal *ulceration gastrointestinal leain and congenital nucrovillus atrophy. Guglietta and Sullvan. tEur. Gwr meal llepol, 2(10). 945-50 (1995). Additionally, EGF has been implicated in hair follicle differentiation-, du Cros. J. Invest Denntol IQI (1 Suppl.). 106S-I 13S (1993). Hillier, Gin. Endocrinol. 427-28 (1990); kidney function, Hanm et al. Semin. NephroL 11 109-15 (1993), Harris. Am. A. Kidney Dis. 627-30 (1991); tear fluid, van Setten et aL. Iiu. Ophihalmol 1j(6); 359-62 (1991); vitamin K mediated blood coagulation, Stnflo a at. Blood 28(7): 1637-51 (1991). EGIF is also implicated various skin disease characterized by abnormal keratinocyte differentiation, psoriasis, epithelial cancers such as squamous cell carcinomas of the lunig, epidcrmold carcinoma of the vulva and gliomas. King et al., Am. Med. Sdi. M2: 154-158 (1988).

20 Of great interest is mounting evidence that genetic alterations in growith factors signaling pathways are closely linked to developmental abnonmalities and to chronic diseases including cancer. Aaronson, Science 2M: 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 overexpressed in hruman brest cancer, K~ing ar al., Science W2: 974-9176 (1985); Gullick, Hormones and their actions, Cooke el aL. eds, 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.

EBRO2 Nephritis is a condition characterized by inflarmnation of the kidney affecting the structure and normal function of the Itidney. Ibis condition can be chronic or acute and is generally caused by infection, degenerative process or vascular disease. In all case, early detection is desirable so that the patient with nephritis can begin treatment of the condition.

An approach to detecting nephritis is to determn the antigens associated with nephritis and antibodies thereto. In rabbit, a tubulointerstitial nephritis antigen MN-ag) has been reported in Nelson, T. et al., I -Biol QM. 27MC7)-16265-70 (July 1995) (GeJBANMM1Z270). This study reports that the rabbit TIN-ag is a basement membrarn glycoprotein having a predicted amino acid seqtxnce 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 I chain of type I collagen, von Willebrand's factor and mucin, indicating stuctural 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 tubulointrstitial 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 iterest 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 hgercin as PR0230, which has homology to tubulointerstitial nephritis antigens.

3. PR0232 Stem cells are undiffrentiated 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 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 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 whaom 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. PR017Z 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 cellular regulators. Their biological effects include proliferation, cheumtaxis and stirmulation of extracethilar matrix production. Growth factors can have both stirnulatory and inhibitory effects. For example. tr-ansforming growth factor (rGF-P) is highly pleuxtropic and can stimulate proliferation in some cells, especially connective tissue, while being a potent inhibitor of proliferation in others, such as lymphocytes and epithelial cells.

Tle physiological effect of growth stimnulation or inhibition by growth factors dependis upon the s tate of devclopmezt and diffrentiation of the target tissue. The mechanism of local cellular regulation by classical endocrine molecules involves comprehends autocrine (same cell), juxtacirine (neighbor cell), and paracrinc (adjacent cells) pathways. Peptd& growth factors are elemns of a complex biological language, providing the basis for intercellular cornxuznication. They permoit cells to convey information between each other, mediate interaction between cells ard cha gem expression. The effect of these multifunctional and pluripotet factors is dependent on the present= or absence of other peptides.

FGF-8 is a member of the fibroblast growth factors (FOGs) which are a family of heparin-binding, potent mogens for both normal diploid fibroblasts and established cell lines, Gospodarowicz det. (1984). Proc. Nat.

Acad. Sa. USA U16963. 'Ilm FOP famifly comnprses acidic FGF (FOP-I), basic FGF (FGF-2). INT-2 (PGF-3), K- FOF/HST (FGF-4), FGIF-S, FGF-6, KGF (FGP-7). AIGF (FGF-8) among others. All FGFs have two conserved 15 cysteir residues and share 30-50% sequence homology at the amino acid level. These factors are mitogenic for a wide variety of normal diploid mesodermn-derived and, neural crest-derived cells, including granulosa cells, adrenal cortical cells, chondrocytes, myoblasts. corneal and vascular endothelial cells (bovine or human), vascular smooth muscle cells, lens, retin and prostatic epithelial cells, oligodendrocytes, astrocytes, chrondocytes. myoblasts and osteoblasts.

.:20 Fibroblast growth factors can also stinrulate a large number of cell typs in a Don-mitogenic manner. These activities include promotion of cell migration into wound area (chemootaxis), initiation of new blood vessel formulation (angiogenesis). modulation of nerve regeneration and survival (neurotrophism), modulation of endocrine functions, :and stimulation or suppression of specific cellular protein expression, extracell ular mtrix production and cell survival. Baird Bolen, Handbook of Excp. Pharmacol. 95(1): 369418, Springer, (1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches to acclerate wound healing, nerve repair, collateral blood vessel formation, and the like. For example, fibroblast growth factors have been suggested to minimnie myocardium damage in heart disease and surgery 4,378,347).

FGF-8, also known as androgen-induced growth factor (AIGP), is a 215 amnino, acid protein which shares 30-40% sequence homology with the other members of the POF family. FGF-8 has been proposed to be under androgenic regulation and induction in the miouse mammary carcinoma cell line SC3. Tanaka at aL. Proc. NaI.

Acad. Sd. USA 12: 8928-8932 (199); Sato er J. &Sevid Bioclzcm. Molec. Bio. 42: 91-98 (1993). As a result, FOP-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 transfected into NIH-3T3 fibroblasts. Kouhara er aL.

Oncogene 2 455-462 (1994). While FGF-8 has been detected in heart brain, lung, idney, testis, prostate and ovary, expression was also detected in the absence of exogenous androgens. Schmitt et J. Stecroid Bloc/ter. Mo. Mot!.

52 173-78 (1996).

F-8 shames the property with several other FOP~s of being expressed at a variety of stages of niurine embryogenesis. which supports the theory that the various FGFs have multiple and perhaps coordinated roles in differentiation and cmbryogenesis Moreover, FGF-8 has also been identified as a protoncogene that cooperates with Wnt-I in the process of mammary tutooigenesis (Shackleford et at.. Proc. Na!. Acad. Sci. USA 2Q, 740-744 (1993); Ileiltinhegmo el al., Mich. Dev. 129-138 (1994)).

In contast to the other FGFs, FGF-8 exists as three protein isoformis, as a result of alternative splicing of the primary transcript. Tanaka et al.. jupra. Normal adult expression of FGF-8 is weak and confined to gonadal tissue, however northern blot analysis has indicated that FGF-8 anRNA is present from day 10 through day 12 or murine gestation, which suggests that FGF-8 is important to normal deveclopment. Heiltinheimto ft al.. Mich Dev.

41(2): 129-38 (1994). Further in sint hybridization assays between day 8 and 16 of gestation indicated initial expression in the surface: ectoderm of the first bronchia 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 hindlirab buds. the nasal its and nasophaxyrkx, the infundibulumn and in the telencephalon diencephalon and ntencepaton. Expression continues in the developing hindlimbs through day 13 of gestation, but is undetectable tbeaftcr. The results suggest that FOP-S has a unique temporal and spatial pattern in emibryogenesis and suggests a role for this growth factor in ultiple regions of ectodermal differentiation in the post-gastnzlation embryo.

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

S. 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 maniputlated to regulate the particular result of the protein-protein interaction. Thus, :dte underlying mechanisms of protein-protein interactions arc of interest to the scientific and medical commnity.

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

Lcucine-rich repeats are short sequence motifs present in a mnber of proteins with diverse functions and cellular locations. The crystal suctur of ribonuicase inhibitor protein has revealed that leucane-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 liochem.- 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 strotna formation. lozzo, R. Crit. Rev. iochem. Mol. Biol., 32C2):141-174 (1997). Others studies implicating leucine rich proteins in wound healing and tissue repair are Dc La Salle, et al.. Vouv.kev Fr erkato (Germany). 37(4):21is-222 (1995), reporting mutations in the leucine rich motif in a complex associated with the bleeding disorder Bemnard-Soulier syndromei and Chlemetson, K. Throsub. Haenost. (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 SWI protein which has been reported to be useful in treting ocuro-degenerative diseases such as Alzheimer's disease. herve damage such as in Parkinson's disease, and for diagnosis of caucr. see. Artavanistsakonas. S. and Rothberg. J. W09210518-Al by Yale, University. other studies reporting on the biological functions of proteins having leucine-rich repeats include: Tayar. ct aL. Hg!L Cell Enocrino., (Ireland). 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor involvement); Miura, et al., tNipn insh (Japan). 54(7):1784-1789 (July 1996) (apoptosis involvement). Harris. P. et al., LAM....r hbjn. 6(4):1125-1133 (Oct. 1995) (kidney disease involvement); and Ruoslahti, E. et al.. W091 10727-A by La Jolla Cancer Research Foundation (decorin binding to transforming growth factor-p involvement for treatment for cancer, wound healing and scarring). Also of particular interest is fibromodulin and its use to prtvet or reduce dermal scarring. A study of fibronindulin is found in U.S. Patcnt No. 5,654,270 to Ruoslalid. et al.

Efforts are therefore being uderulm by both industry and academia to identify new proteins having leucine rich repeats to better understand protein-protein interactions. Of particular interest ame those proteins having leucine rich repeats and homology to known proteins havinig leucine rich repeats such as fibroinodulin, the SLrT protein arnd 0 platelet glycoprotein V. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and memnbrane-bound proteins having leucine rich repeats. We herein describe the identiication and characterization of novel polypeptides having homology to fibroniodulin, herein o~so designated as PRO265 polypeptides.

6. PROZ2 Huma2n matrilin-2 polypeptide is a member of the von ilebrand factor typ A-like module superfamily.

:20 von Wiflcbranl fiuior is a protein which plays an importnt role in the maintenence of hemostasis. More specifically, von Willebrand factor is a protein which is known to participate in platelet-vessel wall interactions at the site of vascular injury via its ability to interac and form a complex with Factor VII.I. The absence of von Willebrand 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 brusing. nose bleed, intestinal bleeding, and the like comprising von Willebrand's disease.

Given the 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 MIlength polypeptide which possesses homology to the humnan matrilin-2 precursor polypeptide.

7. PRQ0a46 The cell surf=c protein HCAR is a membrane-bound protein that acts as a receptor for subgroup C of the adenoviruses and subgroup B of the coxsacievintses. Thus, HCAR may provide a means for mediating viral infection of cells in that the presee 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 specflcially those which serve a cell surface receptor for viruses, efforts are currently being undertaken by both induistry and academia to identify new. native membrane-bound receptor proteins. Many of thesc efforts ame focused on thc screening of mnammalian recomnbinant DNA libraries to icdetfy the coding sequenc for novecl rtceptor proteins. We herein describe a novel memnbrane-bound polypcpdcr (designated lercin as PR0246) having homnology to the cell surthce protein HCAR and to various arroor antigens including A33 and. carcimiembryonic antigen, wherein this polypeptdec may be a novel cell surface virtu receptor or fnnor antigen.

8. PROMS~ There are a number of known seven transmeIbrane proteins and! within this family is a group which includes CD97 and EMRI. CD97 is a sevcn-span trans-nemrane receptor which has a cellular ligand, CD5, DAF.

Hamann, et El. Med. 184C3):1189 (1996). Additionally. CD97 has been reported as being a dediffirenriation marker in hinmn thyroid caznoxms and as associated with inflammation. Aust, et al.. Canccr Re.

57(9):1798 (1997); Gray. ct al., J. Imusunol. 157(12):5439 (1996). CD97 has also been reported as ~being relatod to the secrctin receptor superfamily, but nli known members of that family, CD97 and EMRI have extended extracelular regions that possess several EGF domains at the N-terminus. Hamann 'et al.. Geaicsm 32(l):144 (1996); Harmana, et al.. J.Immuwo1., 155(4):1942 (1995). EMRI is further described in Lin. et al..

Cnmmics, 41(3)-.301 (1997) and Baud, et al.. Genojmicsi. 26CZ):334 (1995). While CD97 and EMRl appear to be relate to the secretin receptors, a known member of the sectin family of G protein-coupled receptors includes the aipbatroxin receptor, latrophilin, which has been described as calcium independent and abundant among neuronal tissues. Ldlianova. et al.. Biol.Chem.. 27234), 21504 (1997); Davletov, et al., J. Biol. Cbe. 271(M8)-23239 (1996). Both members of the aecrtin receptor superfamily and non-members which are related to the scartin receptor superfamily, or CRJF and calcitonin receptors are of interest. In particular, new members of these families, identified by their homology to known proteins, arm of interest.

Efforts are being undertaken by both industry and academia to identify new membrane-bound receptor proteins, particularly transuembrane proteins with EGF repeats arnd large N-terminuses which may belong to the family of seven-transmembrane proteins of which CD97 and EMR1 are members. We herein describe the identification and charactization of novel polypeptides having homology to CD97 and EMRI, designated herein as PR0228 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 nmltifunictional and potent cellular regulatrs. Their biological effects include proliferation, chemotaxis and stimulation of extraceilular matrix production. Growth factors can have both stimulatory and inhibitory effects. For example, transforming growth factors (TGF-P3) is highly pleionopic and can stimulate proliferation in some cells, especially connectiye tissus, uuilc being a potent inhibitor of prolicration in others, such as lymphocytes and epithelial cells.

The physiological effect of growth stimulation or inhibition by growth factors depends upon the state of developmet and diffcerntiation of the Larget tissue. The mechanism of local cellular regulation by classical endocrine WO 99/14328 PCr/US9BII 9330 molecules comprehends autocine (same cell). juxtacine (neighbor Celt), and paracrne (adjacent cell) pathways.

Peptidc growth factors are elenents of a complex: biological 'language, providing the basis for intercellular communication. They permit cells to omvey information between each other, mediate interaction between cells and change gene expression. the effect of these multifunctional and plwipocnt factors is depend=n on (he presence or absence of other pcptides.

Fibroblast growth factors (FGFs) are a family of heparin-binding, potent initogens for both normal diploid fibroblasts and established cell lines Godpodarowicz. D. et iiL (1 984). Pro. Natl. Acad. Sci. USA 8 1: 6983. the FGF family comiprises acidic FGF (FGF- basic FGF (FGF-2). INT-2 (FGF-3). K-FGF/HST (FGF.4). FGF.S, FGF-6. KGF (FGF-7), AIGF (FGF.8) among others. All FGFs have two conserved cysteine residues and share 30-50% sequence homnology at the aino acid level. These factors are niitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-deived cells. inducing granulosa cells, adrenal cortical cells, chrondocytes, myoblasts, cornecal and vascular endothelial cells (bovine or human). vascular smooth muscle cells, lens. retina and prostatic epitheLial cells, oligodendrocytes, astrocytes, chrondocytes myoblasts and osteoblasts.

0 9 Fibroblast growth factors can also stimiulate a large number of cell types in a non-mitogenic; manner. These'

I.L*

:900.0activities include promotion of cell migration into a wound area (chemnotaxs), initiation of new blood vessel formulation 0 15. (angiogenesis), modulation of nerve regeneration and survival (neurotraphism). modulation of endocrine fuinctions, and stimulation or suppression of specific cellular protein expression, octracellular matrix production and cell survival.

0 0Baird, A. Bohlen. Handbook of Exp. PhrmacoL 25 369-418 (1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healinig, ncrve repair. collateral blood vessel formation, and the like. For example, fibroblest growth factors, have been suggested to minimize myocardium 20 damage in heart disease and surgery 4.378.437).

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

PR2 Some of the most important proteins involved in the above described regulation and modulation of cellular .9....processes ame the ezyms which regulate levels of protein phosphorylation in the ceIl For example, it is know that 9 the transduction of signals that regulate cell growth and differentiation is regulated at least in part by phosphorylation 9*9s and dephosphorylation 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 phosphorylation in the cell is thus mediated by the relative activities of these two types of enzymes.

Although manny protein kinas enrymts have beenidentified, the physiological role played by many of these catalytic proteins has yet to be elucidated It is well kown, however, that a number of the known protein kinases function in phosphorylate tyrsin 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 inases since the discovery that many oncogene products and growth factors possess intrinsic protein tyrosine kinas activity. There is, therefore, a desire to identify new members of the protein tyroine kinase family.

Given the physiological importance of the protein kinases, efforts are being undertaken by both industry and WAed to identify new. native kinasc proteins. Many of' these efforts are focused on the screening oftmamnmalian 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 Icinase proteins, designated herein as P*R0245 polypeptides.

11. PRO220. PR0221 and PRO227 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 dhe protein-protein interation. 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.

Lzucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. The crystal structure of ribomiclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they formn a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglubular shape. These two features have been indcatd a reponible for the protein-binding functions of proteins containing lecn-ri crpeats. Se, 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. IozzoR. Crit. Rev. Biochein Mat Riol., 32(2):141-174 (1997). Others studies imeplicating leucine rich proteins in wound healing and tissue repair are Dc LA Salle. et Vauv. Rev- Fir- Hmal (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. Thbm. Haetri. (Germany), 74(l):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 neuro-degenerative diseases such as Alzheimer's disease. nerve damage such as in Parkinson's disease, and for diagnsi of cancer, see, Artavanistsakonas, 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., M-L.

Cel Endocinni., (Ireland). 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor involvemenit); Miura, et al., Njpga.Einsh (Japan), 54(7):1784-1799 (July 1996) (apoptosis involvement); Harris, P. et al..,J Am. Soc.

Ncbm. 6(4):1125-1 133 (Oct. 1995) (kidney disease involvement); and Ruoslahti. E. L. ea al.. W09110727-A by La Jolla Cancer Research Fundxarion (decorin binding to tanforming growth factorfi involvement for treatment for cancer, wound healing and scarring).

Efforts are therefore being undertakien 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 knzown proteins having leucine rich repeats such as the SLIT protein and platelet glycoprotein V.

12. PROMS lmrrumoglobilins are antibody molecules, thc proteins that ftunction both as receptors far antigen on the Bcell membrane and as the secreted products of the plasma cell. Lake all antibody molecules. immunoglobulins perform two major functions: they bind specifically to an antigen and they participate in a limited mnmber of biological effector functions. 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 arc 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. 1Thus, molecules having homology to poliovirus receptors, CRUAM and CD 166 (a ligand for lymphocyte antigen CD6) arc of particular intcrest.

Extracellular and mcembrane-bound proteins play importan roles in the formation. differentiation and maintenance of multicellular organisms. The fate of may individual cells, prolieration, migration.

differentiation or interaction with other cells, is typically governed by information received from other cells and/or *the immediate environment. This information is oftena tansmitted by secreted polypeptides (for instance. mitogenic 0* factors, survival facors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptsides or 15 signaling molecules normally pass throughr the cellular secretozy pathway to reach their site of action in the cxtracellular environment, usually at a membrane-bound receptor protein.

We herein describe the idniation and characterization of novel polypeptides having homology to CRTAM. designated herein as PR0258 polypeptides.

20 13. PRW so. 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.

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

Leucine-rich repeats are short sequence motifs present in a nmber of proteins with diverse fuanctions and cellular o locations. The crystal swmcr of ribottuclease inhibitor protein has revealed that leucine-rich repeats correspond to bera-alpha structural units. Th~ese units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape. These two features have been irndicated as responsible for the protein-binding fuions of proteins containing leucine-rich repeats. See, Kobe and Deisehofer. Trends Bocm. ScL 19(10):415-421 (Oct. 1994).

A study has been reported on leucine-rich proteoglycan 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 atroma formation. lozzo, R. Crit. Rev. iocem. Mol- Riil., 32C2):141-174 (1997). Others snudies implicating leucine rich protens in wound healing and tissue repair are De LA Salle, -ct al.. Vouv. RLv Er- Hmtol. (Germany), 37(4)215-2 (1995). reporting moutations in the leucine rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome and Chlemetson, K. Thromb. Haemost. (Germany).

74(l):lll-116 (July 1995). reporting that platelets have Icucine rch repeats.- Another proteinof particular interest which has been reported to have leucine-rich repeats is the SLIT protein which has been reported to be useful in treating netzro-degenativc diseases such as Alaheimer's disease. nerve damage such as in Parkinson's disease, and for diagnsis ofcancer. see Aavaissakmas. S. aztdRothberg, J. M. W09210518-Al by Yale University. Other studes repoiting on the biological functions of proteins having leucine-ricb repeats include: Tayar, et al, MgL Cel ndoc21o., (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropi receptor involvement); Miura. et al., Nippon inmha (Japan), 54M7: 1784-1789 (July 1996) (apoptosis involvement); Harris, P. et al.. J. Am. So.

Not. 6(4):1125-1 133 (Oct. 1995) (kidney disease involvement); and Ruoslahti, E. L. et al., W09110727-A by La Jolla Cancer Research Foundation (decorin binding to transforming growth factor3 involvement for treatment for cancer, wound healing and scarring).

Efforts are therfor being umiezual= by both industry and academia to identify new proteins having leucine rich repeats to better understand protein-protein interactions, neuronal development and adhesin molecules. Of particular unrest are those proteins having leucine rich repeats and homology to known proteins having leucine rich repeats such as the SUT protein. We herein describe novel polypeptdes having homology to SIrr, designated herein aPR0266 polypeptides.

14. PRO262 Thkrombomodulin binds to and regulates dhe activity of thrombin. It is important in the control of blood coagulation. Thrombomodulin functions as a natural anticoagulant by accelerating the activation of protein C by thrombin. Soluble throinbomodulin may have therapeutic use as an antihronibotic agent with reduced risk for :20 hemorrhage as compared with heparin. Thrombomnodulin is a cell surface trans-maerbrane glycoprotein, present on enidothelial cells and platelets. A smaller, functionally active form of thrombomodulin circulates in the plasma and is also found in urine. (In Haeberti, Human Protein Data, VCH Oub., 1992). Peptides having homology to thrombomnodulin arc particularly desirable.

.We herein describe the identification and characterization of novel polypeptides having homology to thrombomodulin, designaed herein as PR0269 polypeptides.

PROM2J Procollagen C-proteinase enhancer protein binds to and enhances the activity of bone morphogenic protein "BMP I *Irocollagen C-proteinase: (PCP). It plays a role in extracellular matrix deposition. EMPI proteins may be used to induce bone and/or catilage formation and in wound healing and tissue repair. Therefore, procollagen Cproteinase enhane protein, BMIP I and proteins having homnology thereto. arc of interest to the citific and medical communities.

We herein describe the identification and characterization of novel polypeptides having homology to procollagea C-proteinse cahan= protein precursor and procollagen C-proteinase enhancer protein, des ignated herein as PRO287 polypeptides.

16. PR0214 Growth factors are molecular signals or mediators that enhances cell growth or proliferation, &lot= or in concert, by binding to specific cell surface receptors. However, thcre are other cellular reactions than only growth upon expression to growth factor. As a result, growth factors are better characterized as multiurdtonal and potent cellular regulators. T1heir biological effects include proliferation, chemotaxis and stimulation of extracellular matrix production. Growth factors can have both stimulatory and inhibitory effects. For example. trnsfornming growth factor P3 (TGF-4) is highly pleiotipic and can stimulate prolifmizion in somne cells. especially connective tissue, while being a potent inhibitor of proliferation in others, such as lymphocytes and epithelia cells.

The physiological effect of growth stimulation or inhibition by growth factors depends upon the state af development and differentiation of the target tissue. 11r mechanism of local cellular regulation by classical endocrine molecules involves comprehends autocrine (sam cell), juxtacrine (neighbor cell). and paracrine (adjacent cells) pathways. Peptide growth fiaors art elemntms of a complex biological language, providing the basis for intercellular coanmnmication. They permit cells to convey information between each other, mediate: interaction between cells and changec gene expression. T'he effect at these mnultifunictional and phnripotent factors is dependent on the presence or absence of other peptides.

Epidernia growth factor (EGF) is a conventional mitogenic factor that stimulates the proliferation of various types of cells including epithelial cells and fibroblasts. EGF binds to and activates the EGF receptor (EGFR). which initiates intracelular signain and subsequent effects. The EGFR is expressed in neurons of the cerebral cortex.

crebellm and hippocainpus in addition to other regions of the central nervous system (CNS). In addition, EUF is also expresed in various regions of the CNS. Therefore, EGF acts not only on mitotic cells, but also on postmitotic 20 ncurons. In fact mny studies have indicated that EGF has neurotrophic or neuromnodulatory effects on various types of neurons in the CNS. For example, EGF acts directly on cultured cerebral cortical and cerebellar neurons, enhancing ncurte outgrowth and survival. On the other hand. EGF also acts on other cell types, including septal chalinrgic 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 signalig in mitotic cells is bette understood than in postitotic neurons. Studies of cloned pheochromocytoma PCl12 cells and cultured cerebral cortical neurons have suggested that the EUF-induced neurotrophic actions are mediated by sustained activation of the EGFR and nutogecn-activated protein kinase (MAPK) in response to EGF. The sustained intaelular 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 postiiotic neurons.

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

thyroid gland, pituitary gland, and the nervous system, and is found in body fluids such as saliva, blood.

cxebrospina fluid (CSF), urine, aniotic fluid. prosttic fluid, pancreatic juice, and breast milk, Plata-Salaman, CR Peptides 12: 653-663 (1991).

EOF is mediated by its memb~rane specific receptor, which contains an intrinsic tyrosine kinase. Stoscheck CM et Cell Biodzmn. 31: 135-152 (1986). EGF is believed to function by binding to the extraceliular portion of its receptor which induces a transmcmbrarie signal that activates the intrinsic tyrosine kinase.

Purification and sequence analysis of the EGF-like domain has revealed the presence of six conserved cysteine residues which cross-bind to create three peptide loops, Savage CR e Niot Chem. 7669-7672 (1979). Itisi now generally known that several other peptides can react with the EGF receptor which share the same generalized motif X.CXCX,,CX 0 CXCXGX,CX,, where X represents any non-cysteine ammo acid. and n is a variable repeat number. Non isolated peptides having this motif include TGF-a, amphiregulin. schwannoma-derived growth factor (SDGF). hecparin-bindiag EGF-like growth factors and certain virally encoded pepddes Vaccinia virus, Reisner Al, Nature ML: 801-803 (1985). Shope fibroma virus, Chang et al., Mol Cell Biol. 7: 535-540 (1987, Moluscum coutagiurn Porter CD Archard LC. Gen. Vital. 673-682 (1987), amd Myxonia virus, Upton C a al.. Virol. Qi: 1271-1275 (1987). Prigent SA Lemnoine Prog. Growth Factor Res. 4: 1-24 (1992).

EGF-likc domains are not confiried to growth factors but have been observed in a variety of cell-surface and extracelular proteins which have interesting propcrties in cell adhecsion, protein-protein interaction and development, *Laurenice DIR GustersoaflA. Tumor Biol. 11: 229-261 (1990). 7Tese proteins include blood coagulation factors (factors VI. IX, X. XII, prtein C. pnitein S. protein 7, tissue plasminogen activator, urokinase). extracellular matrix components (laninin, cytotactin, entactin). cell surface receptors (LDL receptor, thrombomodulin receptor) and iinmunity-related proteins (complement Clr. uromodulin).

Even more interesting, the general structure pattern of EGF-Iike precursors is preserved through lower organisms as well as in mammalian cells. A number of genes with deveopmnta significance have benidentified in invertebrates with EGF-like repeats. For example, the notch gene of Drosophila encodes 36 tandeinly arranged amino acid repeats which show homology to EGF, Wharton W et al, Cell 1: 557-581 (1985). Hydropathy plots :20 indicatc a putative membrane spanning domain, with the EGF-related sequences being located on the extracellular side of the membrane. Other homeotic genes with EGF-Iike repeats include Delta, 9SF and SZD which were identified using probes based on Notch, and the nematode gene Lin-12 which encodes a putative receptor for a developmnttal signal transmitted between two specified cells.

Specifically, EUF has been shown to have potential in the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic niticosal lesions, Konturek, PC et al.. Eur. J. Gartroenterol Iieparol.

2 933-37 (1995). including the treatment of necrotizing enterocolitis, Zollinger-Ellison syndrome, gastroinrestinl ulceration gasttointstinal ulcerations and congenital microvilius atrophy. A. Guglietta PB Sullivan.

Eur. Gastroenterol Hepatol, Z(10), 945.50 (1995). Additionailly, EOF has been implicated in hair follicle difkrcniano= C.L dii Cros. J. Invest. Dermatol. 1M1(1 Suppl.), 106S-1 13S (1993), SG Hillier, Clin. Enidocrf no!.

427-28 (1990); kidney function, L.L. Hamnm at al.. Senan. Nephrol. ia 109-15 (1993), RC Harris, Am.

J. rjiey Dis. 627-30 (1991); tear fluid. GB van Sctten et Int. Ophtabraolli5(6); 359-62 (1991): vitamin K mediatedi blood coagulation, 1. Stenflo ea Blood 26(7): 1637-51 (1991). EGF is also implicated various skin disease characterized by abnornmal keratinocyte differentiatian psoriasis. epithelial cancers such as squamous cell catcinomia; of the lung, epidermaid. carcinma of the vulva and gliomas. King, LE at al.. Am. J. Med. Sd. 2M6: 154-158 (1988).

Of great interest is mounting evidence that genetic alterations in growth factors signaling pathways are closely linked io developmental abnormalities and to chronic diseass including cancer. Aaronson SA, Science 254: 1146-1153 (1991). For examplc, c-crb-2 (also known as HER-2). a protO-onrce with close structural similarity so EOF reeptor protein, is overexprssed in humnan breast cancer. King et Science 22.9: 974-976 (1985); Oultick, WI. Hormones and their actions. Cooke BA ar al.. eds. Amsterdam. Elsevier. pp 349-360 (1986).

17. PR317 The TOP supege family, or simply TG"-f superfamily, a group of secreted proteins, includes a large number of related growth and differentiation factors expresscd in virtually all Phyla. Superfamily members bind to specific cell surface receptors diat activate signal transduction mechanisms to elicit their mnultifunctional cytokine effects. Kolodziejczyk and Hall, Biochem- Cell -Biol.. 7A- 299-314 (1996); Attisano and Wrana, Cytoknc rrMhi Factor Rev.. 7: 327-339 (1996); and Hill, Cellular Signahing. 1: 533-544 (1996).

Membeus of this family inclue five distinct forms of TGF-fl (Sporn and Roberts. in Peprde Growth Factors and Ibeir Rccetors, Sporn and Roberts, eds. (Springer-Verlag: Berlin. 1990) pp. 419-472). as well -as the differentiation factors vgl (Weeks and Melton, QcL. U: 861-867 (1987)) and DPP-C polypeptide (Padgett et a., Natiure. 3U: 8 1-84 (1987)), the hormones activin and irIWWbi (Mason et al.. Narmm. M.j: 659-663 (1985); Mason et Grot Factors, 77-88 (1987)). the Mullerian-inhibitin substance (MIS) (Cate et al. QCell 41: 685-698 (1986)). the bone morphogenctic, proteins (BMPs) (Wontey et al., Scec 2a: 1528-1534 (1988); PCT WO 8810005 published January 14. 1988; 4,877864 issued October 31, 1989). the developmentally regulated proteins Vgr-I (Lyons ef at., Proc. Nati Acad. Sci USA. Ifi: 4554-4558 (1989)) and Vgr-2 (Jones et al., Mokle Endocrinol 1961-1968 (1992)). the mouse growth differentiation factor (GDP). such as GDF-3 and GDF-9 (Kingsley, Gee e- 1: 133-146 (1994); McPherron and Lee, )-Big]LCbern. 21: 3444-3449 (1993)), the mouse .:20 lefiy/Stral (Meno et al., Nature. In~: 151-155 (1996); Bouillettal., Dv. Bil., 17D: 420-433 (1995)). gtial cell line-derived neurotrophic factor (GDNF) (Ln ra., Iqric=. 2W1: 1130-1132 (1993), neurturin (Katzbauer et al., Natue M. 467-470 (1996)). and aidometrial bleeding-associated factor (EBAF) (Kothapalli ar al., J. Cin.nvest., 22:2342-2350 (1997)). The subset BMP-2A and BMP-2B is approximately 75% homologous in sequence to DPP-C and may represent the mammnalian equivalent of that protein.

The proteins of the TOFP superfamily arc disulfide-linked homo- or heterodimers encoded by larger precursor polypeptide chains containing a hydrophobic signal sequence, a long and relatively poorly conserved Nterminal pro region of several hundred amino acids, a cleavage site (usually palybasic), and a shorter and more highly ****conserved C-terminal region. T7his C-terminal region corresponds to the processed mature protein and contains apprximately 100 amino acids with a chaacteristic cysteirE moti Le.. the conservation of seven of the nine cysteine residues of TOFP amng all known familmubers. 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 evety case from the TGOP consensus C-terminus of Cys-Lys- CYs-Ser. Spamn and Roberts, 1990, sup ra.

There are at least five forms of TOP ca*eslidentified. TOF-Pi. TGF-P2. TGF-fl3, TGP4, and TOFjI5. mm activated form of TGP-fl is a Immodireer formed by dimerization of the carboxy-teminal 112 amino acids of a 390 aino acid precursor. Recombinant TOFPpI has been cloned (Derynck et al.. N~ature, Ml:701-705 (1985)) and expressed in Chinese hamster ovary cells (Gentry et al.. Mo CllBil. 7: 3418-3427 (1987)). Additionally, recombinant human TGF-P2 (dcMartia a aL, EMBOJ.. fi: 3673 (1987)). as well as human and porcine TGF-013 (DeyckekaL. EMO 3737-3743 (1988), ten Dijlcz eraL. Proc. Nad. Acad. Sci. USA, 15: 4715 (1988)) have beta cloned. TGF-f32 has a precursor form of 414 amino acids and is also processed to a homodinmer from the carboxy-terminal 112 amino acids that shares approximately 70% homology with the active form of TOF-fli (Marquardt ar iL- bm 2M: 12127 (1987)). See also EP 200,341; 169,016; 268,561; and 267.463; U.S.

Pat. No. 4.774.322; Cheifctz er aL. f&l. 41: 409-415 (1987); Jakowlew et aL, Molecular Endocrin., 2: 747-755 .(1988); Derynck etal.. I Biol. Chemn. 21: 4377-4379 (1986). Sharples ar at. DNA. fi: 239-244 (1987); Derynck edal, ul-id. Rs.,1: 3188-3189 (1987); Derynck eral., Nuc!. Acids. Res, 15: 3187 (1987). Seycdin egoal..

L.Biol. Chm-3. ZJ1: 5693-5695 (1986); Madisen ar al.. D A, 7: 1-8 (1988); and Hanks at at., Prc al cd SU: 79-82 (1988).

TGF-P4 and TGF-P5 were cloned from a chicken chondrocyte cDNA library (Jakowlew ar al.. Malc Endocrinol.. 2: 1186-1195 (1988)) and from a frog oocyte cDNA library, respectively.

The pro region of TGF-P associates non-covakeuly with dhe mature TGF-P dimer (Wakefield ar al., L. iol.

203: 7646-7654 (1988); Wakefield eal.. Growth Factors. 1: 203-218 (1989)), and the pro regions are found tobeneessary for proper folding and secretion of the active mature dimers of both TGF-P and activin (Gray and Mason, sig M4: 1328-1330 The association between the mature and pro regions of TOrP masks the biological activity of the mature dimer, resulting in formnation of an inactive latent form. Latency is not a constant of dhe TOFP superfamily, since the presence of the pro region has no effect on activin or inhibin biological 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 :20 normal and neoplastic cells. TGF-P i s nmltifinxtional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell fuxxction (Spom and Roberts, rup ra).

One member ot the TG"-f sperfamily EBAF, is expressed in-endonetriun only in the late secretory phase and during abnormal endometrial bleeding. Kothapalli af al., J.Clin. nvest, 22: 2342-2350 (1997). Human andonietriurm is unique in that it is the only tisse in the body that bleeds at regular intervals. In addition, abnormal cirlometria bleeding is one of die most common manifestations of gynecological diseases, and is a prime indication for hysterectomy. In mu hybridization showed that the ruRNA of EBAF was eXpressed in the stroma without any significant mRNA expression in dhe endometrial glands or endothelial cells.

The predicted protein sequence of EBAF showed a strong homology to t protein encoded by mouse leflyls=3a of the TG"-f superfamily. A motif search revealed that the predicted EBAF protein contains most of the cysteinc residues which are conserved among the TGF-p-related proteins and whiich are necssary for the formation of dhe cysteine kot structine. The EBAF sequence contains an additional cysteine residue, 12 amio acids upstream from the first conserved cysteine residue. The only other family members known to contain an additional cysteine residue are TUF-fis, inliibins. and GDF-3. EBAF, similar to LEFI7Y, GDF-3IVgr2, and GDF-9, larks -the cysteine residue that is known to form the intermolecular disulfide bond. Thberefore, EBAF appears to be an additional miember of the TGF-P superfamily with an impaired cysteine residue that may not exist as a dimer. However.

hydrophobic contacts between the two monomer subunits may promote dirner formation. Fluore-scence in siu hybridization showed that the ebaf gene is located on human chromosome I at band q42. 1.

Additional members of the TGF- 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 polypeptides.

18. PR0301 The widespread occurence of cancer has prompted the devotion of comidcrable 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 arc 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 treatent in siru with minima patient description.

15 mAbs can also be used to diagnose during the diagnosis and treatment of colon cancers. For example, when the s c rum 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 scrum A33 antigen levels after surgery would indicare that metastases 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 20 example, U.S.P. 4,579.827 and U.S.S.N. 424.991 199.141) arc directed to therapeutic administration of monoclonal antibodies, the latter of which relates to the application of anti-A33 mAb.

Many cancers of epithelial origin have adenovirus receptors. In fact, adenovirus-derived vectors have been proposed as a means of inserting antisease 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. PR0224 Cholesterol uptake can have serious implications onone'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. Therefore. 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 ihe LDL receptor. These receptors can function to cndocytosc 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 pharnaceucal 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 molcules which enhance endocytosis so as to lower blood cholesterol levels and plaque formation.

It is also desirable to identify molecules which inhibit cndocytosis so that these molecules can be avoided or regulated by individuals having high blood cholesterol. Polypeptides which are homologous to lipoprotein receptors but which do not function as lipoprotin receptors arc 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 apopoproteins: Sawamura, ct al., Nippon Chemiphar Co, Japan patent application J09098787: Novak. S., Sctal., J. Biol. Chem.. 271:C0)11732-6 (1996); Blaas, L irl., 69(11)7244-7 (Nov. 1995); Scott, Inherit.

Metab. Dis. 9/Supp. 1 (3-16) (1986); Yamamoto. ct al., Cell, 39:27-38 (1984); Rebece, ct al., Neurbiol.

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

CcI Mol. iol.. 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 20 and characterization of novel polypeptides having homology to lipoprotein receptors, designated herein as PRO224 Spolypeptides.

PR0222 S* 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 protcolytic enzymes. When activated, complement proteins kill bacteria and other microorganisms, affect vascular permeability, release histamine and attract 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 erythematosus, 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 (CR1). 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 proteins O~b. CO., and iC3b to accelcrate dissociation of C3 coavertases, acts as a cofactor (or Factor [-mediated cleavage of *C3b and CO., and binds iurtune complexes and promotes their dissolution and phagocytosis.

Proteins which have homology to complarm proteins ame of particular interest to the medical and industria Communitics. Often. proteins having homology to each other have similar function. It is also Of interest when proteins having homology do not have similar functions, indicaing that certain structural motifs identify information other than function, such as locality of function.

Efforts arc being undertaken 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 comnpiernent pathway were reviewed in Birmingham DI (1995), Crtcl eiw jamMMoX 15(2):133-154 and in Abbas AK. ct al. (1994) Cellular and Molecular Immunology, 2nd Ed. WA,.

Saunders Company, Philadelphia, pp 295-3 We herein describe the Identification and characterization of novel polypeptides having homology to complement receptors, designated herein as PR0222 polypeptides.

21. PRO34 The successfuil function of many systems within nulticellular organism is dependent on cell-cell interactions. Such interactions are affected by the alignment of particular ligaixis with particular receptors in a manner which allows for ligand-receptor binding and thus; a cell-cell 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. Brandicy f J. Leak. Bio!. 4Q: 917 (1986) and N. Sharon ef at. Sdence 24ti: 227 (1989). Oligosaccharides are well positioned to act as recognition novel lectins due to their cell surface location and structural diversity. Many oligosaecharide structures can be created through the differential activities of a smaller number of glycosyltrans (erases. The diverse structures of oligosaccharides can be generated by transcription of relatively few gene products, which suggests that the oligosaccharides are a plausible mechanism .25 by which is directed a wide range of cell-cell interactions. Examples of differential expression of cell surface cartbydrates and putativ carbohydrate binding proteins (lectina) on interacting cells have been described Dodd T.M. Jessel, Neaurosa. 3278 (1985); LU. Regan a cal., Proc. Natd. Acad. Sci. UiSA 2248 (1986); M.

Constantine.Paton et al.. Nature 3Z4: 459 (1986); and M_ Tiemeyer et al.. J. Biol. Chzem. Ua: 1671 (1989). One interesting member of the lectin family are selectins.

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

Thec types of cell adhesion molecules that are involved in the interaction between leukocytes and the endothelium during an inflammatory response currently stands at four. selectins; (carbohydrate and glycopmoeia) ligands for slctins() itegrins; and integrin listands, which are members of the immunoglobulin gene superfamily.

lTw selectins arm cell adhcuion molecules that arc unified both structualy and functionally. Srucniy, selecrins arc characterized by the inclusion of a domain with homology to a calcium-dependcnt lectin (C-lectins). an epidcrnial growth factor (egf)-like domain and several complement binding-like domains, Bevilacqua. M.P. aet., Soice 2A3: 1160-1165 (1989); Johnston et at., Cell 1033-1044 (1989): Lasky er at, Cell M: 1045-1055 (1989); Siegalman, M. et Science W4: 1165-1172 (1989); Stoolnian, LM.. Cell.U: 907-910 (1989). Functionally.

selretins share t common property of their ability to mediate cell binding through interactions bctwce their lectin domains and cell surface carbohydrate ligands (Brandley. B. di at., Cel! 62. 861-863 (1990); Springer. T. and Lasky, Nature 24. 19-1917 (1991); Bevilacqua, M.P. and Nelson, J. Cm.- Invest. 21 379-387 (1993) and Tedder et J. Exp. Med. Inl: 123-133 (1989).

Therme art three members identified so far in the selectin family of cell adhesion molecules: L-selectin (also called peripheral lymph node horning receptor (pnHR). LEC-CAM-I. LAM-i, gp9OhIEL. gpIM0'L. gpl 10"L'- MEL- 14 antigen, Leu-8 antigen, TQ-I antigen. DREG antigen), E-sdlectin (LEC-CAM-2. LECAM-2. ELAM-i) and P.

selectin (LEC-CAM-3, LECAM-3. OMP-140. PADGEM).

The identification of die C-Icctin domain has led to an intenise effort to define carbohydrate binding ligands for proteins containing such domains. E-selectin is believed to recognize the carbohydrate sequence NeuNAca2- 3GaliP14(Fuca1 -3)GlcNAc (sialyl-Lewis x, or sLc') and related oligosaccharides, Berg ci J. BioL. (2r. W: 14869-14872 (1991); Lowe ar al., Cellfl: 475-484 (1990). Phillips et Science M: 1130-1132 (1990); 7temeyer et al.. Proc. Natt. Acad. Sc:. USA fl- 1138-1142 (1991).

L-selcctin, which comprises a lectin domain, performs its adhesive function by recognizing carbohydratecontaining Ligands on endothelia] cells. L-sclcctin is expressed on the surface of leukocytes, such as lymphocytes.

reutrophils, monocytes and eosinophils, and is involved with the trafficking of lymphocytes to peripheral lymnpboid tissues (Gallatin et Nature 3D3: 30-34 (1983)) and with acute neutrophi-medicated inflammatory responses (Watson, Ature 22164-167 (1991)). The amino acid sequence of L-selectin and t encoding nucleic acid sequence are. for example, disclosed in U.S. patent No. 5,098,833 issued 24 March 1992.

L-selectin (LECAM-i) is particularly interestin because of its ability to block neutrophil influx (Watson et al., Nature M4: 164-167 (1991). It is expressed in chronic lymnphocytic leukemia cells which bind to R1EV (Spertinidca!.. Nafure 2: 691-694(1991). It is also believed that HEY structures; at sites of chronic inflammation arc associated with the symptom of diseases such as rheumatoid arthritis, psoriasis and multiple sclerosis.

E-selectin (ELAM-l). is particularly interesting because of its transient expression on endothelial cells in response to ELI or TNF. Bevilacqua ct al., Science W4: 1160 (1989). The time course of this induced expression (2.8 h) suggests a role for this receptor in initial neutrophil induced extravasation in response to infection and injury.

It has further been reported that ann-ELAM-1I antibody blocks die influx of neutrophils in a primate asthm model and thus is beneficial for preventing airway obstruction resulting from the inflammatory response. Gundel ei J.

Cln. Invest. A_8: 1407 (1991).

The adhesion of circlating ututrophils to stimulated vascular endothelitum is a primary event of the inflammatory response. P-selcin has been reported to recognize die Lewis x structure (Galp1-4(Fuca1-3) G~cNAc), Lars e alc.. Cell 467474(1990). Others report that an additional terminal linked sialic acid is required for high affinity binding. Moore al., Cell. Biol. 12: 491-499 (1991). P-selectin has been shown to be significant in acute lung aniwy. Anti-P-selectin anuibody has been shown to have strong protective effects in a rodent lung injry model.

M.S. Mulligan et aL, J. Cli,,. Invest. 29: 1600 (1991).

Wc herein describe the identification and characterization of novel polypeptides having homology to lectin proteins. herein designated as PR0234 polypeptides.

22. PRl231 It Some of the most imtportant proteins involved in the above described regulation and mnodulation of cellular processes ame 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 dcphosphosylation 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 phosphorylation in the ceil is thus mnediated by the relative activitie ortheae two types of enzymes.

protein phosphatases represent a growing funiy of enzync that are found in many diverse forms, including both membrane-bound and soluble forms. While many protein phosphatases have been describe-d, the functions of only a verny few are beginning to be understood (ronkcs, Semi,,. Cell Biol. 4:373-453 (1993) and Dixon, Recent Prog.

fom. Res. S51:405-414 (1996)). However, in general, it appears that many of the protein phosphatases function to modulate the positive or negative signals induced by various protein kinases. Therefore, it is likely that protein phospharases play critical roles in numerous and diverse cellular processes.

Given the physiological importance of the protein phosphatases, efforts are being undertaken by both :20 industry and academia to identify new, native phosphatase proteins. Many of these eforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel phosphatase proteins.

Examples of sceening methods and techniques are described in the literature [see, for example, Klein et al., Bm NatI. Acad. Sci. 1:710S-7113 (1996); U.S. Patent No. 5,536,637)].

We herein describe the identification and characterization of novel polypeptides having homology to acid phosphatases, designated herein as PR.0231 polypeptides.

23. PRO229 0000 Scavenger receptors are known to protect lgG molecules from catabolic degradation. Riechmann and Hollinger, Nature Biotechnology, 15:617 (1997). In particular, studies of the C312 and CH3 domains have shown that specific sequences of these domains are important in determining the half-lives of antibodies.' Ellerson, et al., J.Linmiil.. 116: 510 (1976), Yasmeen, et al.. Jmmanoi. 116: 518 (1976; Pollock. et al., EllLJmwlmgul.. 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 fuinction, molecules having homology to scavenger receptors are of importance to the scientific and medical community.

Efforts arc being underta=e 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 identify 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 ct al.. Proc. Natl. Acad. Sci., 21:710-7113 (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. PRO238 Oxygen free radicals and antioxidants appear to play an important role in the central nervous system after cerebral ischemia and reperfusion. Moreover, cardiac injury, related to ischaemia and repcrfusion 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 fate of the cells. Furthermore, 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 reasons including for control and prevention of strokes, heart attacks, oxidative stress and hypertension. In this regard, reductases, and particularly, oxidoreductases, are of interest. Publications further describing this subject matr include Kelsey, et al., r. Cancer 76(7):8524 (1997); Friedrich and Weiss, J. Theor. Biol.. 187(4):529-40 (1997) and Picull, ct al., J. Batrio.. 179(18):5684-92 (1997).

Efforts are being urdertaken by both industry and academia to identify new, native secreted and membranebound receptor proteins, particularly secreted proteins which have homology to reductase. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and S. 20 membrane-bound receptor proteins. Examples of screening methods and techniques are described in the literatre [see, for example. Klein et al., Prc. Natl. Acad. Sci.. 23:7108-7113 (1996); U.S. Patent No. 5,536,637)].

We herein describe the identification and characterization of novel polypeptides having homology to reductase, designated herein as PRO238 polypeptides.

25. PRO231 Studies have reported that the redox state of the cell is an important determinant of the fate of the cell.

SFurthermore, reactive oxygen species 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 reasons, including the control and prevention of strokes, heart attacks, oxidative stress and hypertension. Oxygen free radicals and antioxidants appear to play an important role in the central nervous system after cerebral ischemia and reperfusion. Moreover, cardiac injury, related to ischaemia and rcpcrfusion has been reported to be caused by the action of free radicals. In this regard, reductases, and particularly, oxidoreductases, are of interest. In addition, the transcription factors, NF-kappa B and AP- I, are known to be regulated by redox state and to affect the expression of a large variety of genes thought to be involved in the pathogenesis of AIDS. cancer, atherosclerosis and diabetic complications. Publications further describing this subject matter include Kelsey, et al.. Br. Cancer, 76(7):852-4 (1997); Friedrich and Weiss, _L Theor. Biol.. 187(4):529-40 (1997) and Pieull t et Bacteril., 179(18):5684-92 (1997). Given the physiological importariz of rodox reactions in vivo. efforts are currently being under taken to intify new, native proteins which are involved in redox reactions. We describe herein the identification of novel polypeptides which have homology to reductase, designated herein as PR0233 polypeptides.

26, PR0223 The carboxypeptidase family of exopeptidases constitutes a diverse group of enzyn 4 that hydrolyze carboxyl-terminal amide bonds in polypeptides, wherein a large nm~ber of mammalian tissues produce these cnzyRUC3. Many of the carboxypeptidase enzymes that have been identified to date exhibit rather strong cleavage specificities for certain amino acids in polypeptides. For example. carboxypepadase enzymes have been identified which prefer lysine. argimne. secme or amino acids with either aromatic or branched aliphatic side chains as substrates at the carboxyl terminus of the polypeptide.

With regard to the acmen carboxypeptidases such amino acid specific enzymes have been identified from a variety of different nur iln and non-mammalian organisms. The mammaliant serine carboxypeptidase enzymes play important roles in many different biological processes including, for example, protein digestion, activation, :.::inactivation, or modulation of peptide hormone activity, and alteration of the physical properties of proteins and In light of the physiological importance of the serine carboxypeptidases, efforts are being undertaken by both industry and academia to identify new, native secreted and membrane-bound receptor proteins and specifically novel carboxypeptidases. Many of these efforts are focused on the screening of mammalian recombinant DNA libraries to idetif the codivg sequetnces for novel secreted and membrane-boiund receptor proteins. We describe herein novel polypeptides having homology to one or more seie carboxypeptidase polypeptides, designated herein as PR0223 polypeptdecs.

*27. PRO235 Plexin was first identified in Xenopus tadpole nervous system as a membrane glycoprotein which was shown to mediate cell adhesion via a homophilic binding mechanism in the presence of calciumn ions. Strong evolutionary conservation between Xenopus. mouse and human homologs of plexin has been observed. [Kaneyamna et aL.

Biochem. And Biophys. Res. Comm. 226: 524-529 (1996)]. Given the physiological importance of cell adhesion mechanisms in vo, efforts are currently being under taken to identify new, native proteins which are involved in cell adhesion. We describe herein t Identification of a novel polypeptide which has homology to plexin. designated herein as PRO235.

28. PRO236 and PR262 P-galactosidasc is a well known enzymatic protein which functions to hydrolyze 1-galactoside molecules.

1-galactosidase has been employed for a variety of different applications. both in viro 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 P-gaactosidasc: polypeptide.

Given the strong interest in obtaining novyl polypeptides having homology to P-galactosidase, efforts are currently being undertaken by both industry and academia to identify new, native k-alactosidase bomolog proteins.

Many of these efforts ame focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel P-gahactosidase-like proteins. Examples of screning methods ad techniques arc described in the literate [see, for example, Klein et al., Proc. Nag, Acad- Sci- 912:7109-7113 (1996); U.S. Patent No.

5.536.637)). We herein describe novel poylpeptides having siginificant homology to the IP-galactosidase enzyme, designated herein as PR0236 and PR0262 polypeptides.

29. ERfl29 Densin is a glycoprotein which has been isolated from the brain which has all the ha~lmrks of an adhesion molecule. it is highly concentrated at synaptic sites in die brain and is expressed prominently in dendzitic processes in developing neurons. Densia has been characterized as a member of the 0-linked sialoglycoproteins. Dentsin has relevance to medically important processes such as regeneration. Given the physiological importance of synaptic processes and cell adhesion mchlanisms in vivo, efforts are currently being under taken to identify new, native proteins which arc involved in synaptic miachinery and cell adhesion. We describe herein the Identification of novel polypeptides which have homology to denim, designated herein as PR0239 polypeptides.

*30. PRO257 Ebnerin Is a cell surface protein associated with von Ebner glands in mammals. Efforts are being undertaken by both industry and academia to identify new, native cell surface receptor proteins and specifically those which *:20 possess sequence homology to cell surface proteins such as ebnerin. Many of these efforts are focused on the screening of mamnmalian recombinant DNA libraries to identify the coding sequences for novel receptor proteins.

We herein describe the identification of novel polypeptides having significant homology to the von Ebner's gland- :associated protein ebnerin, designatcd herein as PR0257 polypeptides.

31. PROZ2 Fucosidases are enzymes that remove futcose: residues; from fucose containing proteoglycans. In somec pathological conditions, such as cancer, rheumatoid arthritis, and diabetes, there is an abnormal fuicosylation of serum proteins. Therefore, fucosidases, and proteins having homology. to fucosidase, are 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.YViro..

71(9):6796 (1997). Aktogu. et al.. Monaldi. Arch- Cest Dis. (Italy), 52(2):l 18 (1997) and Focarelli, et al., Richm Biophvs. Res. Commun. 234(l):.54 (1997).

Efforts are being undertaken by both indlustry and academia to identify new, native secreted and membranebound receptor proteins. Of particular interest are proteins having homology to the aipha-l-fucosidase precursor.

Many efforts are focused on the screening of mammalian recomnbinant 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 literatare for example. Kleine &i L, Proc. Nad. Acad. Sci 21:7108-7113 (1996); U.S. Patent No. 5.536.637)).

We herein describe the Identification and characteriaton at novel polypcptidc-s having homology co hucosidases, designated herein as PR0260 polypeptides.

32. PRQ263 CD44 is a cell surface adhesion moicwie involved in cell-elU and cell-matrix interactions. Hyaluronic acid.

a component of the catracellular matrix is a major ligand. Otlher ligands include collagen, fibronectin, Luminin, d~ulrdoitin sulfate. mucosal addrcssin. gerglycmn and osteoponin. CD44 is also important in regulating cell traffic, lymph node homing, transmission of growth signals. and presentation of chemnokincs and growth factors to traveling cells. CD44 surface proteins are associated with metastatic tumrs and CD44 has been used as a marker for HIV infection. Certain splice variants are associated with metastasis and poor prognosis of cance patients. Therefore.

molecules having homology with CD44 ame of particular intrest, as their homology indicates that they may have funtions related to those functions of CD44. CD44 is ftrtr described in U.S. Patent Nas. 5,506.119, 5,504.194 and 5,108,904; Gerberick. et al.. Taxicod A2nal Pharol., 146(l):1 (1997): Wittig, et al., Imnl. Lette~rs (Netherlands), 57(1-3):217 (1997); and Oliveira and Odell, Orl nL co (England), 33(4):260 (1997).

Efforts arc being undertaken by both indusay and academia to identify, new, native secreted and membranebound receptor proteins, particularly transmemnbrae proteins with homology to CD44 antigen. Many efforts are focused an 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 arc described in :20 the literature [see, for example, Klcin et al.. Proc. Natl. Acad. Set, 2:710&-7113 (1996); U.S. Patent No.

5,536,637)].

We herin describe the identification and characterization of novel polypeptides having homology to CD44 .antgen, designated herein as PR0263 polypeptdecs.

33. PRO270I Thioredoxins effect reducticm-caidation (redox) statte. Many diseases are potentially related to redox state and reactive oxygen species may play a role in many important biological processes. The transcription factors, NF-kappa B and AP-l1, are regulated by redox state and are known to affect the expression of a large veriety of genes thought to be involved in the pathogenesis of AIDS, cancer, atherosclerosis and diabetic complications. Such proteins may also play a role in cellular antioxidant defense, and in pathological conditions involving oxidative stres such as stroke and inilammation in additioni to having a role in apoptosis. Therefore, thioredoxins. and proteins having homology thereto.

are of interest to the scientific and medical commnmities.

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

34. PRO271 The proteoglycan link proten is a protein which is intimately associated with various extraceltlar matixx proteins and mnore specifically with proteins such as collagen. For example. one primary component of collagen is a large proteoglycan called aggrecan. This molecule is retained by binding to the glycosaminoglycan hyaluronan throgh the amino terminal GI globular domain of thc core protein. This binding is stabilized by the protcoglycaji link protein which is a protein that is also associated with other tissues Containing hyaluronan binding proteoglycans such as versican.

Link protein has been identified as a potential target for mntoimmune antibodies in individuals who suffcr fmjuvenile rewatoid arthritis (see Guerassimov et al.. J. Riacwnarology 24(5):959-964 (1997)). As such, there is stzong 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.

PRZ272 Reticulocalbin is an endoplasmic reticula protein which may be involved in protein transport and luntinal protein processing. Reticulocalbin resides in the lumen of the endopladsmic reruiculum, is known to bind calcium.

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

.:20 36. PRO294 Collagen, a naturally occurring procein, finds wide application in industry. Chemically hydrolyzed natural collagen can be denatured and renatured 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 characterizatinn of a novel polypeptide which has homology to portions of the collagen molecule, designated herein as PROM9.

37. PROQ295 The integrins comprise a supergene family of cell-surface glycoprotein receptors that promote cellular adhesion. Each cell has numerous 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 inuegrins arc of particular importance in regulating movement and function of immune system cells The platelet [lb/lilA integrin complex is of particular importance in regulating platelet aggregation. A member of the integnin fan*ly integrin P-6, is expressed on epithelial cells and modulates epitheLial inflammation. Another integrin, leucocyte-associated antigen-I (LFA-l) is important in the adhesion of lymphocytes during an immune response. Tie integrins ame expressed as heterodimers of noncovalently associated alpha and beta subunits. Given the physiological importance of cell adhesion mechanisms in "vo efforts arm 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. PR0293 Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As morc is known about the structural and functional mechanisms underlying protein-protein iteractioni, protein-protein .nrcjn can be more easily manipulated to regulate the particular result of the protein-protein interaction. Thus.

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

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

Leucine-rich repeats arc short sequence motifs present in a number of proteins with diverse functions and cellular locations. 71e crystal structure of ribornuclease 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 unuasual. nonglubular shape. Tese two features have been izxlica~edas respotsible forthet protein-binding functions of proteins containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends Biochen.- Sci., 19(10):415A421 (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 tumnor stroma formation. lozzo. R. Cu-it. Rev. iohem. Mol- Biol 32C2):141-174 (1997). Others studies imnplicating Ieucine rich proteins in wound healing and tissue repair are Dc La Salle. et al.. Vouv. Rev.

Fr Hernatol. (Germany). 37(4)215-222 (1995), reporting moutations in the leucine rich motif in a complex associated 20 with the bleeding disorder Bernard-Soulier syndrome and Chiemeison, K. 33ironb. Haernost. (Germany), 74(l):lll-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-degenrative diseases such as Alzheimuer's disease, nerve damage such as in Parktinson's disease, and for diagnosis of cancer. see, Artavanistsakocns, S. and Rodhberg. J. W09210518-Al by Yale University. Other studies reporting on the biological functions of proteins having leucine-rich repeats include: Tayar, ct al.. M2L £QIjnd,. (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotrojin receptor involvement); Miura, et al., *Njogn insho (Japan). 54(l):1784-1789 (July 1996) (apoptosis involvement); Harris. P. et al.. L m S tigpbmj.. 6(4):1125-1133 (Oct. 1995) (kidney disease involvement); and Ruoslahti. 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 undettaken by both industry and academia to identify new proteins having leucine rich repeats to bette understand protein-protein interactions. Of particular interest are those proteins having Icucine rich repeats and homology to ktnown meronal leucid rich repeat proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and nMembrane-bouind proteins having leucine rich repeats. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. NaI Acad. Sci, 22:7108-7113 (1996); U.S. Patent No. 5,536.637)].

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

39. PRO 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. protcin-protein interactions can be more easily manipulated to regulate the particular result of the protein-proteitn 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.

Leucine-rich repeats arc short sequence motifs present in a aanber of proteins with diverse functions and cellular locations. The crystal structure of ribonuclease inhibitor protein has revealed that leucine-tich 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, nonghabular shape. These two featurcs have been indcatedas 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, tisse repair, andl tumor stroma formation. lozzo, Crit. Rev. iochem, Mol. Biol'.. 32C2):141-174 (19917). Others studies implicating leucine rich proteins in wound healing and tise repair are De La Salle, ct al., Voy- Rev,~ Er. knlatl. (Germnany). 37(4):215-222 (1995). reporting mutations in the leucine rich motif in a complex associated *:20 with the bleeding disorder Bernard-Soulier syndrome and Chlcmtson, YI ]bzgmb J32gm (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-degenerative diseases such as Alzheimer's disease, nerve damage such as in Paikinson's disease, and for diagnosis of cancer, see, Artavanistiakonas, S. and Rothberg, J. W09210519-Al by Yale University. Other studies reporting on the biological functions of proteins having leucire-rich repeats include: Tayar, et al., MoL, Cell ndocinol., (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotopin receptor involvement): Mura, et al., Nip* J nsh (Japan), 54(7):1784-1789 (July 1996) (apoptorsis involvemnent); Harris, P. et al..,J LAz.So Ncpbrl., 6(4):1125-1 133 (Oct. 1995) (kidney disease involvement). aduolhiE..,t 11027- by IA Jolla Cance Research Foundation (decorin binding to tansforming growth factorp involvement for treatment 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 rmolecule. It is highly concentrated at synaptic sites; in the brain and is expressed 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 arc currently being under taken to identify new, native proteins which are involved in synaptic machinery and cell adhesion. Densin isfather described in Kennedy, M.B, Trends Ncwzosi. (England), 20(6)-.264 (1997) and Apperson, et al.. 1. Nusci., 16(21):6939 (1996).

Efforts ame therefore being undcntakin by both industry andl academia to identify new proteins having leucine richi repeats to better understand proteim-protein interactions. Of particular interest arc those proteins having leucine: rich repeats and homology to known proteins having leucine rich repeats such as KIAAO23I and densin. Many effrts arc focused on the sc ening of mmmal an recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound proteins having Icucine rich repeats. Examples of screening methods and tchtniques are described in the literature (see, for exaniple, Klein et al.. Proc. Nail. Acad. SL 1:7108-7113 (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 PRO343 Proteases are enzymatic protein which are involved in a Large number of very important biological processes in mammalian aid non-mammalian organism. Numerous different protease enzymes from a variety of different mammalian and non-marmmalian organisms have been both identified and characterized. The mammalian' protease enzymes play important roles in many differcent biological processes including, for example, protein digestion activation. inactvation, 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, natve protease homologs. Many of these 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. Nat]. Acad. ScL, 9-:7108-7113 (1996); U.S. Patent No.

5.536,637)). We herein describe t identification of novel polypeptides having homology to various protease :enzymes, designated herein as PR0302. PR0303. PR0304. PR0307 and PRO343 polypeptides.

41. PRO328 The GLIP protein family has been characterized as comnprising zinc-finger proteins which play important roles in embryogenesis. T7hese proteins may function as transcriptional regulatory proteins and are known to be amplified in a subset of ]human tumors. Glioma pathogienesis protein is struicturally related to a group of plant pathogenesis-related proteins. It is highly expressed in glioblastomn. See US Par. Nos. 5,582,981 (issued Dec. 1996) and 5.322.801 (issued June 21, 1996), Ellington, A.D. ct al., Nature, M:818 (1990), Grindley. J.C. et al., De2 188(2):337 (1997). Marine, J.C. et al., Mch Dev1f:2 11 (1997), The CRISP or cysteine rich secretory protein family arc a group of proteins which are also structurally related to a group of plant pathogenesis proteins. [Schwidetzky, BiochemJ.. M2:325 (1997), Pfisterer. Mol.Cell Riol-. 1=lf:6160 (1996), Kratzschnmar. .r BdiorL, 827 (1996)]. We describe herein the identification of a novel polypeptide which has homology to GLIP and CRISP, designated herein as PR0328 polypeptides.

42. PROMS5. PR0331 and~ PRO326 Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the structural and functional mechanism underlying protein-protein interactions, protein-protein interactions can be more easily manipulated to regulate the particular result of the protein-protein interaction. Thus.

dhe underlying mechan sms of protein-protein interactions are of interest to the scientific and medical community.

A]l proteins containing leucine-rich repeats are thought to be involved in protein-protein interactions.

Leucine-rich repeats ame short sequence motifs present in a number of proteins with diverse functions and cellular locations. Ile crystal structure of ribonuclease inhibitor protein has revealcd 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 dhe 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, Trendq fiochem Sci.. 19(10):415-421 (Oct. 1994).

study has been reported on leucine-rich protoglycans 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. Biochcmn. Mot. Biol., 32C2):141-174 (1997). Others studies iniplicating leucine rich proteins in wound healing and tissue repair are De La Salle, et al., Voy Rv Er. Hmatol. (Germany), 37(4):215-2 (1995). reporting mutations in the leucine rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome, Chlemetson, K. 1]rmb gikHaemos. (Germany). 74(1): 111 116 (July 1995), reporting that platelets have leucine rich repeats and Ruoslahti. E. L. ct al.. W091 10727-A by La Jolla Cancr Research Foundation reporting that decorin binding to transforming growth factorp has irvolverment in a tremmxent for cancer, wound healing and scarring. Related by funtion 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, skint and bone; in promoting body growth in humans and animals; and in stimulating other growth-related processes. The acid labile subunic of IGF (ALS) is also of interest in that it increases the half-life of IGF and is part of the JOF complex invivo.

Another protein which has been reported to have leucine-rich repeats is the SLIT protein which ha been reported to be useful in treatin neuro-degenerative diseases such as Alzheimer's disease, nerve damage such as in *Parkinson' s disease, and for diagnosis of cancer. see, Artavanistsakonas. S. and Rothbcrg, 1. W09210518-Al by Yale University. Of particuilar interest is UG-! a membrane glycoprotein that is expressed specificafly in glial cells in the mouse: brain. and has leucine rich repeats and! immoglobulin-like domains. Suzuki, et al., I. Biol.

Omarn. 271(37)2222 (1996). Other studies reporting on die biological functions of proteins having leucine rich repeats include: Tayar. et al., Mol. Cell Endcriiol., (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor involvement); Miura, et al.. Njpin-RinsW (Japan), 54C7):1794-1789 (July 1996) (apoptosis involvemen); Harris. P. ct a, J. Am. Soc, Norol., 6(4):1 125-1133 (Oct. 1995) (idnecy disease involvement).

Efforts arc therefore bein undertakien 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 leticine 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 identif the coding sequences for novel secreted and mnembrane-bound proteins having Icucine rich repeats. Examples of screening methods and teius are describe in the literature (see. for exarnplc. Kleinet Proc. Nati. Acid. Sci., 9:7109-7113 (1996); U.S. Patent No. 5,536,637)].

We describe herein the identification and characterization of novel polypeptddes which have homology to proteins of Owe leucitie rich repeat superfamily, designated herein as PROM.5 PR0331 and PR0326 polypeptides.

43. EM=~ Secreted proteins comprising a repeat characterized by an arrangemnen of conserved leucine residues (leucine-rica repeat otif) have diverse biological roles. Certain proteoglycans, such as biglycan, fibromodulin and decorin, are. for example, characterized by the presence of a leucine-rich repeat of about 24 amino acids [Ruosiahti, 10 Ann- Rev.Ceti. Biot- A229-255S(1988) Oldberg er al.. -EN4B J. 2601-2604 (1989)]. In general. proteoglycans amc believed to play a role in regulan extraceihilar matrix, cartilage or bone function. The proteoglycan decorin binds to collagen type I and H and affects the rate of fibril formation. Fibromodulin also binds collagen and delays ~fibril formation. Both fibromodulin and decorin inhib~it the activity of transforming growth factor beta (TGF-f3) (U.S.

Patent No. 5.583,103 issued December 10. 1996). TGF-P is knwn to play a key role in the induction of extraccllular matrix and has been implicated in the development of fibrotic diseases, such as cancer and glomeniloiwphritis. Accordingly, proteoglycans have been proposed for the treatment of fibrotic cancer, based upon their ability to inhibit TGF-f3s growth stimulating activity on the cancer cell. Proteoglycans have also been described as potentially useful in the treatment of other proliferative pathologies, including rheumatoid arthritis. arteriosclerosis, adult respiratory 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 injuries, or cosmetic or reconstruictive surgery Patent No. 5,654,270, issued August 5, 1997).

We describe herein the identification and characterization of novel polypeptides wich have homology to proteins of the leucine rich repeat superfamily, designated herein as PR0332 potypeptides.

44. PRO33 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 skin which has been damaged from aging, injuries or the sun. Fibrillin microfibrils define the contirnious elastic network of skin, and are present in dermis as microfibril bundles devoid of measurable elastin extending from the dermal-epithelial junction and as components of the thtick elastic fibres present in the deep reticular dermis. Moreover, Marfan syndrome has been linked to mutations which interfere with multimerization of fibrilin monomers or other connctive tisse elements.

Fibulin-l is a modular glycoprotein with amino-terminal. anaphiatoxin-like modules followed by nine epidermal growth factor (EGF)-like modules and, depending on alternative splicing, four possible carboxyl termini.

Fibulin-2 is a novel extracellular matrix protein frequently found in close association with microfibrils containing either fibronectin or fibrillin. Thus, fibrillin, fihulin. and molecules related thereto are of interest, particularly for the use of preventing skin from being damaged from aging, injuries or the sun, or for restoring sin damaged from samie. Moreover. drse molcuiles are generally of irteret in die study of connective tissue and attachment molecules and related mechanisms. FibmiIn, fihiLin and related molecules are further described in Adams, et LMLDjWk..

272(2):226-36 (1997); Kielty and Shuttleworth, Microsc. Res- Tech.. 38(4):413-27 (1997); and Child_.1.Card.

SPIiL. 12(2Supp.):131-5 (1997).

Currently, efforts arm being undertaken by both industry and academia to identify new, native secreted and membrane-bumid receptor proteins, particularly secreted proteins which have homnology to fibuhin and librilhin. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and mem wanc-bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc- Nat]- Arad. Sci., 23:7108-7113 (1996); U.S. Patent No.

5,536,637)].

We herein describe the Identification and characterization of novel polypeptides having homology to fibuhln and fibrillin. designated herein as PR0334 polypeptides.

*45. PR0346 I'me 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 (mAbs) which arc specific to tumor antigens. Such mAbs. which can distinguish between normal and cancerous cells are useful in t diagnosis, prognosis and treaunent of the disease. Particular antigens are known to be associated with neoplastic diseases, such as colorecta 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 rnAbs can be used for treatment in situ with minimal paient description.

Carcinoembryonic antigen (CEA) is a glycoprotein found in human colon cancer and the digestive organs a 2-6 month human embrtyos. 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 serum levels of CEA are elevated int a patient, a drop of CEA levels after surgery would indicate the tumnor resection was successful. On the other hand, a subsequent 9 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 my also be a target for mAb, antisense nucleotides PR6.68 Protein disufide isomease is an enzymatic protein which a involved in the promotion of correct refolding of proteins through the establishment of correct disulfide bond formation. Protein disulfide isomnerase was initially identfied based upon its ability to catalyze the renaturaion of reduced denatured RNAse (Goldberger et al., J. Bio.

OumL 239:1406-1410 (1964) and Epstein et al.. Cold Spring Harbor S)m. Quant. Biol. 28:439-449 (1963)). Protein disulfide isomerase has been shown to be a resident enzyine of the endoplasmic reticulum 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, efforts'are 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 isonmrase homologs. We herein describe a novel polypeptide having homology to protein disulfide isomerase. designated herein as PRO268.

47. PRO330 Prolyl 4-hydroxylase is an enzyme which functions to post-translationally hydroxylate proline residues at the Y position of the amino acid sequence Gly-X-Y, which is a repeating three amino acid sequence found in both collagen and procollagcn. Hydroxylarion 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 threedimensional triple-helical conformation. If bydroxylation does not occur, synthesized collagen polypeptides remain non-helical, are poorly secreted by cells and cannot assemble into stable functional collagen fibrils. Vuorio et al., Proc. Nal. Acad. Sci. USA 89:7467-7470 (1992). Prolyl 4-hydroxylase is comprised 15 of at least two different polypeptide subunits, alpha and beta.

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 membranc-bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Natl. Acad. Sci., 20 21:7108-7113 (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 PRO330.

48. PR0339 and PRO310 Fringe is a protein which specifically blocks serrate-mediated activation of notch in the dorsal compartment of the Drosophila wing imaginal disc. Fleming, et al.. Develoment. 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. PR0244 Lctin 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-1l gp90MEL, gplOML, 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 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 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 25 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 PRO211 and PR0217 EGFlike homologuc polypptides. 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. Applcants have identified a cDNA clone that encodes a novel polypcptidc, wherein the polypeptide is designated in the present application as "PRO230". I 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 PRO230 polypcptide having amino acid residues 1 through 467 of Figure 6 (SEQ ID NO:12), or is complementary to such encoding S 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 [D NO:12).

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

3. PRO23 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PRO232.

*20 In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO232 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO232 polypeptide having amino acid residues 1 to 114 of Figure 9 (SEQ ID NOe18), 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 PRO232 polypeptide. In particular, the invention 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 polypcptide, designated in the present application as PR0187".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO187 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO187 polypeptide of Figure 11 (SEQ ID N0O23), 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 nuceic 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 PRO 187 polypcptidc. 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.

S. PR0265 Applicants have identified a cDNA clone that encodes a novel polypcptide, wherein the polypcptide is designated in the present application as *PR0265".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0265 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0265 polypcptide having amino acid residues I to 660 of Figure 13 (SEQ ID NO:28), 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 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 NO28). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0265 polypeptide.

6. PRQ219 20 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 nuckic 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 1 to 915 of Figure 15 (SEQ ID NO:34).

7. PRO24 Applicants have identified a cDNA clone that encodes a novel polypcptide, 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 isolated PRO246 polypeptide. In particular, the invention provides isolated native sequence PR0246 polypeptde, 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 extracelular domain of a PR0246 polypeptide.

8. PRO228 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to CD97, EMR1 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 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO228 polypeptide having amino acid residues 1 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.

In another embodiment, the invention provides isolated PR0228 polypeptide. In particular, the invention provides isolated native sequence PRO228 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 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 PR0228 polypeptide.

In another embodiment, the invention provides an expressed sequence tag (EST) comprising the nucleotide sequence of SEQ ID NO:50, designated herein as DNA21951.

9. PRO533 Applicants have identified a cDNA clone (DNA49435-1219) that encodes a novel polypeptide, designated in the present application as PR0533.

In one embodiment, the invention provides an isolated nucleic acid molecule having at least about sequence identity to a DNA molecule encoding a PRO533 polypeptide comprising the sequence of amino acids 23 to 216 of Figure 22 (SEQ ID N059). or the complement of the DNA molecule of(a). 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 (amino acids 23 to 216 of Figure 22, SEQ ID NO:59). In a further embodiment, the isolated nucleic acid molecule comprises DNA encoding a PR0533 polypcptide having amino acid residues 1 to 216 of Figure 22 (SEQ ID NO:59), 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 of the full length protein of clone DNA49435-1219. deposited with the ATCC under accession number ATCC 209480.

In yet another embodiment, 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 residues 23 to 216 of Figure 22 (SEQ ID N0:59). Native PR0533 polypeptides with or without the native signal sequence (amino acids I to 22 in Figure 22 (SEQ ID NO:59)), and with or without the initiating methionine are specifically included. Altcrnatively, the invention provides a PRO533 polypcptide encoded by the nucleic acid deposited under accession number ATCC 209480.

10. PR024S Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0245".

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 PRO245 polypeptide having amino acid residues 1 to 312 of Fig. 24 (SEQ ID NO:64), 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 PR0245 polypeptide. In particular, the invention S. provides isolated native sequence PR0245 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 312 of Figure 24 (SEQ ID NO:64).

11. PRO220. PR0221 and PRO227 Applicants have identified cDNA clones that each encode novel polypeptides, all having leucine rich repeats.

These polypeptides are designated in the present application as PR0220, PRO221 and PR0227.

In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA respectively 20 encoding PR0220, PRO221 and PR0227. respectively. In one aspect, provided herein is an isolated nucleic acid comprises DNA encoding the PRO220 polypeptide having amino acid residues I through 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 comprises DNA encoding the PRO221 polypeptide having amino acid residues 1 through 259 of Figure 28 (SEQ ID NO:71), 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. Moreover, also provided herein is an isolated nucleic 50o00 acid comprises DNA encoding the PRO227 polypeptide having amino acid residues 1 through 620 of Figure 30 (SEQ ID NO:73), 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 PR0220. PRO221 and PRO227 polypeptides. In particular, provided herein is the isolated native sequence for the PRO220 polypepide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 708 of Figure 26 (SEQ ID NO:69). Additionally provided herein is the isolated native sequence for the PR0221 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 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 1 to 620 of Figure 30 (SEQ ID NO:73).

12. PR0259 Applicants have identified a cDNA done that =,odes a novel polypeptide having homology to CRTAM and poliovims 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 PROMS polypeptide. In one aspect, the isolated nrucleic acid comprises DNA encoding the PR0r258 polyp eptide having amino acid residues I to 398 of Figure 32 (SEQ ID NO:84). or is complementary to such-cnodig nucleic acid sequence, and remains stab'ly bound to it under at least moderate, and optionally, under high stringency conditions.

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

13. PRO2 Applicants have identified a eDNA clone that encodes a novel polypeptide. wherein the polypeptide is designated in the present application as *PR0266'.

In one embodiment, the invention provides an isolated nuecic acid molecule comprising DNA encoding a PR0266 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO266 polypeptide having amino acid residues I 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 mnoderate, and optionally, under high stringeny 0 cnditonIn 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 I to 696 of Figure 34 (SEQ ID NO:91).

14. PR.Z2 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 PR0269 polypeptide having amino acid meidues I to 490 of Fig. 36 (SEQ ID NO:96), or is complementaty 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 cmprising residues I to 490 of Figure 36 (SEQ ID NO:96). An additional embodiment of the present invention is directed to an isolated extracellular domain ofta PR0269 polypeptide.

is. £E.QZ2 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptidc is designated in the present application as "PR0287.

In one embodiment, the invention provides an isolated nucl~ic acid molecule comprising DNA encoding a PR0287 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0287 polypeptide haigamno adresidues I to 4 15of Fig. 3 (SEQ lNN: 04.o icomplemayosuencoignucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high strngency conditions.

In another embodiment, the invention provides isolated PR0287 polypeptde. In particular, the invention provides isolated native sequence PR0297 polypeptide, which in one cznbodimznt. includes an amino acid sequence 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 "PR0214".

0o:.00In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0214 polyetide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0214 polypeptide of ::o*Fig. 40 (SEQ IM NO: 109). or is complementary to such encoding nrucleic acid sequence, and remains stably bound o to it under at least moderate. and optionally, under high stringency conditions. [n 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.l 191. deposited with 20 ATCC under accession numiber ATCC 209385.

In yet another embodiment, the invention provides isolated PR0214 polypeptide. In particular, the invention provides isolated native sequence PR0214 polypde, which in one embodiment, includes an amino acid sequence .0 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. PRO317 Applicants have identified a cDNA clone that encodes a novel polypeptde,. designated in the present application as "PR0317".

In one embodiment. the invention provides an isolated nucleic acid molecule comprising DNA encoding PR0317 polypeptide. Inone aspect, the isolated nucleic acid comprises DNA (SEQ ID NO: 113) encoding PR0317 polypepuide having amo 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 modlerate. and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0317 polypptde. In particular, the invention provides isolated nadve-sepence PR0317 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 366 of Figure 42 (SEQ ID NOA 14).

In yet another embodiment, the invention supplies a method of detecting the presence of PR0317 in a sample, the method comprising: a) contacting a detectable ani-PRO317 antibody with a sample suspected of containing PRO317; and b) dctcting binding of the antibody to the 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 still further embodiment a method is provided for determining the presence of PR0317 mRNA in a sample, the method comprising: a) contacting a sample suspected of containing PRO317 mRNA with a detectable nucleic acid probe that hybridizes under moderate to stringent conditions to PRO317 mRNA; and b) detecting hybridization of the probe to the sample.

Preferably, in this method the sample is a tissue sample and the detecting step is by in situ hybridization, or the sample is a cell extract and detection is by Northern analysis.

Further, the invention provides a method for treating a PRO317-associated disorder comprising administering to a mammal an effective amount of the PRO317 polypeptide or a composition thereof containing a carrier, or with an effective amount of a PR0317 agonist or PR0317 antagonist, such as an antibody which binds specifically to PRO317.

15 18. PR0301 Applicants have identified a cDNA clone (DNA40628-1216) that encodes a novel polypeptide, designated *in the present application as "PRO301".

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 20 28 to 258 of Fig. 44 (SEQ ID NO:119). or the complement of the DNA molecule 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 28 to 258 of Fig. 44 (SEQ ID NO:119). Preferably, the highest degree of sequence identity occurs within the exracellular domains (amino acids 28 to 258 of Fig. 44, SEQ ID NO:119). In a further embodiment, the isolated nucleic acid molecule comprises DNA encoding a PRO301 polypcptide having amino acid residues 28 to 299 of Fig. 44 (SEQ ID NO:119), 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 of the full length protein of clone DNA40628-1216. deposited with the ATCC under accession number ATCC 209432, alternatively the coding sequence of clone 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 extracelular domain residues 28 to 258 of Figure 44 (SEQ ID NO: 119). Native PRO301 polypeptides with or without the native signal sequence (amino acids 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 transmabranc domain (residues 236 to about 258 in Figure 44; SEQ ID NO: 119) and/or the intraccllular domain (about residue 259 to 299 in Figure 44; SEQ ID.NO:119). Alternatively, the invention provides a PRO301 polypeptide encoded by the nucleic acid deposited under accession number ATCC 209432.

19. PR0224 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 comprising DNA encoding a PR0224 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO224 polypcptide having amino acid residues 1 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 least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0224 polypeptide. In particular, the invention provides isolated native sequence PRO224 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 282 of Figure 46 (SEQ ID NO:127).

0 00* 20. PR022 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 PR0222 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0222 polypcptide having amino acid residues 1 to 490 of Fig. 48 (SEQ ID NO:132), or is complementary to such encoding nucleic acid 20 sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

*In another embodiment, the invention provides isolated PR0222 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).

21. PRO234 Applicants have identified a cDNA clone that encodes a novel lectin polypeptide molecule, designated in the present application as "PR0234".

S In 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 PR0234 polypeptides having amino acid residues 1 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 PRO234 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 isolain genomnic and cDNA nucleotide sequences.

nl. 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 "PR0231'.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0231 polypeptide. In one aspect, the isolated nuecic acid comnprises DNA ecoding the PROM3 polypeptide having aino acid residues I to 428 of Fig. 52 (SEQ WD NO: 142). or is comnplementary to such enicoding nucleic acid sequence, andi remins; 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 iMoated native sequence PR0231I polypeptide, which in one embodiment. includes an amino acid sequence .e:.comprising residues I to 428 of Figure 52 (SEQ ID NO:.142).

23. PRO229 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'.

he one embodiment. the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0229 polypeptide. he one aspect. the isolated nucleic acid comprises DNA encoding the PR0229 polypeptide having amino acid residues I to 347 of Figure 54 (SEQ ID NO: 148). or is complementary to such encoding nucleic :20 acid sequence. and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

he another embodiment, the invention provides isolated PR0229 polypeptdec. he particular, the invention :provides isolated native sequence PR0229 polypeptide. 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 eDNA clone that encodes a novel polypepidec having homology to reductase, wherein the polypeptide is designated in the present application as "PR0238".

he 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 amin acid residues I to 310of 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 PR0238 polypeptide. In particular, the invention provides isolated native sequence PR0238 polypeptide. which in one embodiment, includes an amino acid sequence comprising residues 1 to 3 10 of Figure 56 (SEQ ID NO: 153).

PR0233 Applicants have identified a cDNA clone that encodes a novel polypcptide, 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 anino acid residues 1 to 300 of Figure 58 (SEQ ID NO:159), or is complementary to suchtencoding 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 polypcptide. 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. PRO223 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to scrine carboxypeptidase polypcptides, 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 polypeptide. 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.

20 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).

*PRO

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

28. PRO236 and PR0262 Applicants have identified cDNA clones that encode novel polypeptides having homology to P-galactosidasc, wherein those polypcptides are designated in the present application as 'PRO236' and *PR0262".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO236 polypeptidc. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO236 polypeptide having amino acid residues I to 636 of Figure 64 (SEQ ID NO:175), 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 an isolated nucleic acid molecule comprising DNA encoding a PRO262 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0262 polypeptide having amino acid residues 1 to 654 of Figure 66 (SEQ ID NO: 177). 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 PRO236 polypcptide. In particular, the invention provides isolated native sequence PRO236 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 636 of Figure 64 (SEQ ID NO:175).

In another embodiment, the invention provides isolated PR0262 polypcptide. 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. PRO239 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as 'PR0239".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a S" PRO239 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0239 polypeptide having amino acid residues 1 to 501 of Figure 68 (SEQ ID NO:185), 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 PRO239 polypeptide. In particular, the invention provides isolated native sequence PRO239 polypcptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 501 of Figure 68 (SEQ ID NO:185).

PR0257 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypcptide is designated in the present application as "PR0257".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0257 polypcptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0257 polypcptide having amino add 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 PR0257 polypeptidc. In particular, the invention provides isolated native sequence PRO2S7 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 extracellular domain of a PRO257 polypcptide.

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

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0260 polypcptidc. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0260 polypeptide having amino acid residues 1 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.

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. PRO263 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 "PR0263".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0263 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO263 polypeptide S" having amino acid residues 1 to 322 of Figure 74 (SEQ ID NO:201). 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 PR0263 polypeptide. In particular, the invention Sprovides 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 PRO263 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 PR0270 polypeptide. In one aspect, the isolated nucleic acid comprises DNA whivch includes the sequence encoding the PRO270 polypeptide having amino acid residues 1 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 polypeptide. 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 polypeptide 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 add residues I to 360 of Figure 78 (SEQ ID N0213). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency o. conditions.

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

PRO272 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is 20 designated in the present application as "PR0272".

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 conditions.

In another embodiment, the invention provides isolated PRO272 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 erbodiment, includes an amino acid sequence carprising residues 1 to 550 of Figure 82 (SEQ ID NO:227).

37. PR295 Applicants have identified a cENA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0295".

In one erbodiment, the invention provides an isolated nucleic acid molecule ccnprising ENA encoding the PR0295 polypeptide. In one aspect, the isolated nucleic acid carprises ENA encoding the PRO285 polypeptide having amino acid residues 1 to 350 of Figure 84 (SEQ ID o ND:236), 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 PRO295 polypeptide. In particular, the invention provides isolated native sequence PR0295 polypeptide, which in one embodiment, includes an amino acid sequence ccnprising residues 1 to 350 of Figure 84 (SEQ ID NO:236) 38. PRO293 Applicants have identified a cENA 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 carprising ENA encoding a PR0293 polypeptide. In one aspect, the isolated nucleic acid carprises IA encoding the PRO293 polypeptide having amino acid residues 1 to 713 of Figure 86 (SEQ ID NO:244), or is caoplementary 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 PR0293 polypeptide. In particular, the invention provides isolated native sequence PRO293 polypeptide, which in one enbodiment, includes an amino acid sequence ccaprising residues 1 to 713 of Figure 86 (SEQ ID N3:244).

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

49 39. PR0247 Applicants have identified a cENA clone that encodes a novel polypeptide having leucine rich repeats wherein the polypeptide is designated in the present application as "PR0247".

In one embodinent, the invention provides an isolated nucleic acid molecule carprising NA encoding a PR0247 polypeptide. In. one aspect, the isolated nucleic acid cuprises EA encoding the PR0247 polypeptide having amino acid residues 1 to 546 of Figure 88 (SBQ ID NO:249), or is carplerentary 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 PRO247 polypeptide. In particular, the invention provides isolated native sequence PR0247 polypeptide, which in one embodiment, includes an amino acid sequence ccprising residues 1 to 546 of Figure 88 (SBQ ID N0:249).

An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0247 polypeptide.

PR0302, PR0303, PR304, PRO307 and PR0343 Applicants have identified cEA clones that encode novel polypeptides having horology to various proteases, wherein those polypeptide are designated in the present application as "PRO302", "PRO303", "PRO304", "PR0307" and "PR0343" polypeptides.

In one embodiment, the invention provides an isolated nucleic 25 acid molecule ccaprising CA encoding a PRO302 polypeptide. In one aspect, the isolated nucleic acid coaprises INA encoding the PRO302 polypeptide having amino acid.residues 1 to 452 of Figure 90 (SBQ ID ND:254), or is carplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least noderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides an isolated nucleic acid nolecule comprising ENA encoding a PR0303 polypeptide. In one aspect, the isolated nucleic acid carprises ENA encoding the PR0303 polypeptide having amino acid residues 1 to 314 of Figure 92 (SEQ ID N0:256), 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 yet another e rodiment, the invention provides an isolated nucleic acid nolecule carprising ENA encoding a PRO304 polypeptide. In one 50 aspect, the isolated nucleic acid molecule ccrprises ENA encoding a PR0304 polypeptide having amino acid residues 1 to 556 of Figure 94 (SEQ ID N0:258), 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 an isolated nucleic acid nolecule coprising MNA encoding a PRO307 polypeptide. In one aspect, the isolated nucleic acid coaprises ENA encoding the PR0307 polypeptide having amino acid residues 1 to 383 of Figure 96 (SEQ ID N0:260), 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 enbodiment, the invention provides an isolated nucleic acid nolecule coaprising INA encoding a PR0343 polypeptide. In one aspect, the isolated nucleic acid ccnprises INA encoding the PR0343 polypeptide having amino acid residues 1 to 317 of Figure 98 (SEQ ID NO:262), 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 anrbodinment, the invention provides isolated PR0302 polypeptide. In particular, the invention provides isolated native sequence PRO302 polypeptide, which in one enbodiment, include an amino acid sequence ccaprising residues 1 to 452 of Figure 90 (SED ID NO:254).

In another e bodiment, the invention provides isolated PR0303 polypeptide. In particular, the invention provides isolated native sequence PR0303 polypeptide, which in one embodiment, includes an amino acid sequence ccaprising residues 1 to 314 of Figure 92 (SBQ ID NO:256).

In another embodient, the invention provides isolated PRO304 polypeptide. In particular, the invention provides isolated native sequence PR0304 polypeptide, which in one embodirent, includes an amino acid sequence carprising residues 1 to 556 of Figure 94 (SEQ ID N0:258).

In another eatodiment, the invention provides isolated PRO307 polypeptide. In particular, the invention provides isolated native sequence PR0307 polypeptide, which in one embodiment, includes an amino acid sequence carprising residues 1 to 383 of Figure 96 (SEQ ID NO:260).

In another enbodiment, the invention provides isolated PRO343 polypeptide. In particular, the invention provides isolated native sequence PR0343 polypeptide, which in one eabodiment, includes an amino acid sequence carprising residues 1 to 317 of Figure 98 (SEQ ID N0:262).

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

In one eabodiment, the invention provides an isolated nucleic acid molecule ccuprising MA encoding a PRO328 polypeptide. In one aspect, the isolated nucleic acid ccnprises EA encoding the PRO328 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 remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another eAbodinent, the invention provides isolated PRO328 polypeptide. In particular, the invention provides isolated native 15 sequence PRO328 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 463 of Figure 100 (SEQ ID ND:284).

An additional embodiment of the present invention is directed to an isolated extracellular damain of a PRO306 polypeptide.

42. PP0335, PRO331 and PR0326 Applicants have identified three ciiA clones that respectively encode three novel polypeptides, each having leucine rich repeats and hoology to LIG-1 and ALS. These polypeptides are designated in the present application as PRO335, PRO331 and PRO326, respectively.

25 In one etodinent, the invention provides three isolated nucleic acid molecules cmaprising EA respectively encoding PRO335, PRO331 and PRO326, respectively. In one aspect, herein is provided an isolated nucleic acid carprising 09 encoding the PRO335 polypeptide having amino acid residues 1 through 1059 of Figure 102 (SEQ ID N0:289), 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. Also provided herein is an isolated nucleic acid comprises NA encoding the PR0331 polypeptide having amino acid residues 1 through 640 of Figure 104 (SEQ ID N1:291), or is cnaplementary 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 ccprises EIA 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 enbodiment, the invention provides isolated PRO335, PR0331 and PR0326 polypeptides or extracellular domains thereof. In particular, the invention provides isolated native sequence for the PR0335 polypeptide, which in one enbodiment, includes an amino acid sequence ccprising residues 1 through 1059 of Figure 102 (SEQ ID NO:289). Also provided herein is 'the isolated native sequence for the PR0331 polypeptide, which in one embodiment, includes an amino acid sequence ccanprising 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 embodiment, includes an amino acid sequence conprising residues 1 through 1119 of Figure 106 (SEQ ID NO: 293).

15 43. PRO332 Applicants have identified a cENA clone (EIA40982-1235) that encodes a novel polypeptide, designated in the present application as "PRO332".

In one embodiment, the invention provides an isolated nucleic acid molecule ccaprising EA having at least about 80% sequence identity to a INA molecule encoding a PR0358 polypeptide carprising the sequence of amino acids 49 to 642 of Fig. 108 (SEQ ID NO:309), or the cmaplement of the E1A olecule of The sequence identity preferably is about .m nore preferably about 90%, most preferably about 95%. In one aspect, the S"9 25 isolated nucleic acid has at least about 80%, preferably at least about nore 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 N3:309). 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 uolecule conprises IMA encoding a PRO332 polypeptide having amino acid residues 49 to 642 of Fig. 108 (SEQ ID NO:309), 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 PRO332 polypeptides. In particular, the invention provides isolated native sequence PRO332 polypeptide, which in one embodiment, includes an amino acid sequence ccaprising residues 49 to 624 of Figure 108 (SEQ ID ND: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. PR0334 Applicants have identified a cENA clone that encodes a novel polypeptide having harology to fibulin and fibrillin, wherein the polypeptide is designated in the present application as "PR0334".

In one ebodinment, the invention provides an isolated nucleic acid molecule ccnprising IMA encoding a PRO334 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. N:314), or is carplenentary to such encoding nucleic acid 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 PRO334 0. polypeptide. In particular, the invention provides isolated native sequence PR0334 polypeptide, which in one embodiment, includes an amino acid sequence ccaprising residues 1 to 509 of Figure 110 (SEQ ID NO:314) 45. PRo346 Applicants have identified a cBEA clone (1NA44167-1243) that encodes a novel polypeptide, designated in the present application as "PRO346".

In one embodinent, the invention provides an isolated nucleic acid molecule oo having at least about 80% sequence identity to a IA molecule encoding a PRO346 polypeptide caromprising the sequence of amino acids 19 to 339 of Fig. 112 (SEQ ID NO:319), or the carplement of the E molecule of The sequence identity preferably is about 85%, nore preferably about set$ 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 N0:319). Preferably, the highest degree of sequence identity occurs within the extracellular doma ins (amino acids 19 to 339 of Fig. 112, SEQ ID N0:319). In alternative embodiments, the polypeptide by which the hanology is measured caomprises 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 comprises ENA 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 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 of the full length protein of clone ENA44167-1243, deposited with the ATCC under accession number ATCC 209434, alternatively the coding sequence of clone [NA44167-1243, deposited under accession number AT 209434.

In yet another embodiment, the invention provides isolated PR0346 polypeptide. In particular, the invention provides isolated native sequence PR0346 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 19 to 339 of Figure 112 (SEQ ID N0:319).

Native PR0346 polypeptides with or without the native signal sequence .(residues 1 to 18 in Figure 112 (S=Q ID NO: 319), with or without the initiating methionine, with or without the transumbrane domain (residues 15 340 to 360) and with or without the intracellular dcmain (residues 361 to o 450) are specifically included. Alternatively, the invention provides a PR0346 polypeptide encoded by the nucleic acid deposited under accession number ATC 209434.

46. PR0268 Applicants have identified a cMA clone that encodes a novel polypeptide having homology to protein disulfide iscmerase, wherein the polypeptide is designated in the present application as "PRO268".

°e In one ebodiment, the invention provides an isolated nucleic 25 acid molecule carprising EA encoding a PRO268 polypeptide. In one aspect, the isolated nucleic acid comprises ENA encoding the PR0268 polypeptide having amino acid residues 1 to 280 of Figure 114 (SEQ ID NO:324), or is crmplementary 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 PRO268 polypeptide. In particular, the invention provides isolated native sequence PRO0268 polypeptide, which in one embodiment, includes an amino acid sequence comrprising residues 1 to 280 of Figure 114 (SEQ ID N0:324).

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

47. PRO330 Applicants have identified a cEINA clone that encodes a novel 54a polypeptide having haorlogy 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 nolecule ccmprising ENA encoding the PR0330 polypeptide. In one aspect, the isolated nucleic acid nolecule ccnprising EIA encoding a PR0330 polypeptide having amino acid residues 1 to 533 of Figure 116 (SEQ ID N0:331), or is ccnplenentary to such encoding nucleic acid sequence, and renains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another enbodinent, the invention provides isolated PRO330 polypeptide. In particular, the invention provides isolated native sequence PR0330 polypeptide, which in one erbodiment, includes an amino acid sequence ccaprising residues 1 to 533 of Figure 116 (SEQ ID NO:331) 48. PR0339 and P0310 Applicants have identified two cIA clones wherein each clone encodes a novel polypeptide having horology to fringe, wherein the polypeptides are designated in the present application as "PR0339" and 20 "PRO310".

""In one erbodiment, the invention provides isolated nucleic acid molecules ccnprising EA encoding the PR0339 and/or a PRO310 polypeptide.

In one aspect, the isolated nucleic acid canprises MNA encoding the PRO339 polypeptide having amino acid residues 1 to 772 of Figure 118 (SEQ ID 25 ND:338), or is caiplementary 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 comprises MA encoding the PRO310 polypeptide having amino acid residues 1 to 318 of Figure 120 (SEQ ID ND0:340), 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 isolated PR0339 as well as isolated PRO310 polypeptides. In particular, the invention provides isolated native sequence PR0339 polypeptide, which in one embodinent, includes an amino acid sequence cacprising residues 1 to 772 of Figure 118 (SEQ ID NO: 338). The invention further provides isolated native sequence PRO310 polypeptide, which in one enbodiment, includes an amino acid sequence carprising residues 1 to 318 of Figure 120 (SEQ ID ND:340).

54b 49. PR0244 Applicants have identified a clIA clone that encodes a novel polypeptide, designated in the present application as "PRO244".

In one embodinent, the invention provides an isolated nucleic acid molecule comprising EIA encoding PRO244 polypeptide. In one aspect, the isolated nucleic acid conprises ENA encoding PRO244 polypeptide having amino acid residues 1 to 219 of Fig. 122 (SEQ ID N3:376), or is ccrplementary 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 carprising residues 1 to 219 of Figure 122 (SEQ ID NO:376).

50. Additional Ebodizments In other embodiments of the present invention, the invention Sprovides vectors comprising ENA encoding any of the above or below described polypeptides. A host cell ccnprising any such vector is also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast. A process for producing any of the above or below described polypeptides is further provided and ccnprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide fran the cell culture.

25 In other enbodiments, the invention provides chimeric molecules ccprising any of the above or below described polypeptides fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule carprises any of the above or below described polypeptides fused to an epitope tag sequence or a Fc region of an 3 0 inumnoglobulin.

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 monnoclonal antibody.

In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genanic and cMNA 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.

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) of a native sequence PR0230 cDNA, wherein SEQ ID NO:11 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:11 shown in Figure 25 Figure 7 shows a nucleotide sequence designated herein as DNA20088 (SEQ ID NO:13).

*ee Figure 8 shows a nuckodde sequene (SEQ ID NO: 17) of a nativc sequence PR0232 CDNA. wherein SEQ ID NO: 17 is a clone designated herein as 'UNQ2o6' and/or "DNA34435-1140 Figure 9 shows the amino acid sequence (SEQ ID NO:1IS) derived from the coding sequence of SEQ ID NO. 17 shown in Figure 8.

Figure 10 shows a iszclnxide sequezcc: (SEQ ID 140:22) of anative sequence PRO187 cDNA. wherein SEQ ID NO:22 is a clone designated herein as 'UNQ161r and/or *D1A2786-1ISS*.

Figure 11I shows t amino acid sequence (SEQ ED N0:23) derived from the coding sequence of SEQ ID N0:22 shown in Figure Figure 12 shows a isiclodde sequence (SEQ ID 14027) of a native sequence PR0265 clDNA, wherein SEQ ID 140:27 is a clone designated herein as LJNQ232' and/or ODNA36350-11S8".

Figure 13 shows the amino acid sequence (SEQ ID NO:28) derived from the coding sequence of SEQ ID N4027 shown in Figure 12.

Figures 14A-B show a rjicleotde sequence (SEQ MD NO:33) of a native sequence PR0219 eDNA, wherein SEQ ID 140:33 is a clonec designated herein as "U14Q193" and/or 'DNA32290-l 164'.

Figure 15 shows the amino acid sequence (SEQ IM 10:34) derived from the coding sequence of SEQ ID 140:33 shown in Figures 14A-B.

Figure 16 shows a rnucloeode sequence (SEQ I138): of a native sequence PR0246 cDNA, wherein SEQ ID 140:38 is a cdone designated heren as 'U1Q220' and/or IDNA35639-1172'.

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

Figure 18 shows a nudeotide sequeoce (SEQ ID 140:48) of a native sequence PRO228 cDNA, wherein SEQ ID 140:4 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 sequenice of SEQ ID 140:48 shown in Figure 18.

Figure 20 shows a nucleotide sequence designated herein as DNA21951 (SEQ ID 140:50).

Figure 21 shows a nucodde seqee(SEQD M 0.58)of anative sequence PR0533 cDNA, wherein S EQ ID N0538 is a clone designated herein as OUNQ344' and/or *D1A49435-1219".I Figure 22 shows dhe amino acid sequence (SEQ ID NO:S9) derived from the coding sequence of SEQ ID NO:58 shown in Figure 21.

Figure 23 shows a oickodde sequence (SEQ MD NO:63) of a native sequence PRO245 elDNA, wherein SEQ MD 10:63 is a clone designated herein as OUNQ2l9* and/or ODNA3563 B-1141 0.

Figure 24 shows the amino acid sequence (SEQ ID NO:64) derived from t coding sequence of SEQ ID 140:63 shown in Figure 23.

Figuire 25 shows a maiciodde sequence (SEQ H) 140:68) of a native sequcnce PR0220 clDNA. wherein SEQ ID NO:68 is a clone designated herein as OUNQl94' and/or 'D14A32298-1 132'.

Figure 26 shows the amino acid sequece (SEQ ID 140:69) derived from the,coding sequence of SEQ ID NOM6 shown in Figure Figure 27 shows a ncooddc sequenc (SEQ I NO.70) of a nativec sequence PR0221 cDNA. wherein SEQ ID NO:70 is a clone designated herein as 'JNQI95* and/or ODNA33089-1 132'.

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

Figure 29 shows a m~ics&dd sequece (SEQ ID NO:72) of a native sequence PR0227 cDNA. wherein SEQ M I NO;72 is a clone designated herein as "LJNQ201" and/or *DNA33786-1 132'.t.

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

Figure 31 shows a zmciodde sequence (SEQ ID NO:83) of a native sequence PR0258 eDNA, wherein SEQ ID NO:83 is a clone designated herein as 'UNQ225* and/or 'DNA3591 8-1174'.

Figure 32 shows the amino acid sequence (SEQ ID NO:94) derived from the coding sequence of SEQ ID NO: 93 shown in Figure 31.

Figure 33 shows a miclewdde sepence (SEQ ID NO:90) of a native sequence PR0266 eDNA, wherein SEQ ID NO:90 is a clone designated herein as "UNQ233Y and/or 'DNA371- 178'.

Figure 34 shows the amino acid sequence (SEQ ID NO:91) derived from the coding sequence of SEQ ID NO:90 shown in Figure 33.

Figure 35 shows a micloodde sequence (SEQ 13) NO:95) of a natve sequence PR0269 cDNA. wherein SEQ ID NO:95 is a clone designated herein as 'UNQ236' and/or DNA38260-1 180'.

Figure 36 shows the amino acid sequence (SEQ ID NO:96) derived from the coding sequence of SEQ ID shown in Figure Figure 37 shows a nucleotide sequence (SEQ ID NO:103) of a native sequece= PR0287 cDNA. wherein SEQ ID NO0:103 is a clone designated herein as JNQ2So" and/or *DNA39969-l 185*.

Figure 38 shows the amino acid sequence (SEQ ID NO: 104) derived from the coding sequence of SEQ ID *:NO:0103 shown in Figure 37.

Figure 39 shows a nucleotide sequence (SEQ ID 140:108) of a native sequence PR0214 cDNA. wherein SEQ ID NO: 108 is a clone designated herein as *U1Q188" and/or *DNA32286-1191'.

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 sequece= (SEQ ID NO: 113) of a native sequence PR0317 cDNA. wherein SEQ ID NO:1 13 is a clone designated herein as "U1Q278" and/or 'DNA33461-1199'.

Figure 42 shows the amino acid sequenice (SEQ ID NO: 114) derived from the coding sequence of SEQ ID NO: 113 shown in Figure 41.

Figure 43 shows a nucleotidc sequence (SEQ ID NO: 118) of a native sequence PRO301 cDNA, wherein SEQ ID NO: 118 is a clone designated herein as NUNQ264 *and/or *DNA40628-1216*.

Figure 4 shows the amino acid sequence (SEQ ID NO: 119) derived from the coding sequence of SEQ ID NO:118 shown in Figume43.

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 9NQ 198 "and/or "DNA33221-l 133*.

Figure 46 shows the amino acid sequence (SEQ ID 80:127) derived from the coding sequence of SEQ ID NO: 126 shown in Figure Figure 47 shows a nueceocide sequence (SEQ MD NO: 13 1) of a native sequence PR0222 cDNA. whcrein SEQ ID NO: 131 is a clone designated herein as *UNQ196' and/or 'DNA33107-1135'.

Fgur 48 shows the amino acid sequence (SEQ ID NO: 132) derived from the coding .s .equence of SEQ [D NO:131 shownin Figure 47.

Figure 49 shows a nuclotde sequence (SEQ ID NO: 136) of a native sequence PR0234 eDNA, wherein SEQ ID N0: 136 is a clone designated herein as 'UNQ2OS" andor "DNA35557-1137".

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

Figur 5 1 shows a nucleotide sequence (SEQ MD NO: 14 1) of a native sequence PIZ0231 cDNA, wherein SEQ ID NO: 141 is a clone designated herein as 'UNQ2OS' and/or 'DNA34434-1 139'.

Figure 52s a h amin adid sequence (SEQ ID N0: 142) derived from the coding sequence of SEQ ID NO: 141 shown in Figure 5 1.

Figure 53 shows a nucleotide sequence (SEQ ID N0: 147) of a native sequence PR0229 cDNA, wherein SEQ ID NO: 147 is a clone designated herein as 'UNQ203" and/or NDNA33100.1159".

Figure 54 shows the amino arid sequence (SEQ ID 80:148) derived from the coding sequence of SEQ ID NO: 147 shown in Figure 53.

Figure 55 shows a nucleotide sequence (SEQ ID N0: 152) of a native sequence PR0238 eDNA, wherein SEQ ID NO: 152 is a clone designated herein as "UNQ2l2" and/or "DNA35600-1162'.

Figure 56 shows the amino acid sequence (SEQ ID NO:;153) derived from the coding sequence of SEQ ID NO: 152 shown in Figure Figure 57 shows a nuclootide sequence (SEQ ID N0: 158) of a native sequence PR0233 eDNA, wherein .:SEQ ID N0: 158 is a clone designated herein as 'UNQ207" and/or "DNA34436-1238'.

Figure 58 shows the amfino adid sequence (SEQ ID NO: 159) derived from the coding sequence of SEQ ID 80: 158 shown in Figure 57.

Figuire 59 shows a nucleotide sequence (SEQ ID NO: 163) of a native sequence PR0223 eDNA. wherein SEQ ID NO: 163 is a clone designated herein as 'UNQ197' and/or "DNA33206-1165'.

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

Figure 61 shows a nucleotide sequence (SEQ ID NO: 169) ofta native sequence PR0235 eDNA. wherein SEQ ID N0: 169 is a clone designated herein as U8MQ209* and/or "DNA35558-1167*.

Figure 62 shows the amino acid sequence (SEQ ID 80: 170) derived from the coding sequence of SEQ ID N0: 169 shown in Figure 61.

Figure 63 shows a nucleotide sequence (SEQ ID N0: 174) of a native sequence PR0236 eDNA, wherein SEQ ID NO: 174 is a clone designated herein as UNQ210O and/or 'DNA35599-1 168'.

Figure 64 shows the amino acid sequence (SEQ ID NO: 175) derived from the coding sequence of SEQ ID N0: 174 shown in Figure 63.

Figure 65 shows a nucleodde sequence (SEQ ID NO: 176) of a native sequence PR0262 cDNA. wherein 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 S EQ nI) NO: 176 shown in Figure Figure 67 shows a nucleotide sequence (SEQ ID NO: 184) of a native sequence PR0239 cDNA. wherein SEQ ID NO: 194 is a clone designated herein as OUNQ2130 and/or *DNA34407-1169'. t' Figure 68 shows the amino acid sequence (SEQ ID NO:IM5 derived from the coding sequence of SEQ ID NO: 184 shown in Figure 67.

Figure 69 shows a nucleotide sequence (SEQ ID NO: 189) of a native sequence: PR0257 cDNA wherein SEQ ID NO: 199 is a clone designated herein as "UNQ224" and/or "DNA35841-1173'.

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

Figure 71 shows a nucleotide sequence (SEQ ID NO: 194) of a native sequence PR0260 cDNA. wherein SEQ ID NO: 194 is a clone designiated herein as 'UNQ227' and/or "DNA33470-1 175'.

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 nucleodec sequence (SEQ ID NO :200) of a native sequence PR0263 cDNA, wherein SEQ ID NO:.200 is a clone designated herein as "UNQ230" anid/or "DNA34431-1 177T.

Figure 74 shows die amino arid sequence (SEQ 1I) NO:201) derived from the coding sequence of SEQ ID NO:200 shown in Figure 73.

Figure 75 shows a nucicotide sequence (SEQ ID NO:206) of a native sequence PR0270 cDNA. wherein .4.,SEQ ID NO:206 is a clone designated herein as "UNQ237* and/or *DNA3951-1181'.

Figure 76 shows t amino acid sequenc (SEQ ]D NO:207) derived from the coding sequence of SEQ ED NO:206 shown in Figure Figure 77 shows a nucleotde sequence (SEQ ID NO1:212) of a native sequence PR0271 cDNA. wherein SEQ ID NO:212 is a clone designated herein as 'UNQ238' and/or "DNA39423-1lST".

Figure 78 shows the amino acid sequence (SEQ ID NO :213) derived from the coding sequence of SEQ ID NO:212 shown in Figure 77.

Figure 79 shows a nucleotide sequence (SEQ ID NO:220) of a native sequence PR0272 eDNA, wherein SEQ WD NO:220 is a clone designated herein as 'UNQ239' and/or "DNA40620-1 193.

Figure 90 shows t amino acid sequence (SEQ MD 11:221) derived from the coding sequence of SEQ MD NO:220 shown in Figure 79.

Figure 81 shows a nucleotide sequence (SEQ ID NO:226) of a native sequence PR0294 eDNA, wherein SEQ ID NO:226 is a clone designated hercin as "UNQ2S7* and/or "DNA40604-1197".

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

Figure 83 shows a nuecide sequence (SEQ ID NO:235) of a native sequence PR0295 cDNA. wherein SEQ ID NO:235 is a clone designated herein as OUNQ258' and/or 'DNA38268-1188".

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

Figures 85A-B show a nucleotide sequence (SDEQ ID N:243) of a native sequence PRO293 cl\, wherein SEQ ID N3:244 is a clone designated herein as "UNQ256" and/or C1NA37151-1193".

Figure 86 shows the amino acid sequence (SEQ IND:244) derived froman the coding sequence of SEQ ID MD:244 shown in Figures Figures 89A-B show a nucleotide sequence (SED ID NO:248) of a native sequence PRO247 ctlA, wherein SEQ ID N:248 is a clone designated herein as "UN2221" and/or DENA35673-1201".

Figure 88 shows the amino acid sequence (SE2 ID 1 D 249) derived fran the coding sequence of SEQ ID 13:248 shown in Figure 87.

Figure 89 shows a nucleotide sequence (SEQ ID N1:253) of a native sequence PR0302 clr, wherein SEQ ID N1:253 is a clone designated herein as "UNQ265" and/or "IEA40370-1217.

Figure 90 shows the amino acid sequence (SED ID NO:254) derived fran the coding sequence of SED ID 13:253 shown in Figure 89.

Figure 91 shows a nucleotide sequence (SDQ ID N1:255) of a native sequence PR0303 cENA, wherein SEQ ID NO:255 is a clone designated herein as "'UNQ266 and/or -IaA42551-1217.

Figure 92 shows the amino acid sequence (SE ID NO:256) derived fran the coding sequence of SEQ2 ID MO:255 shown in Figure 91.

Figure 93 shows a nucleotide sequence (SEQ ID ND:257) of a native sequence PRO304 cEINA, wherein SEQ ID 13:257 is a clone designated herein as "UNP267 and/or "ENA39520-1217.

Figure 94 shows the amino acid sequence (SEM ID NO:258) derived ~fran the coding sequence of SEQ ID 1N:257 shown in Figure 93.

Figure 95 shows a nucleotide sequence (SED ID 1:259) of a native sequence PRD307 cINA, wherein SEQ ID 13:259 is a clone designated herein as *UNQ2700 and/or "TENA41225-12170.

Figure 96 shows the amino acid sequence (SEQ ID NO:260) derived fran the coding sequence of SEQ ID NO:259 shown in Figure Figure 97 shows a nucleotide sequence (SEQ ID NO:261) of a native sequence PRO343 cE1l, wherein SEQ ID 1:261 is a clone designated herein as "UNQ302 and/or 1IiA43318-1217.

Figure 98 shows the amino acid sequence (SED ID NO:262) derived froman the coding sequence of SEQ ID 10:262 shown in Figure 97.

Figure 99 shows a nucleotide sequence (SEQ ID NO:283) of a native sequence PRO328 clNA, wherein SEQ ID 13:283 is a clone designated 60 herein as "IUNQ289 and/or "EM40587-1231'.

Figure 100 shows the amino acid sequence (SEQ ID NO:284) derived froman the coding sequence of S=2 ID NO:283 shown in Figure 99.

Figures 101A-B show a nucleotide sequence (SEQ ID ND:288) of a native sequence PR0335 cINA, wherein SED ID ND:288 is a clone designated herein as "UbN287- and/or "lAE41388-1234.

Figure 102 shows the amino acid sequence (SEQD2 ID NO:289) derived froman the coding sequence of SEQ ID ND:288 shown in Figures 103A-B.

Figure 103 shows a nucleotide sequence (SEQ ID Nm:290) of a native sequence ER0331 cE1, wherein SEQ ID m:290 is a clone designated herein as "UND292" and/or "IA40981-1234'.

Figure 104 shows the amino acid sequence (SEQ ID N:291) derived froman the coding sequence of S2EQ ID NO:290 shon in Figure 103.

~Figure 105A-B show a nucleotide sequence (SEQ ID 1: 293) of a 15 native sequence PR0326 cCIA, wherein SE2 ID 1N:293 is a clone designated herein as "UNQ287 and/or "EN37140-1234".

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

Figures 107A-B show a nucleotide sequence (SEQ ID 0:309) of a native sequence PR0332 cIA, wherein SEB ID NO:309 is a clone designated herein as "UNQ2930 or "ENA40982-1235'.

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

Figure 109 shows a nucleotide sequence (SEQ ID ND:314) of a 25 native sequence PR0334 cIEN, wherein SEQ ID NO:314 is a clone designated herein as "UNQ295 or '"II41379-1236".

Figure 110 shows the amino acid sequence (SEQ ID NO:315) derived fran the coding sequence of SE2 ID NO:314 shown in Figure 109.

Figure 111 shows a nucleotide sequence (SEQ ID NOD:319) of a native sequence PR0346 cmA, wherein SED ID 1O:318 is a clone designated herein as "UNQ305" or "IIr44167-1243".

Figure 112 shows the amino acid sequence (SE ID NO:319) derived froman the coding sequence of SEQ ID ND:318 shown in Figure 111.

Figure 113 shows a nucleotide sequence (SED ID N0:32 3 of a native sequence PR268 cdU, wherein SEM ID ND:323 is a clone designated herein as "U1235 or 'IN39427-1179".

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 N1:330) of a 61 native sequence PRO330 cENA, wherein SEID NO:330 is a clone designated herein as "UNQ290" or "IiA40603-1232".

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

Figure 117 shows a nucleotide sequence (SEQ ID ND:337). of a native sequence PRO339 clA, wherein SEQ ID ND:337 is a clone designated herein as "UNQ9229" or "I2A43466-1225".

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

Figure 119 shows a nucleotide sequence (SEQ ID NO:339) of a native sequence PR0310 crA, wherein SP ID N0:339 is a clone designated herein as uUN273 or 'IJM43046-1225.

Figure 120 shows the amino acid sequence (SE ID N3:340) derived froman the coding sequence of SEQ ID NO:339 shown in Figure 119.

Figure 121 shows a nucleotide sequence (SED ID 10:375) of a native sequence PR0244 cEZA, wherein SEQ ID 1NO:375 is a clone designated herein as "UNQ218 or "IN35668-11 7 1".

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

DEL ED DESnCRIP'ICN OF TIRE PREFERBE EMBODIMEN1S I. Definitions The terms -PRO polypeptidef and "PROf as used herein and when immediately S:followed by a numerical designation refer to various polypeptides, wherein the carplete designation PR/nurder) refers to specific polypeptide sequences as described herein. The terms "PRO/nrber polypeptide" and "PRO/nuriber as used herein enrcapass native sequence polypeptides and polypeptide variants (which are further defined herein) The PRO polypeptides described herein may be isolated fran a variety of sources, such as franom humran tissue types or frcn another source, or prepared by recabinant or synthetic rethods.

A "native sequence PRO polypeptidev ccaprises a polypeptide having the sane amino acid sequence as the corresponding PRO polypeptide derived fran nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recanbinant or synthetic ireans. The term "native sequence PRO polypeptide" specifically enccarpasses naturallyoccurring truncated or secreted forms of the specific PRO polypeptide and extracellular dmnain sequence), naturally-occurring variant fornrms of alternatively spliced forms) and naturally-occurring 61a allelic variants of the polypeptide. In various entodiments of the invention, the native sequence PRO211 is a mature or full-length native sequence PRO211 polypeptide ccnprising amino acids 1 to 353 of Figure 2 (SBQ ID NO:2), the native sequence PRO217 is a mature or full-length native sequence PR0217 polypeptide comprising amino acids 1 to 379 of Figure 4 (SEQ ID Np:4), the native sequence PRO230 is a nature or full-length native sequence PR0230 polypeptide ccuprising amino acids 1 to 467 of Figure 6 (SEQ ID 3N:12), the native sequence PRO232 polypeptide is a mature or fulllength native sequence PRO232 polypeptide ccnprising amino acids 1 to 114 S 10 of Figure 9 (SEQ ID N0:18), the native sequence PRO187 is a mature or fulllength native sequence PR0187 ccuprising amino acids 1 to 205 of Figure 11 (SEQ ID NO:23), the native sequence PRO265 polypeptide is a mature or fulllength native sequence PRO265 polypeptide is an extracellular d~nain of the full-length PRO265 protein, wherein the putative transmatirane domain of the full-length PRO265 protein is encoded by nucleotides beginning at nucleotide 1969 of SEQ ID N0:31, the native sequence PR0219 polypeptide is a mature or full-length native sequence PR219 polypeptide corprising amino acids 1 to 915 of Figure 15 (SEQ ID NO:34), the native sequence PR0246 is a nature or full-length native sequence PRO246 polypeptide ccaprising amino acids 1 to 390 of Figure 17 (SEQ ID NO:39) or the native sequence PR0246 polypeptide is an extracellular dmnain of the full-length PR0246 protein, wherein the putative transmembrane domain 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 couprising amino acids 1 to 690 of Figure 19 (SDQ ID NO: 49) or the native sequence PR0228 polypeptide is an extracellular dmnain of the full-length PRO228 protein, the native sequence PR0533 is a mature or full-length native sequence PR0533 ccaprising amino acids 1 to 216 of Figure 22 (SEQ ID N0:59), with or without the N-terminal signal sequence, and with or without the initiating methionine at position 1, the native sequence PR0245 polypeptide is a mature or full-length native sequence PRO245 polypeptide co~szgamino acids 1 to 312 of Figure 24 (SEQ ID NO:64). the native sequence of each PR0220, PR0221 and PR0227 polypeptides is a mature or fuall-length native sequence PR0220, PR0221 and PR0227 polypcptidc compriing amino acids I dirough 708ofFagur 26 (SEQ ID N0:69). I through 259of Figure 28(SEQ ID 40:7 1), &W 1 through 620 of Figur 30 (SEQ MD NO:73). the native sequence PRO259 polypeptidec is a mature or full-length intive sequecew PR0258 polypeptide comprising amino acids 1 to 398 of Figure 32 (SEQ ID) 10:84) or the native sequence PR0258 polypeptidc is an extracclIular domain of the full-length PR0258 protein, wher'ft the putative transineibranc domain of the full-length PRO258 protein is encoded by nucleotides beginning at nucleotide 1134 of SEQ ID NO:83, the native sequence PR0266 polypeptide is a mature or full4ength native sequence PR0266 polypeptide comnprising amino acids I to 696 of Figure 34 (SEQ ID 140:91) or the native sequence PR0266 polypeptide is an ecxnllular domain of the full-1agth PR0266 protein, wherein the putative transtmembmane domain of the full-length PRO266 protein is encoded by zaiceotides beginning at about rusloodde 2009 of SEQ MD NO: 104'.

the native sequnc PR0269 polypeptide is a mature or ful-lngth native sequence PR0269 polypeptide comprising *amino acids I to 490 of Figure 36 (SEQ ID 10:96) or die native sequence PR0269 polypeptide is an extracellular domain of the full-length PR0269 protein, whierein the putative transmembraedmn of the full-length PR0269 V -0 protein is encoded by nucleotides beginning at nucleotide 1502 as shown in Figure 35. the native sequence PRO29 polypeptide is amature or fullegth naive sequence PRO287 polypeptide comprising amino acids I to 415 of Figure 38 (SEQ ID NO: 104), the native sequence PR0214 is a mature or full-length natve sequence PR0214 comprising 0 ~alyino acids I to 420 of Fig. 40 (SEQ ID 140:109), the native-sequec PR0317 is afull-length native-pre-sequence PR0317 comprising amino acids 1 to 366 of Fig. 42 (SEQ ID NO. 114) or a mature native-sequence PR0317 comprising amino acids 19 to 366 of Fig. 42 (SEQ ID NO0:114). the native sequence PR0301 is a mature or fulllength native sequence PRO301 comprising amino acids 1 to 299 of Fig. 4 (SEQ ID 140:119). with or without the N-terminal signal sequence, with or without the initiating methionine at position 1. with or without the potential tsansmembrane 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 mature or full-length native sequence PR0224 polypeptide comprising amino acids 1 to 282 of Figure 46 (SEQ ID 140:127). dhe native sequence PR0222 polypeptidc is a mature or full-length native sequence PR0222 polypeptide comprising amino acids 1 to 490 of Figure 48 (SEQ ID NO-132), the native sequence PR0234 is a mature or full-length native sequence novel lectin comprising amino acids I to 382 oftFig. 50 (SEQ ID NO: 137), the native sequence PR0231 polypeptide is a mature or funl-length native sequence PR0231 polypeptide comprising amino acids I to 428 of Figure 52 (SEQ ID 140:142), the native sequence PR0229 polypeptide is a mature or full-lazthnative sequence PR0229 polypeptide comprising amin acids I to 347 of Figure 54 (SEQ ID NO:148), the native sequence PR0238 polypeptide is a mature or full-length native sequence PR0239 polypeptide comprising amino acids I to 310 of Figure 56 (SEQ ID 140:153), the native sequence PR0233 polypeptide is a matre or full-length native sequea PR0233 polypeptide comprising amino acids 1 to 300 of Figure 58 (SEQ ID 140:159), the native sequence PR0223 polypeptide is a smatre or full-length native scquence PR0223 polypeptide comprising amin acids I to 476 of Figue 60 (SEQ ID 140:164), the native sequence PR0235 polypptde is a mature or full-length native sequ~ee PRO235 polypeptide comprising amino acids I to 552 of Figure 62 (SEQ ID NO: 170). the native sequence PR0236 polypep6&d is a mature or full-length native sequence PR0236 polypeptide comprising 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 ccrprising amino acids 1 to 654 of Figure 66 (SEQ ID N0:177) the native sequence PRO239 polypeptide is a mature or full-length native sequence PRO239 polypeptide cczprising amino acids 1 to 501 of Figure 68 (SED ID M0:185) the native sequence PRO257 polypeptide is a nature or full-length native sequence PR0257 polypeptide ccarprising amino acids 1 to 607 of Figure 70 (SEQ ID NO:190) or the native sequence PRO257 polypeptide is an extracellular darain of the full-length PRO257 protein, wherein the putative transmarbrane dcmain of the full-length PRO257 protein is encoded by nucleotides beginning at nucleotide 2668 as shown in Figure 69, the native sequence PRO260 polypeptide is a mature or full-length native sequence PRO260 polypeptide carprising amino acids 1 to 467 of Figure 72 (SEQ ID N10:195), the native sequence PRO263 polypeptide is a mature or full-length native sequence PRO263 polypeptide carprising amino acids 1 to 322 of Figure 74 (SED ID NO:201) or the native sequence PRO263 polypeptide 0 is an extracellular danain of the full-length PRO263 protein, wherein the putative transmntrane darain of the full-length PRO263 protein is encoded by nucleotides beginning at nucleotide 868 of SEM ID NO:200, the native sequence PRO270 polypeptide is a mature or full-length native sequence PRO270 polypeptide carprising amino acids 1 to 296 of Figure 76 (SEQ ID 10:207), the native sequence PR0271 polypeptide carprising amino acids 1 to 296 of Figure 76 (SEQ ID N:207), the native sequence PRO271 polypeptide is a mature or full-length native sequence PRO271 ocnprising amino acids 1 to 360 of Figure 78 (SEQ ID ND:213), the native sequence PRO272 polypeptide is 25 a mature or full-length native sequence PRO272 polypeptide ccrprising amino acids 1 to 328 of Figure 80 (SEQ ID NO:221), the native sequence PRO294 polypeptide is a mature or full-length native sequence PRO294 polypeptide ccuprising amino acids 1 to 550 of Figure 82 (SEQ ID 13:227), the native sequence PRO295 polypeptide is a mature or full-length native sequence PRO295 polypeptide ccuarprising amino acids 1 to 350 of Figure 84 (SEQ ID 10:236), the native sequence PRO293 polypeptide is a nmature or full-length native sequence PRO293 polypeptide ccarprising amino acids 1 to 713 of Figure 86 (SEQ ID N1:244) or the native sequence PRO293 polypeptide is an extracellular darain of the full-length PR0293 protein, wherein the putative trandantane dmuain of the full-length PRO293 protein is encoded by nucleotides beginning at nucleotide 2771 of SEQ ID N:243, the native sequence PR247 polypeptide is a mature or full-length native sequence PRO247 polypeptide caprising amino acids 1 to 546 of Figure 88 (SED ID N0:249), the native sequence PRO302 polypeptide is a mature or full-length 64 native sequence PRO302 polypptide ccsaprising amino acids 1 to 452 of Figure 90 (SEX? ID NO:254), the native sequence PR0303 polypptide is a nature or full-length native sequience PR0303 polypeptide ccuiPrising amino acids 1 to 314 of Figure 92 (SE)2 ID N:256), the native sequence EiR)304 polypptide is a mature or full-length native sequence PRO)304 polypeptide ccnprising amino acids 1 to 556 of Figure 94 (SE12 ID ND:258).,. the native sequence PR0307 polypeptide is -a mature or full-length native sequence PR0307 polypeptide crprising amino acids 1 to 383 of Figure 96 (SE? ID NO:260), the native sequence PRO343 polypeptide is a mature or full-length 10 native sequence PR0343 polypeptide caiprising amino acids 1 to 317 of Figure 98 (SE2 ID NO:262), the native sequence PR0328 polypeptide is a mature or full-length native sequence PR0328 polypeptide cciiprising amino acids 1 to 463 of Figure 100 (SEX? I NJ:284) or the native sequence PRO)306 *e polypeptide is an extracellular dcxnain. of the full-length PRO)306 protein.

wherein the putative extracellular dcmain. of the full-length PRO3O6 protein, the native sequence PR0335 polypeptide is a nature or full-length native sequence PR0335 polypeptide cciprising amino acids 1 through 1059 of Figure 102 (SEX? ID ND: 289), the native sequence- PR0331 polypeptide is a mature or full-length native sequence PR033 1 polypeptide ccmprising amino acids 1 through 640 of Figure 104 (SEQ ID NO:291), the native sequence PR0326 polypeptide is a mature or full-length native sequence PR0326 *'polypeptide ccnprising amxino acids 1 through 1119 of Figure 106 (SEX? ID NJ :293), wherein additional enbodinents include wherein the trarnszrnbrane regions are deleted or the peptides are truncated, so as to not include the transrronbrane regions for each of PR033 5, PROM33, and PR0326, the native sequence PR0332 is a mature or full-length native sequence PRO332 ccziprising amino acids 49 to 642 of Figure 180 (SEX? M NJ:309), with or without the N-terminal signal sequience, and with or without the initiating methionine at position 1, the native sequence PRO334 polypeptide is a mature or full-length native seqence PR0334 polypeptide ccmprising amino acids 1 to 509 of Figure 110 (SEX? ID NO: 314), the native sequence PRO346 is a nature or full-length native sequence PR0346 wiprising amino acids 19 to 339 of Figure 112 (SEQ ID NJ:319), with or without the N-terminal signal sequence, with or without the initiating nethionine, with or without the trnnnbaediain at positions 340 to 360 and with or wiEhout the intracellular dcxrain at positions 361 to 450, the native sequence PR0268 polypept ide is a mature or full-length native sequence PRO268 polypePtide ccuprising amino acids 1 to 280 of Figure 114 (SEX? ID ND:324) or the native sequence PRO268 polypeptide is an extracellular dairain of the full-length 64a i a a 9 0

S**

C a C C *C

C

C.

0*

*O

9~e o •9 eoe* oo oo PRO268 protein, wherein the putative tranaembrane domain of the fulllength PRO268 protein is encoded by nucleotides beginning at nucleotide 559 as shown in Figure 113, the native sequence PR0330 polypeptide is a nature or full-length native sequence PR0330 polypeptide comprising amino acids 1 to 533 of Figure 116 (SEQ ID N3:331), the native sequence PRO339 polypeptide is a mature or full-length native sequence PRO339 polypeptide ccprising amino acids I to 772 of Figure 118 (SEQ ID NO:338), the native sequence PRO310 polypeptide is a mature or full-length native sequence PRO310 polypeptide cenprising amino acids 1 to 318 of Figure 120 (SEQ ID N:340) and the native sequence PRO244 is a mature or full-length native sequence PRO244 conprising amino acids 1 to 219 of Figure 122 (SEQ ID NO:376), wherein the mature, full-length native sequence PRO244 protein conprises a cytoplasmic dmiain (about amino acid positions 1 to 20), a transnmbrane damain (about amino acid positions 21 to 46), and an extracellular damain (about amino acid positions 47 to 219). Within the extracellular domain, the C-lectin domain is between about amino acid positions 55 and about amino acid position 206. Native sequence PRO244 as shown in Figure 122 maps to chrroosome 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 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.

"PRO317 variants" or "PRO317 sequence variants" as defined herein mean biologically active PRO317s as defined below having less than 100% sequence identity with the PRO317 isolated from recombinant cell culture or fram mammalian fetal kidney tissue having the deduced sequence described in Figure 42. Ordinarily, a biologically active PRO317 variant 3 5 will have an amino acid sequence having at least about 70% "amino acid sequence identity with the PRO317 of Figure 42, preferably at least about nore preferably at least about 80%, still mnore preferably at least about 85%, even mnore preferably at least about 90%, and most preferably at least about 95% 65 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 PRO317 shown in Figure 42.

Other preferred PRO317 fragments include those produced as a result of chemical or enzymatic hydrolysis or digestion of the purified PR0317.

A "chimeric PR0317" is a polypeptide comprising fulllength PRO317 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 PRO317. 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 PRO317 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 PRO317 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 "PRO317-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 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 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 obtain at least 15 residues of N-terminal or interal amino acid sequence by use of a spinning cup sequenaror. or to homogeneity by SDS-PAGE under nonreducing or reducing conditions using Coomassic blue or. preferably, silver stain. Isolated polypeptide includes polypeptide In siru within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

An "isolated" PRO polypeptide nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant ucleic acid molecule with which it is ordinrily associated in the natural irce of the PRO potypeptide 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 polypcptide 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 ncleic add 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.

'Southern analysis" or "Southern blotting" is -a method by which the presence of DNA sequences in a S* rsaiction cnd nuclease digest of DNA or a DNA-coaining composition is confirmed by hybridization to a known, labeled oligonucleotide or DNA fragment. Southern analysis typically involves electrophorctic separation of DNA 15 digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nitrocellulose. nylon, or another suitable membrane support for analysis with a radiolabeled, bioinylated, or enzymelabeled probe as described in sections 937-9.52 of Sambrook et 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 "20 a known probe such as an oligonucleoide, DNA fragment, cDNA or fragment thereof, or RNA fragment. The probe is labeled with a radioisotope such as mP, or by biotinylation, 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 er al., supra.

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, Sinclude a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic aid 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 seme and specifically covers single anti-PRO polypeptide monoclonal anibodis (Cmnluding agonist. antagonist, and netralizing antibodies) and and-PRO polypeptide 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 naturaly-occurring mutations that may be present in minor amounts.

"Active" or activity" for the purposes herin refers to form(s) of PRO polypeptde which fetain the biologic and/or immunologic activities of the specific native or maturally-occurring PRO polypeptide. The activity of a PRO332 polypcptide preferably involves the regulation of extracellular matrix, cartilage, or bone function.

"PRO317-associated disorder" refers to a pathological condition or disease wherein PR0317 is over- or underexpressed. Such disorders include diseases of the female genital tract or of the endometrium of a mammal.

including hyperplasia, endometriis, endometriosis, wherein the patient is at risk for infertility due to endomctrial factor, endomctrioma, and cndomerial cancer, especially those diseases involving abnormal bleeding such as a gynecological disease. They also include diseases involving angiogcncsis, wherein the angiogenesis results in a pathological condition, such as cancer involving solid tumors (the therapy for the disorder would result in decreased 0O 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 heart, including patients with coronary arterial disease who are not optimal candidates for angioplasty and coronary artery 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 20 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.

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 pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable 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 glycine, glutamine.

asparagine, arginine or lysinc: monasaccharides, disaccharides, and other carbohydrates including glucose, marmose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEW", polyethylene glycol (PEG), and PLURONICS".

The term "agonist" is used to refer to peptide and non-peptide analogs of the native PRO polypcptides (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 polypeptdec. Preferably, the agonists of the present invention retain dhe gulitative binding recognition properties and receptor activation properties of the native PRO polypeptide.

The term *antagonist' is used to refer to a molecule inhibiting a biological activity of a native PRO polypeptide of the present invention wherein native PRO polypeptide refers to pro-PRO poltpeptide. pre-PRO polypeptide. prepro-PRO polypeptide, or mature PRO polypeptide. Preferably, the antagonists herein inhibit the binding of a native PRO polypeptde of the present invention. Preferred antagonists essentially completely block the binding of a native PRO317 polypeptide to a PRO317 polypeptide receptor to which it otherwise binds. Such receptors may inchide the Type I and Type U, and possibly Type In receptors identified for the TOF- superfamily.

KolodzieJczyk and Hall, supra. 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 polypeptde). Such molecules can be screened for their ability to competitively inhibit PRO polypeptide receptor activation by monitoring binding of native PRO polypeptide in the presence and absence of the test antagonist molecule. for example. Examples of PR0317 polypeptide antagonists include neutralizing antibodies against F-2. An antagonist of the invention also encompasses an antisense polynucleotide against the PRO polypeptide gene. which antisense polynucleotide blocks transcription or translation of the PRO polypeptide gene, thereby inhiibiting its expression and biological activity.

'Stringent conditions" means employing low ionic sutregt and high temperature for washing, for example. 0.015 sodium chloride/0.0015 M sodium citrate/0.lI sodium dodecyl sulfate at 50*C. or employing during hybridization a denaturing agent, such as formarnide, for example, 50% (vol/vol) formoamide with 0.1 bovine serum albuniin/0. I Ficoll/0.l polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mnM sodium chloride, 75 miM sodium citrate at 42*C. Another example is use of 50% formamaide, 5 x SSC (0.75 M NaCI. 0.075 M sodium citrate). 50mrM sodium phosphate (pH 0.1 sodium pyrophosphate, 5 x Denhardt's solution. sonicated salmon sperm DNA (50 pug/mi), 0. 1% SDS, and 10% dextan sulfate at 420C, with washes at '42*C in 0.2 x SSC and 0.1% SDS. Yet another example is hybridization using a buffer of 10% dextran sulfate. 2 xSSC (sodium diloride/sodium citrate) and 50% formanide at SS*C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 550C.

'Moderately stringent condiions' are described in Sambrook et supra, and include the use of a washing solution and hybridization conditions temperatre, ionic strength, and %SDS) less stringent than described above. An example of moderately stringnt conditions is a condition such as overnight incubation at 37*C in a solution comprising: 20% forinmick, 5 x SSC (150 mM NaCI, 15 mnM trisodimn citrate), 50 mM sodium phosphate (pH 5 x Denhardt's solution, 10% dextran sulfate. and 20 mg/nil denatured sheared salmon sperm DNA, followed by washing the filters in I x SSC at about 37-50*C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc., as necessary to accommaodate factors such as probe length andthe like.

11. Comootmonor andi Methodt of the Invenition- 1. Fall-Iength PRO2I1 and PR27Pletde The present invention provides newly identiflcd and isolated nucleobide sequences encoding polypeptides refenedso in dz present application as PRO211 and PRO2 17. In particular, Applicants have identificd and isolated cDNA eccoding PRO211 and PRO217 polypepcides. as disclosed in further detail in the Examples below. Using BLAST (FastA format) acquence alignment computer programs. Applicants found that cDNA s'uences encoding full-length native sequence PRO211 and PR0217 have homologies to known proteins having EGF-like domains.

Specifically, tecDNA sequne DNA32292-1131 (Figure 1, SEQ ID NO:1) has 36% identify and a Blast score of 209 with PAC6 RAT and 31 identif and a Blast score of 206 widL Fibulin-l, isoform c precursor. The eDNA sequence DNA33094-1131 (Figure 3. SEQ ID NO.3) has 36% identity and a Blast score of 336 with eas mr newt amscin, andI 37% Identity and a Blast score of 331 with human tcnascip-X precursor. Accordingly. it is presently believed that PRO211 and PR0217 polypeptides disclosed In the present application are newly identified members of the EGF-like family and possesses properties typical of the EUF-like protein family.

2. Full-length PRO230 Povuep!1des The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROM3. In partcular, Applicants have identified and isolated cDNA encoding a PR0230 polypeptide. as disclosed in further detail in the Examples below. Using known programs such as BLAST and FastA scquenice alignment computer program. Applicants found that a clDNA sequence encoding full- :length native sequence PR0230 has 48% amino acid identity with the rabbit tubulointerstitial nephritis antigen 20 precursor. Accordingly, it is presently believed that PR0230 polypeptide disclosed in the present application is a newly identified memnber of the tubulointerstitial nephritis antigen family and possesses the ability to be recognized by human autoantibodies in certain forms of tubulointerstitial nephritis.

3. Full-lenpth PRO232 Polypoies The present invention provides newly identified and isolated nucleotdec sequences encoding polypeptides referred to in the present application as PROM3. In particular, Applicants have identified and'isolated, cDNA encoding a PR0232 polypcpdde. as disclosed in futher detail in die Examples below. Using BLAST and FastA sequec alignment computer programs, Applicants found that a portion of die full-length 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 presently believed that the PR0232 polypeptide disclosed in the present application may be a newly identified stem cell antigen.

4. Full-lenpth P-RO187 Povnetides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR.0187. 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 sequence alignmient computer programis. Applicants foundl that a fu-length native sequence PROW1S (shown in Figure has 74% amino acid sequence identity and BLAST score of 310 with various andirogen-iniduced growth factors and FOP-8. Accordingly, it is presently believed that PRO 197 polypeptide disclosed in thc present application is a newly identified member of the FOP-8 protein family and may possess identify activity or property typical of the FGF-8-like protein family.

5, Fulijlpnith PR0265 Pob'netfdes Ile present invention provides newly identified and isolated ucleotide sequences encoding polypeptides referred to in dhe present application as PROW6. In particular, Applicants have identified and isolated cDNA encoding a PR0265 polypeptide. as disclosed in further detail in the Examples below. Using programs such as BLAST and FastA sequence alignment computer programs. Applicant found that various portions of the PR0265 polypeptide have signifiant homology with the fibromodulin protein and fibromodulin precursor protein. Applicants have also found doa the DNA encoding the PR0265 polypptide has significant homology with platelet glycoprotein a aner of the leucine ridh related protein family involved in skin 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 andi possesses protein protein binding capabilities, as well as be involved in skin and wound repair as typical of tis family.

6. Full-ennth PRO219 Polvueptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROM 1. In particular, Applicants have identified and isolated cDNA encoding a PR0219 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 PR0219 polypeptide have significant homology with the mouse and human nuailin-2 precursor polypeptides. Accordingly, it is presently believed that PR0219 polypeptide disclosed in the present application is related to the niatrilin-2 precursor polypeptide.

7. Full-length-PR024 Polvptides IlTe 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 cDNA encoding a PR0246 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 PR10246 polypeptidle has significant homology with the hwnan cellsrfac proteinFHCAR. Accordngly, it is presently believed that PR0246 polypeptide disclosed in the present application may be a newly identified membrane-boundl virus receptor or minor cell-specific antigen.

8. Full-lenieth PRO228 Polypentides The present invention provides newly identified and isolated nuclcotide sequences encoding polYPeptides referred to in the present application as P110228. In particular, Applicants have identified and isolated cDNA encoding a PR0228 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 PR0229 polypeptidc have significant homology with the EMRI protein. Applicants have also found that the DNA encoding t PR0228 polypeptide has significant homnology with latrophilin, acrphage-resticted cell surface glycoprotein. B0457.1 and leucocyte antigen CD97 precursr. Accordingly. it is presently believed that PR0228 polypepde disclosed in the present application is a newly identified member of the seven transitembrae superfamily ind possesses characteristics and functional properties typical of this family. In particular, It Is believed that PR0228 is a new member of the subgroup within this family to which CD97 and EMRI belong.

9. F'ulenv'th PROS33 Polvneptides 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 cDNA encoding a PRO533 polypeptide, as disclosed in further detail in the Examples below. Using BLAST-2 and FastA sequne aliginmers computer programs. Applicargs found that a full-length native sequence PR0533 (shown in Figure 22 atul SEQ ID NO:59) has a Blast soore of 509 and 53 amino acid sequence identity with fibroblast-growth factor (FGP). Accordingly, it is presently believed that PR0533 disclosed in the present application is a newly identified *memnber of the fibroblast growth factor family and may possess activity typical of such polypeptides.

Fufllength PR245 Poiyptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides 20 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 sequce at;inen computer programs, Applicants found that a portion of the amino acid sequence of the PR0245 polypeptide has 60% amino acid identity with the humnan c-myb protein. Accordingly, it is presently believed that the PR0245 polypeptide disclosed in the present application may be a newly identified member of the transmnembrane protein tyrosine kinase family.

11. Full-length PR0220. PR221 and PR227 Polynentides The present invention provides newly Identified and isolated ueeotide sequences encoding polypeptides referred to in the present application as PR0220. PR0221 and PR0227. In particular, Applicants have identified and isolated cDNAt encoding a PR0220. PR0221 and PR0227 polypeptide, respectively, as disclosed in further detail in dhin Eamples below. Using BLAST sand FastA sequence alignment computer programs, PR0220 has *mn adid identity with din amino acid sequence of a Inicir rich protein wherein the Identity is 87 PR0220additionally has amin acid identity withi the iciuronal Iecdne rich protein wherein the identity is 55%. The neuronal leucitie rich protein Is further described in Taguchi, eciat., Mol.LBrain Res 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 his amino acid identity with the amn acid sequence of platelet glycoprocein V precursor. The sanz results were obtained for hulman glycoproteln V. Different portions of these 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 and that each possesses protein-protein bindin capabilities typical of de leucine rich repeat: protein superfamily. It is-4lso believed that they have capabilities similar to those of SUT, the leucine rich repeat protein and human glycoprotein V.

12. Full4ength PRO258 Pobyptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO2SS. In particular. Applicants have identified and isolated cDN{A encoding a PR0258 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PRO2SS polypeptide have significan homology with the CRTAM and poliovinas receptors. Accordingly, it is presently believed that PR0258 polyeptde disclosed in the present application is a newly identified member of the Ig superfamily and possesses virus .15 receptor capabilities or regulates inmune function as typical of this family.

13. Fullength PRO266 Polynentides '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 further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that varioas 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 biding activity and neuronal development typical of tlis family. SLIT has been shown to be useful in the study and treatment of Alzheimer's disease, srpra. and thus, PR0266 may have involvement in the *:study and cure of this disease.

14. Full-length PRO269 Povnentides 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 sequene alignment computer programs. Applicants found that the aminto acid sequence encoded by nueceotides 314 to 1783 of the full-length native seqcep PR0269 (shown in FRgure 35 and SEQ ID NO:95) has significant homology to human urinary thrombonmodulin and various thronmboznodali analogues respectively, to which it was aligned.

Accordingly, it is presently believed doat PR0269 polypeptWl disclosed in the present application is a newly Identified member of the thrombomodulin family.

IS. Fullenueth PRO297 Polvneptides The present invention provides newly identifie and isolated nucleotide sequensces encoding polyeptides referred to in the present application as PR0287. In particular. Applicants have identified and isolated cDNA encoding a PRO28 polypepde. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer program. Applicants found that various portions of dhe PRO2S7-polypeptide have significant botnology with dan type I procollagen C-pmuteinase enhancer protein precursor and type 1 'procollagen Cproteanase enhancer protein. Accordingly. it is presently believed doa PRO28 polypcptdc disclosed in dhe present application is a newly Identified member of the C-proteinase enhancer protein family.

16. Mleng-th PRO214 PoIYIecldes Then prjesent invention provides newly identified and isolated nucleotide sequences enicoding polypetides referred to in the present application as PR0214. In particular, Applicants have identified and isolated cDNA encoding a PR0214 polypepude, as disclosed in further detail in die Examples below. Using BLAST and FastA :0 sequence alignment computer programs. Applicants found that a Full-length native sequence PR0214 polypeptide (shown in Figure 40 and SEQ ID NO: 109) has 49% amino acid sequenc identity with HTf protein, a knwn member of the EGF-faxily. The comparison resulted in a BLAST scorc of(920, with 150 matching micleotides. Accordingly, it is presently believed that the PR0214 polypeptide disclosed in the present application is a newly identified member of dhe Lunily comprising EGF domains and may possess activities or properties typical of dhe EGF-domain containing family.

17. Full-lenffth PR0317 Polvept ides The present invention provides newly identified and isolated nucleodde sequences encoding polypeptides referred to in the present application as PROW1. In particular. eDNA encoding a PR0317 polypeptide has been klwtifed and isolated. as disclosed in further detail in the Examples below. Using BLAST' 4 and FastA"' sequence alignnscat computer programs. it was found that a full-length native-sequence PR0317 (shown in Figure 42 andl SEQ ID NO:114) has 92% amino acid sequence identity with EBAF-l. Further, it is closely aligned with many other members of die TGP. superfamily.

Accordingly, it is presently believed that PR0317 disclosed in the present application is a newly identified member of the TGP- superfatmily and may possess properties that are therapeutically useful in conditions of uterine bleeding. etc. Hene, PR0317 may be useful in diagnosing or treating abnormal bleeding involved in gynecological diseases, for example, to avoid or lessen the need for a hysterectomy. PR0317 nay also be useful as an agent that affects angiogenesis in general, so PR0317 may be useful in andi-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 werc found in normal tissues (uterus, prostate, colon. and pancreas), in several tumors (colon, brain (twice), pancreas, and mullerian cell), and in a heart with ischernia. PR0317 has shown up in several tissues as well, but it does look to have a greater concentration in uterus. Hence. PR0317 may ha~ve a broader use by the body than EEAF-l. It is contemplated that, at least for some indications. PR0317 may have opposite effects from EBAF-1.

18. Full-length PRO301 Pglynentdeg The present hnventio provides newly identified and isolated rul'otide sequences encoding polypeptides referred to in the present application as PRO301. In particular. Applicants have identified and isolated cDNA encoding a PRO3OI polypeptide, as disclosed in further detail in die Examples below. Using BLAST and PastA sequence aligntn computer programs. Applicants francd that a full-length native sequence PRO301 (shown in Figure 44 and SEQ ID NO:l 19) has a Blast score of 246 corresponding to 30% amino acid sequence Idaity with human A33 antigen precursor. Accordingly. it is-presently believed that PRO301 disclosed in the present application is a newly identified member of the A33 antigen protein fatmily and may be expressed in human neoplastic diseases such as colorcetal cancer.

19. Full-lerwth PRO224 JP&lvuenides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0224. In particular. Applicants have identified and isolated eDNA encoding a PRO224 polypeptide, as disclosed in further detail in the Examples below. Using known programs such as BLAST and FastA sequence alignent computer programs, Applicants found that full-length native PR0224 (Figure 46, SEQ MD NO:127) has amino acid identity with apolipoprotein E receptor 2906 from bomno sapiens. The allgmnents of differnt portions of these twopolypeptides show amino acid identities of 37%. 36%. 30%, 44%, 44% and 28% respctively. Full-length native PR0224 (Figure 46. SEQ ID NO:127) also has amino acid identity with very low-density lipoprotein receptor precursor from gall. 7the alignments of different portions of these two polypeptides show amnino acid identities of 38%, 37%, 42%, 33%. and 37% respectively. Additionally, full-lenigth native PR0224 (Figure 46, SEQ ID NO: 127) has amino acid identity with the chicken oocyte receptor P95 from Gallus gailus. The alignments of different portions of these two polypeptides show amino acid identities of 38%.

37%. 42%, 33%, and 37% respectively. Moreover, full-length native PR0224 (Figure 46. SEQ ID 140:127) has amino acid Identity with very low density lipoprotein receptor short formn precursor from humans. The alignmrents of different portions of these two polypeptides show amino acid identities of 32%, 38%, 34%. 45 and 31 respectively. Accordingly, it is presently believed thit 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 functional ability to recognize and endocyt ose low density lipoproteins typical of the low density lipoprotain recepto rfamily. (The alignments described above used the following scoring parameters: T=7, S S2=36, Matrix: BLOSUM62.) FulIlength PR.0222 Po]vueriides The present invention provides newly identified and isolated nuclcotide 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 further detail i de Examples below. Using BLAST and FaStA sequence aligrnent computer programs, Applicants founod that a sequence encoding full-length native sequence PR0222 (shown in Figure 48 and SEQ ID NO: 132) has 25-26 amnino acid identity with mouse complement factor It precursor, has 27-29% amino acid identity with complement receptor, has 25-47% amino acid identity with mouse compement COb receptor type 2 long farm precursor. has 4o% amino acid identity with hjuman hypothetical protein kiaaO247. Accordingly, it is presently believed that PRO22 polypeptude disclosed in the present application is a newly identified romber of the canplctent reeptor family and possesses activity typical of the complement receptor family.

21. Full-length PRO234 Pal)MVj;WZS~L The present invention provides newly identified and isolated nucleodde sequences encoding polypeptides referred to in the present application as PR0234. In particular, Applicants have identified and isolated cDNA encoding a PR0234 polypptd&, as disclosed in further detail in the Examples below. Using BLAST (FastA-format) sequence alignment computer programs, Applicants found that a cDNA equence encoding full-length native sequenc PR0234 has 31 identity and Blast score of 134 with E-eelectin precursor. Accordingly, it is presently believed that the PR0234 polypeptides disclosed in the present application are newly identified members of the lectin/selectin family and possess activity typical of the lectin/sclectin family.

22. FulI-length PR10231 Plvnentdes 15 Th present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0231. In particular, Applicants have identified and isolated cDNA encoding a PR0231 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequne alignment computer programs, Applicants found that the full-length native sequence PRO231I polypeptide (dxrwn in igure52 and SEQl1D)NO: 142) has 3 %and 31 amino acid identity with humnnandratprostatic acid 20 phosphatase precursor proteins. respectively. Accordingly, it is presently believed that the PR0231I polyetide disclosed in the present application may be a newly identified member of the acid phosphatase protein family.

Fult4englh PR10229 Polvnetideg The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0229. In particula, Applicants have identified and isolated cDNA encoding a PR0229 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that various portions of the PR0229 polypeptide have significan homology with antigen wcl M130 antigen. T cell surface glycoprotein CD6 and CD6. It also is related to Sp-alphz. Accordingly, it is presently believed that PR0229 polypeptide disclosed in the present application is a newly identified member of the family containing scavenger receptor homology, a sequence motif found in a number of proteins involved in immune function and thus possesses immune fumction and /or segments which resist degradation, typical of this family.

24. Fultleneth PR10239 Polyneintides The present invention provides ncwly identified and isolated nucleotidc sequences encoding polypeptides referred to in the present application as PRO23S. In particular, Applicants have identified and isolated cDNA encoding a PR0238 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequecwe aliganent computer programs. Applicamt fournd that various portions of the PR0238 polypeptidc have significant homology with reductases, including oxidoreduciase and fatty acyi-CoA reductase. Accordingly, it is presently believed that PR0238 polypeptide disclosed in the present app!lication is a newly identified member of the reductase family and possesses reducing activity typical of the reductase family.

25. lFulI-leneh PRO233 Polvneotdes The present invention provides newly identified and isolated 'nucleotide sequences encoding polypcpddcs referred to in the present application as PROZ33. 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 sequence-alignment computer programs, Applicants found that various portions of the PR0233 polypeptdec have sigttificant homology with the reduciase protein. Applicantfs have also found that the DNA encoding the PRO233 polypeptide has significant homology with proteins from Caenwhd~is elegans. Accordingly, it is presently believed doat PR0233 polypeptide disclosed in the present application is a newly identified member of the reducis family and possesses die ability to effct the redox state of the cell typical of the reductase family.

26. Full-length P!R(223 Pol=rietdes The present ivention 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 PRO22 polypeptide, as disclosed in further detail in dr. Examples below. Using BLAST and FastA sequenceahignnient comiputer programs, Applicants found that the PR0223 polyetide 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 PRO235 Po]vr=ptid&; The present invention provides newly identified and isolated nuclcotide sequences encoding polypeptides referred to in the present application as PROW3. In particular, Applicants have identified and isolated cDNA encoding a PR0235 polypeptide, as disclosed in further detail in die Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0235 polyetide have significamt homology with the various plexti proteins. Accordingly, it is presently believed that PR0235 polypcptde 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-leogth PR236 sind PR0262 Poloenides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0236 and PR0262. In particular, Applicants have identied and isolated cDNA enicoding PR0236 and PR0262 polypeptds. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence aligrnent computer programs, Applicants found that various portions of the PR0236 and PR0262 polypeptides have significant homology with various JI-galactosldaze and P-galactosidase precursor polypcpddes. Accordingly. It is presently believed that tie PR0236 and PR0262 polypeptides disclosed in the present application are newly identified 1-galactosidase hosnologs.

29. FulIlength PR=29 Polvpeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeprides referred to in the present application as PROM3. In particular. Applicants have identified &rnd-isolated cDNA encoding a PR0239 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found tha various portions of t PRO239 polypeptide have significant homology with densin proteins. Accordingly, it is pr=etly believed that PR0239 polypeptide disclosed in the present applicarion is a newly identified miember of the densin family and possesses cell adhesion and the ability to effect synaptic processes as is typical of dhe densin family.

30. Fullenah PROM5 Poh'nqitfdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in die present application as PR0257. In particular, Applicants have identified and isolated cDNA encoding a PR0257 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0257 polypeptide have significant homology with the ebinrin precursor and ehuerin protein. Accordingly, it is presently believed that PRO257 polyepide disclosed in dhe present application is a newly identified protein nmember which is related to the ebnerin protein.

31. Full-Ienrth PR260 Polypetticex :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 beo.Ug porm such as BLAST and PastA sequenc alignment computer programs. Applicants found that various portions of the PR0260 polypeptde have significant homology with the alpha-lfucosidase precursor. Accordingly, it is presently believed that PR0260 polypeptide disclosed in the present application is a newly identified member of the fucosidase family and possesses enzymatic activity related to fuicose residues typical of the fucosidase family.

32. Full-length PR263 Pyptides The present invention provides newly identified and isolated aucleotide seqluences encoding polypeptides referred to in the present application as PROM6. In particular, Applicants have idcantiid and isolated CDNA encoding a PR0263 polypeptide, as disclosed in furthe detail in the*Examples; below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of die PR0263 Rolypeptide have significant 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 least one of the properties associated with these antigens. ie.. cancer and HIV marker, cell-cell or cellnatrix linsactjons. regulating cl traffic, lymph node homting, transmission of growth signals, and presentation at chcmokines and growth (acons to traveling cells.

33. Full-leneh PROZ270 PolvL&Pfides 71hc present invention provides newly identified and isolated nucleodde sequences encoding polypcptidCs referred in in the present application as PROM7. In particular. Applicants have identified and-isolated cDNA encoding a PR0270 polypeptide. as disclosed in furthier detail in the Examples bclow. Using BLAST. FatA and sequence alignment computer programs, Applicants found that that various portions of the PRO270 polypeptide have significant homology with various ihioredoxin 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 io effect reduction-oxidation (redox) state typical of the thioredoxin family.

34. Full-lneth PRO271 Polvnentides The present invention provides newly identified and isolated nucleotdec scquences encoding polypeptides referred 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 aliginent computer programs. Applicants fandthat the PR0271 polypeptide has significant homology with various link proteins andI preurors thereof. Accordingly. it is presently believed that PR0271 polypetide disclosed in the present application is a newly identified link protein homlog.

35. Full-lengyth PR272 Polyneneides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred 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 sequencc.alignment computer programs. Applicants found that various portions of dhe PR0272 polypeptide have significant homology with the human reticulocalbin protein and its precursors. Applicants have also found that the DNA encoding dhe PR0272 polypeptide has significant homology with the maouse reticulocalbin precursor protein.

Accrdngl, t s pesntl blived that PRO272 polypeptide disclosed intepresent aplction is a ewly idetfie member of die reticulocalbin family and possesses the ablitiy to bind calcium typical of the renilocaibin family.

36. Full-lenrth PR0294 olyeatddu 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 polypeptde. as disclosed in further detail in the Examples below. Using BLAST and PastA sequence alignment computer programs. Applicants foun that various portions of the PR0294 polypeptide have significarst homology with the various portions of a nmmcr of collagen proceins. Accordingly. it is presently believed that PR0294 polypeptide disclosed in the present application is a newly identified member of the collagen family.

37. Full-lerngth P!RO29S Pobypnides The present invention provides newly identified and isolated nuclootide sequences encoding polypeptides referred to in the present application as PROM9. In particular, Applicants have identified and isolated cDNA encoding a PRO295 polypeptide. as disclosed in futher detail in the Examples below. Using BLAST and FastA sequence #lignment compuzter program, Applicants found that various portions of dhe PR0295 polypepti dc have significant homology with uwegrn proteins. Accordingly, it is presently believed that PR0295 polyetide disclosed in dfe present application is a newly identified memrber of the integrin family and possesses cell adhesion typical of the integrin family.

38. Full-lengh PRO293 Polvpnides Thie present invention provides newly identified and isolated nuddcld sequences ecoding polypetides referred to in dhe present application as PR0293. In particular. Applicants have identffed and isolated cDNA encoding a PR0293 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence uligruncat compete programs. Applicants found that portions of the PR0293 polypeptide have significant homology with the neuronal leucine rich repeat proteins 1 and 2, (NLRR-l and NLRR-2), particularly NLRR-2.

Accordingly. it is presently believed that PR0293 polypqxide: disclosed in the present application is a newly identified meber of the neuronal leucine rich repeat protein family and possesses ligand-ligand binding activity typical of the NRLL protein family.

:39. Full-leneth PR247 Polvocottdes 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 cDNA :::encoding a PR0247 polypeptide. as disclosed in faither detail in the Eixamples; 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 diat the DNA encoding the PR0247 polypeptide has significant homology with a number of other proteins, including KIAA023I. Accordingly. it is presendy believed that PR0247 polypeptide disclosed in die present application is a newly identified member of the leucine rich repeat family and possesses ligand binding abilities typical of this family.

Full-ength PR0302. PROM,3 PR304. PR307 and PRO343 Polnpntdes The present invention provides newly identified arnd isolated nudceotide sequences encoding polypeptides referred to in the presen application as PR0302. P10303. PR0304. PR0307 and PROW4. In particular. Applicants have identified and isolated cDNA encoding PR0302. PR0303. PR0304, PR0307 and PR0343 polypeptides, as disclosed in bartber detail in the Examples below. Using BLAST and FastA sequence alignment computer programs.

Applicants found that various portions of the PR0302. PR0303, PR0304, PR0307 and PR0343ypolypeptider. 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 newly identified protease proteins.

80 41. Full-Length PRo328 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PFRO328. In particular, Applicants have identified and isolated cEA encoding a PR0328 polypeptide, as disclosed in further detail in the EKanples below. Using BLAST and FastA sequence aligment carlputer programs, Applicants found that various portions of the PR0328 polypeptide have significant harmlogy with the humran glioblastana protein ("GLIP).

Further, Applicants found that various portions of the PEO328 polypeptide have significant harology with the cysteine rich secretory protein ("CRISP) as identified by BLASr harology (ECRISP3_1, S68683, and CRS3_HUAN]j. Accordingly, it is presently believed that PR0328 polypeptide disclosed in the present application is a newly identified nanber of the GLIP or CRISP families and possesses transcriptional regulatory activity typical of the GLIP or CRISP families.

42. Full-rangth P335, PO331 and P1RO326 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present .i 20 application as PRO335, PRO331 or PR0326. In particular, Applicants have identified and isolated cEIA encoding a PRO335, PR0331 or PR0326 polypeptide, as disclosed in further detail in the EKanples below. Using BLASP and FastA sequence aligrment canputer programs, Applicants found that various portions of the PRO)335, PRO331 or PRO326 polypeptide have significant hcmology with LIG-1, AS and in the case of PRO331, additionally, decorin. Accordingly, it is presently believed that the PRO335, PRO331 and PRO326 polypeptides disclosed in the present application are newly identified marbers 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 PE332 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 cZA encoding PR0332 polypeptides, as disclosed in further detail in the Ekarrples below. Using BLAST and FastA sequence alignment caiputer programs, Applicants found that a full-length native sequence PR0332 (shon in Figure 108 and SED ID NO:309) has about 30-40% amino acid sequence 80a identity with a series of known proteoglycan sequences, including, for eanpie, fibra~alin and fibrmoudulin precursor seuences of various species (F14)O BUIN. FM)DCHICK, H)DJWAr, FIMUJISE, RVDMN N, PJU6773), ostecrdulin sequences (AB00011C1, AB007848_1), decorin sequences (CR183141_1, 0CU03394_1 PR42266, P..R42267, P.R42260, PR89439), keratan sulfate proteoglycans (BIU483601, AF0228901), corneal proteoglycan (AF022256_1), and bone/cartilage proteoglycans and proteoglycane precursors (FGSLBOBVINE, EGS2_F= MSE, E,3S2_KIN).

Accordingly, it is presently believed that PfRO332 disclosed in the present application is a new proteoglycan-type iolecule, and may play a role in regulating extracellular mratrix, -cartilage, and/or bane function.

44. Full-ength PR0334 Polypetldes The present invention provides newly identified and isolated nuCleotidc 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 the PR0334 polypeptdec have significant homology with fibulin and fibuihtin. Accordingly, it is presently believed that PR0334 polypcptidc disclosed in tbn present application is a newly identified member of the epidemal growth factor family and possesses properties and activities typical of this family.

Full~length PRO346 PolvppntfdCS The present invention provides newly identified and isolated nocleotde sequences encoding polypeptides referred to in the present application as PR0346. In particular, Applicants have identified and isolated cDNA encoding a PR0346 polypepide. as disclosed In futher detail in the Examples below. Using BLAST and FastA sequenice alignmint computer programs. Applicants found that a fuli-length native sequence PR0346 (shown in Figure 112 and SEQ ID NO:320) has 28 amino acid sequence identity with carcinoemiJryonic antigen. Accordingly, it e.15 is presently believed that PR0346 disclosed in the present application is a newly identified memnber of the carcinoembzyonic protein family and may be expressed in association with neoplastic tissue disorders.

46. Full-length PR0269 PolvonIdeSg :Thc present invention provides newly identified and isolaed nucleotide sequences encoding polypeptides o 20 referred to in the 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 alignmnent computer programns, Applicants found that portions of the PR0268 polypeptide have significant 5..homology with die various protein disulfide isomerase proteins. Accordingly, it is presently believed that PR0268 polypeptide disclosed in the present application is a hotnolog of the protein disulfide isomnerase p5 protein.

So&:47. Full-length PRO330 Poivnetides 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 die niurine prolyl 4-hydroxylase alpha-li subunit protein. Accordingly, it is presently believed that PR0330 polypeptdec disclosed in the present application is a novel prolyl 4-hydroxylase subunit polypeptide.

48. Full-length PR0 n PR 310 PoLyjeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the presen application as PR0339 and PRO310. In particular, Applicants have identified and isolated 82 cENA encoding a PR0339 polypeptide, as disclosed in further detail in the Exanples below. Applicants have also identified and isolated cNA encoding a PRO310 polypeptide, as disclosed in further detail in the Exanples below.

Using BLAST and FastA sequence alignment caTputer programs, Applicants found that various portions of the PR0339 and PR0310 polypeptides have significant homology with small secreted proteins from C. elegans and are distantly related to fringe. PR0339 also shows haoology to collagen-like polymers. Sequences which were used to identify PRO310, designated herein as INA40533 and INA42267, also show harology to proteins frnm C. elegans.

Accordingly, it is presently believed that the PRO339 and PRO310 polypeptides disclosed in the present application are newly identified member of the family of proteins involved in development, and which may have regulatory abilities similar to the capability of fringe to regulate S. serrate.

49. Full-Length PR244 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding C-type lectins referred to in the present application as PRO244. 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 carputer programs, Applicants found that a full-length native sequence PRO244 (shown in Figure 122 and SEQ ID NO:376) has 43% amino acid sequence identity with the hepatic lectin gallus gallus (LEXBH-CICK), 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 immune function, apoptosis, or in the pathogenesis of atherosclerosis. In addition, PRO244 may be useful in identifying tumor-associated epitopes.

PRO Polypeptide Variants In addition to the full-length native sequence PRO polypeptides described herein, it is contenplated that PRO polypeptide variants can be prepared. PRO polypeptide variants can be prepared by introducing appropriate nucleotide changes into the PRO polypeptide MA, or by synthesis of the desired PRO polypeptide. Those skilled in the art will appreciate that amino acid changes nay alter post-translational processes of the PRO polypeptide, such as changing the number or position of glycosylation sites or altering the manbrane 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 way be a substitution, deletion or insertion of one or more codons encoding the PRO polypeptide that results in a change in the amino acid sequence of the PRO polypeptide as compared 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 more of the dcains of the PRO polypeptide. jGuidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by ccuparing the sequence of the PRO polypeptide with that of homologous known protein molecules and minimizing the numiter of amino acid sequence changes made in regions of high homology. 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 S" with a serine, i.e., oeoeoo° conservative amino adid replacements. Insertions or deletions mnay optionally be in the range of I to 5 amino acids.

The variation allowed ay be determined by systermatialy nuking insertions. deletions or'substitutions of amino acids in the sequence and testing t e resulting variants for activity in the in vitro assay described in the Examples below.

The variations can be made using methds known in the art such as oligonuct ode-niediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., 8izcl. A gi-, Rs fl14331 (1986), Zollcr ct al., iNcl- Aids Fes .IQ:6487 (1987], cassette mucagenesis [Wells et al.. fi= 34:315 (1985)]. restriction selection nurtagencsis [Wells et al.. Pliilos- Tans. R. Soc. Lodon SerA, MI:1 (1986)] or other 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 seqene. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids inle alanne, glycirie, 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. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton. Jbc Protmiu. Freeman Co., Chothia, L.

MoL. iol-,I50:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

S1. Modifications or-PRO Poloeptides Covalent niodifications of PRO polypeptides are included within the scope of this invention. One type of 20 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. Derivatiztion with bifunctional agents is usefu, for instance. for crosslinking a PRO polypeptide to a water-insohuble support miatrix or suface for use in the method for purifying anti-PRO potypeptide antibodies, and vice-versa. Commonly used crosslinking agents include, I ,l-bis(diazoacetyl)-2-phenylcthan, glutaraldehyde.

N-bydroxysuccirnmide esters, for example. esters with 4-azidosalicylic acid, homobifiinctional irnidoesters. including disuecinimidyl esters such as 3 -dithiobis(succinimzdylpropionate), bifunetional maleimides such as his-Nsalcimido-l ,8-octane and agents such as med yl-3-[j aidophenyP~dthio]propioinidatc.

Other modifications include deanidation of glutaminyl and asparaginyl residues to the corresponding ghmcmyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains IT.E.

Creighton, proteins- Structure and Moleculal PoeaM;ies, WI!. Freeman Co.. San Francisco, pp. 79-86 (1983)].

acctylation of the N-terminal amine, and amidation 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 glycosylation 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 amino acid sequence. The alteration may be made, for example. by the addition of, or substitution by. one or more scrine or throninc residues to the native sequence PRO polyepide (for 0-linked glycosylation sites). The PRO polypeptidc ammD acid sequenc may optionally be altered through changes at the DNA level. particularly by mutatin the DNA encoding the PRO polpetd& at preselected bases such that codons are generated that will translate into the desired amino acids. t Another means of increasing the numnber of carbohydrate moieties on the PRO polypephide polypeptide is by chemical or enzymatic coupling of glycosides to the polypepide. Such methods are described in the an, in WO 87/05330 published II September 1987. and in Aplin and Wriston. CRC Crit. Rev- Biacbern., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or en zymatically or by mutational substiturtion of codons encoding for amino acid residues that serve as- targets for glycosylasoa Ciemnical deglcoylatim techiniques are known in the art and described, for imtance byllakimuddin.

.et al.. Arch. Siochem- inhys- W:52 (1987) and by Edge et al., Anl-..Djchem-. 111:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides. can be achieved by the use of a variety of endo- and exoglycosidases as described by Thotakura et al.. Meth. Et~MoL, 1:350 (1987).

Another type of covalent modification of PRO polypptides of the invention comprises liniking the PRO polypeptide to one of a variety of nonproteiniaceous 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.

*20 IlcPOplppie ftepeetivninmyalob oiidi a ofr hmrcmlcl comprising a PRO polypeptide fused to another, heterologous polypeptide or amino acid sequence. In onc embodiment, such a chimeric molecule comprises-a fusion of the PRO polyetide with a tag polypeptide which :provides an epitope to which an anti-sag antibody can selectively bind. 71e epitope tag is genecrlly placed at the amino- or carboxyl- terminus of the PRO polypeptide. The presence of such epitope-tagged forms of the PRO polypepdec can be detected using an araWdy against the rag polypeptide. Also. provision of the epitope tag enables the PRO polypeptide to be readily purilled by affinity purificatiort using an anti-Wa antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the ehim ric molecule may comprise a fusion of the PRO polypepdec with an inmmoglobulia or a particuilar region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgO molecule.

Various tag polypeptides and their respective antibodies are well known in the art. Examples include polyhistidine (poly-Is) or poly-blstidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CAS (Field et al.. Mot, Cell. fiol.. 8-2159-2165 (1988)]; t c-myc tag and the 8179. 3C7, 6E10, G4. B7 and 91110 antibodies therto [Evan et Wl., Molecular and Cellular Biology, 1:3610-3616 (1985)]; and the Herpes Simplex. virus glycoprotein D (gD) tag and its antibody IPaborsky et al..,~oenEgncig 1(6):547-553 Other tag polypeptides include the Flag-peptdec [Hoppert al., Bio~echnl .6:1204-1210(1988)1; the KT3 epitope peptide [Martinct al., Science,2:92-94 (992)1; ancz-tubulinecpitope peptide: (Skinner etal., 1 l- 12LC L, 2:5163- 15166 (1991)], and the 717 gene 10 protein peptide tag (L=t-Freyernmth et al., Proc. Nail. Acad. Sci. UISA, 1:6393- 6397 (1990)1.

52. Modification of PRO317 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.

deletions from, or insenions or substitutions of. residues within the amino acid sequence shown for human PRO317 in Figure 42. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided tha the final construct possesses the desired characteristics. The amino acid changes also may alter posttranslational processes of the PRO317, such as changing the number or position of glycosylation sites. Moreover, like most mammalian genes, PR0317 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 characteristic(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 S 15 PRO317, EBAF-.1 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 selections 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 PRO317 polypeptide that are 20 preferred locations for mutagnesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Sciene, 24: 1081-1085 (1989). Here, a residue or group of target residues are identified charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably S. alanimn 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 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 med not be predetermined. For example.

o to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at St" he 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 arc variants from the Figure 42 sequence, and may represent naturally occurring alleles (which will not require manipulation of the PRO317 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. EBAF-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 PR0317 more significantly. The number of consecutive deletions will be selected so as to preserve the tertiary structure of PRO317 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 polypcptides 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 numbe 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 maturePR0317 in recombinant cell culmre, and fusion of a heterologous N-terminal signal sequence to the N-terminus of the mature PRO317 molecule facilitate the secrtion 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 TGFsuperfamily. Suitable sequences include STII or pp 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 PR0317 molecule include the fusion to the N- or C-termimns of PRO317 of immunogenic polypeptides, bacterial polypeptides such as beta-lactamase or an enzyme encoded by the E.

coli tp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobulin 20 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 i. residue in the PR0317 molecule removed and a different residue inserted in its place. The sites of greatest interest for substituonal mutagenesis include sites identified as the active site(s) of PRO317 and sites where the amino acids fund in the known analogues are substantially different in terms of side-chain bulk, charge, or hydrophobicity, but where there is also a high degree of sequence identity at the selected site within various animal PR0317 species, or wh er e 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-1 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 arc 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, denominatcd exemnplary substitutions in Table 1. or as further described below in referece to amino acid classes, are introduced and the products screened.

TahbcI Original Ala (A) Arg (R) Asn (M Asp (D) Cys (C) Gin (Q) Gfu (E) Gly G) His (to le (1) Exemplary Substitutions Preferred Substitutions a LcU (L) Val; ICU; ile Ins gin; asn gin; his; lys; arg ser asn asp pro; ala asn; gin; lys; arg leu; val; mnet; ala. phe; norleucine norleucine; ile; val;.

flet; ala; phe arg; gin; asn 1w; phe; Hle 1wu; val; ile; ala; tyr ala thr ter tyr; phe trp; phie: thr; ser ile; 1wu; met; phe; ala; norleucine Lys (K) Met (M) Phe (F) Pro (P) Ser (S) Thu, CD Trp(W Tyr M Val MV Substantial modifications in function or immnitological identity of the PRO317 are accomplished by selecting substitutions that differ sitgnfeai in their effect on maintaining the structure of the potypeptidi backbone in the area of the substitution, for examplc, as a sheet or helical conformation. the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain. Naturally occurring residues arc divided into groups based on conmon side-chain properties: hydrophobic: norleucine, met, ala, val. l1w. ile; neutral hydrophilic: cys, ser, thr; acidic: asp. gin;.

basic: asn, gIn, his, b's. arg; residues that influence chain orientation: gly, pro; and aromatic: trp, tyr, pin.

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

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

In one cmbodizn=n of the invention, it is desirable to inactivate one or more protease cleavage sites that arc present in the molecule. 7hese sites arc identified by inspection of the encoded amino acid sequence, in the case of ftypsiA eg.. for an arginyt or lysinyl residue. When protease cleavage sites are identified. they are rendcred inactive to proteolytic cleavage by substituing the targeted residue with another residue, preferably a basic residue such as glutamine or a hydrophilic residue such as sermne; by deleting the residue. or by insenung a prolyl residue immediately after the residue.

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

Any cystcine residues not involved in maintaining the proper conformation of PR0317 also may be substituted, generally with serine. to iniprove the oiddative stability of the molecule and prevent aberrant crosstinking.

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 ntatural source (in the case of naturally occurring amino acid sequence variants) or preparation by otigonuclotde-mediated (or site-directed) utagcnesis, PCR niutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of PRO317.

Oligonucleotide-mediated nmtngenecsis is a preferred method for preparing substitution, deletion, and insertion variants of PRO317 DNA. This technique is well known in the art as described by Adelman et aI.. PHA.

2: 193(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 PROW 1. After hybridization, a DNA polymecrase 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 corapletely complemenmary to the template on either side of the nucleotide(s) coding for the muttion. This ensures that the oligonucleotidc will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et aL. Proc- NaI. Acad. Sci. SA. 7J: 5765 (1978).

The DNA template can be generated by those vectors that are either derived from bacteriophage M13 vectors (the comnmercially available Ml3mpl8 arnd Ml3mpl9 vectors are suitable), or those vectors that contain a singie-stranded phage origin of replication as described by Viera at at. M~et nrL, M. 3 (1987). Thus, the DNA that is to be mutated may be in~serted into one of these vectors to generate sin~gle-stranded template. Production of the single-stranded template is described in Sections 4.21-4.41 of Sambrook e aL. ua: Alternatively. single-stranided DNA template may be generated by denaturing double-stranded plasniid (or other) DNA using standard techniques.

For alteration of the native DNA sequence (to generate amino acid sequence variants, for eXamnple), the oligonucleodde is hybridized to the single-stranded template under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of DNA polymerase 1, is tn added to synthesize the complementary strand of the template using the oligonucleotide 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 PRO317. 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 oligonucleotide 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 an appropriate host.

The method described immediately above may be modified such that a homoduplcx 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 deoxyribonuleotides, dcoxyriboadenosine (dATP). deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP).

is combined with a modified thio-doxyribocytosine called dCTP-(aS) (which can be obtained from the Amersham Corporation). This mixture is added to the template-oligonuceotidc 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 endonucleasc digestion.

15 After the template strand of the double-stranded bcteroduplex is nicked with an appropriate restriction aenyme, the template strand can be digested with Exoll nuclease or another appropriate nuclease past the region that contains the site(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 presene of all four dcoxyribonucleotide triphosphates. ATP, and DNA ligase. This homoduplex molecule can then 20 be transformed into a suitable host cell such as E. coi JM 101. 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 cose together in the polypeptide chain, they may be mutated sinultaneously 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 acids), it is more difficult 1o 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 oligonucleotdes 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 fist round is as described for the single mtants: wild-type DNA is used for the template, an oligonucleotide 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 amnino acid variants of PRO3 17. While the following discussion refers to DNA. it is understood that the techaique also finds application with RNA. The PCI technique generally refers to the following procedure (see Erlich. &IR-Tm2Iolgy, (Stockton Press. NY. 1989). the chapter by R. Higuchi. p. 61-70): When small amounts of template DNA are used as starting material ina PCi, 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 bragment that differs from the template sequence only at the po,#tions where the prnrs dife fromn the template. For introduction 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 plasmoid, 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 mtcleotides 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 using 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 et al., Gene, JA: 315 (1985). The starting material is the plasmid (or other vector) comprising the PR0317 DNA to be ma ae. The codon(s) in the PR0317 DNA to be mnutated are identified. There must be a unique restriction endonuclease site on each side of the identified mtation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-tnediated mutagenesis method to introduce them at appropriate locations in t PRO317 DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-strdd oligonucleotide 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 synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide referred toas thecassette. This cassette is designed to have 3'and 5'ends that are comnpatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated PR0317 DNA sequence.

Covalent modifications of PR0317 ame also included within die 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- termini' residues of the PRO3 17. Derivatization with bifunactional agents is useful, for instance, for crosslinking PR03 17 to a water-insoluble support matrix or surface for use in the method for purifying anti-PR0317 antibodies, and vice-versa. Commonly used crosslinking agents incluh 1.1-bis(diazoacetyl)-2-phenylethane. glutaraldehyde. N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid. homobifunctional inidoesters. including disuccinimidyl esters such as 3y3-dithiobis- (succininnidylpropionate), bifunctional maleiniides such as bis-N-maleimido-l .1-octane, and agents such as niethyl-3- ((p-azidophenyl~dtho)propioimidate.

Other modifications include deamidation of ghusaminyl and asparaginyl residues to ihe corresponding glutantyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxYl groups of seryl or threonyl residues, methylation of the '-amxino groups of lysine. arginine. and histidine side chains (T.E.

Creigltton. Proteins- Structure and Molecular Proprties, W.H. Freeman Co.. San FrAncisco, pp. 79-86 (1983)).

acetyladion of' dhe N-termintal amine, and amidation of any C-terminal carboxcyl group.

Another type of covalent modification of the PR0317 polypepdde included within the scope of this invention comprises alterig the native glycosylation pattern of the polypeptide. 'Altritig dic native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native-sequence PRO polypeptide. and/or adding one or more glycosylation sites that are not present in the native-sequence PRO polypeptide. The deduced amino acid sequence of PR0317 shown in Figure 42 (SEQ ID NO: 114) has one predicted N-linked glycosylation site at residue 160.

Addition of glywosylaiion sites to the PR0317 polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the niative-sequence PR0317 (for 0-linked glycosylation sites). The PR0317 amino acid sequenc may optionally be altered through changes at the DNA evel. particularly by mutating the DNA encoding the PR0317 polypeptde at preselected bases such that codons are generated that will tranislate into the desired amino .acids.

Another means of increasing the number of carbohydrate moieties on the PR0317 polypeptd& is by chemical -or enzymatic coupling of glycosides to the polypepitide. Such methods are described i te art, in WO 97/05330 published 11 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 enzymnatically or by mutational substitution oif codons encoding amino acid residues that serve as targets for gbwcsylauion. Chemical deglycosylation ichlnkque ame known in the art and described, for instance, by Hairniddin, ei al., Arch, Biochem. Biophvs.. M:5.2 (1987) and by Edge er al.. Anal. Biohm, LLB: 131 (198 Enzymatic *~.cleavage of carbohydrate moieties on polypeprides can be achieved by the use of a variety of endo- and exoglycosidases as described by Thotakur at al., Meth..Enzynxl.. flS: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 inivention may also be modified in a way to form a chimeric molecule comprising PR0317 fuised to another. heterologous polypeptide or amino acid sequence. In one embodiment, such a chiimeric molecule comprises a fusion of the PR0317 with a tag polypeptide which provides an epitope to which an anti-tag atiibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of the PR0317.

The presence of such epitope-tagged forms 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 cimreric molecule may comprise a fusion of the PR10317 with an immunoglobulin or a particular region of an imnmnoglobulin. For a bivaleix form of the chimeric molecule, such a fusion could be to tie Fc region of an IgG molecule.

Various tag polypeptides and their respective antibodies are well known in the art. Examples include polyhistidine (poly-his) or poly-histidine-glyciie (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CM5 (Field a al., Mol.Ccl-iol 3:2159-2165 (1988)): the c-myc tag and the SF9. 3C7. 61110. G4. B7, and 9E10 antbodies thereto (Evan er al., Molecular and Cellular BioloZv, 5-3610-3616 (1985)); ad the Herpes Simplex virus glycoprocin D (ID) tag and its antibody (Pabotaky eral.. Prti Egncit 2(6):547-553 (1990)). Other tag polypeptides include the Flag-pepdde (Hopp et al.. Bipffechnolory A: 1204-1210 (1988)); the K73 epitope peptide (Martin e al. Sigi. 25:192-194 (1992)); an *-tubulin epitope peptdec (Skinner et al., J.Bigl-Chiem 2f:15 163- 15166 (1991)); and the Ti gene 10 protein peptd& tag (Lmz-Frcycrmuth er al.. Proc. adl. Acad-Sci- USA- 8:6393- 6397 (1990)).

53. Prearation of PRO Polvueptides Thec description below relates primarily to production of PRO polypeptides by culturing cells transformed or transfacted with a vector cmontann the desired PRO polypeptide nucleic acid. It is. of course, contemplated ta= alternative methods, which are well known in the art, may be employed to prepare the PRO polypeptide. For ~instance, the PRO polypeptde secqes. or portions thereof, may be produced by direct peptide synthesis using solidphase techniques [see. Stewart ar al.. Solid-Pbase PEnride S3nthesis, W.H. Freeman Co.. San Francisco. CA :5(1969); Merrifield, J- Am. Chem, Soc. U :2149-2154 (1963)). In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis snay be accomplished, for instance, using an Applied Binosysterns Pcpride Synthesizer (Faster City, CA) using manufacturer's insructions. Various portons of the desired PRO polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the ful-ength PRO polypeptide.

A. Isolation or DNA Engadine PRO Polypsntides a DNA encoding PRO polypeptides may be obtained from a cDNA library prepared from tissue believed to possess the desired PRO polypeptdec rnRNA and to express it at a detectable level. Accordingly, humnan PRO polypetde DNA can be converienitly obtained from a cDNA library prepared from humnan tissue, such as described in the Examples. The PRO polypeptide-encoding gene may also be obtained from a genomic library or by oligonucicotide synthesis.

libraies can be screened with probes (such as antibodies to the desired PRO polypeptide or oligonueceotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning- A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press.

1989). An alternative means to isolate the gene encoding the desired PRO polypeptide is to use PCR methodology (Sambrook et al.. Dieffenbach et al., PCR Pritner:A Laborator MAaa (Cold Spring Harbor L.aboratory Press, 1995)].

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

biotinyladi or enzyme labeling. Hybridization conditions, including mnoderate stringency and high stringency, are provided in Sambrook et al., V=p.

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

Sequence iatity (at either the amino adid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programns such as BLAST. ALIGN. DNAstr. and INHERIT which employ various algorithm to measure homology.

Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genornic libraries using the deduiced amino acid sequence disclosed herein for the first time, and, if necessary, using' conventional primer extension procedures as described in Sambrook et al.. immp, to detect precursors and processing intermediates of znRNA that may not have been reverse-trnscribed into cDNA.

B. Selection and Transformation of Host Cells *Host cells art uzansfectod or trantsformed with expression or cloning vectors described herein for PRO **polypeptide production and cultured in conventional nutrient media modified as appropriate for Inducing protnoters, selecting transforrnants. or amnplify'ing the genes encoding the desired sequences. The culture conditions, such as media, temperature. pH andl the lime can be selected by the skilled artisan without undue experimentation. In gencral, principles, protocols, and practical techniques for maximizin the productivity of cell cultures can be found in Mammalian -Cell -Biotcclmologv* a Pratical Aporoach, M. Butler, ed. (IRL Press, 1991) and Sambrook ct al..

Methods of transfection 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.

e T1he calcium treatment employing calcium chloride, as described in Sambrook et al., L=.a or electroporation is generally used for prokaryocs or othe cells that contain substantial cell-wall barriers. Infection with Agro bacterium nnqmsefaio Is used for transformnation of certain plant cells, as described by Shaw et al., G=,c 2.1315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calciurn phosphate precipitation method of Graham and van der Eb. irology. 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transformations have been described in U.S. Patent No. 4.399.216. Transforrmations C ~~into yeast are typicallyarried ct aording to the method of Van Solingen et al., J. Bact., 212:946 (1977) and Hsiao.

et al.. Prac. Nail. Acad Sci. (USA), 263829 (1979. However, other methods for introducing DNA into cells, such as by nuclear nucroinjection, electroporation, bacterial1 protoplast fusion with intact cells, or polycations. e.g..

polybrenc. polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al, Methods in EravmologX JU527-537 (199) and Mansour et al.. kutgA 2:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in (he vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include hut are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriacze such as E. co/i. Various E. coil strains are publicly available, such as E. co/iK12 strain MM29 (ATCC 31.446); E. coli X1776 (ATCC 31,537); E. co/i strain W31 (ATCC 27.325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacceriaceac such as Eic4Neridzia. E. co/i, Emuervbacrer, Erwinia, KiebsielLa, Proteus, Saknonella, SaL'nonella ryphimurium, Serratia, Serrataa mxrcant.j and Shigeia, as well as Bacili such as B. subtfi and B. lfclaeufonnis B.

licheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa. and Streptonycas. Various E. coil strains are publicly available, sruch as E. coil K12 strain MM294 (ATCC 31.446); E.

coil X 1776 (ATCC 31,537); E. coil strain W31 10 (ATCC 27.325); and K5 772 (ATCC 53.635). These examples are i~luswr4ve rather than limiting. Swain W3 110 is one particularly preferred host or parent. host because it is a common host strain (or recombioant DNA product fermentations. Preferably, the host Cell secretes miinimal -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. coiL W31 10 strain IA2, which has the complete genotype tond E. coil W31 10 strain 9134. which has the complete genotype tonA pr; E. co&l W31 10 strain 27C7 (ATCC 55.244), which has the complete genotype boAL ptr3 p/WA EI5 (argF-lac)169 degP anpTkn' E. colf W3110 strain 37D6. which has thc complete genotype toad4 prr~pzoA EI5 (argF-Ioac)169 degP cnpT Ibs7ilvG kad; E. coil W31l10 strain 40B4, wbidi is strain 37D6 with a non-kanarnycin resistant degP deletion mutation; and an E. coii strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in Wry methods of cloning, PCR or other nucleic acid polymerase reactions, arc suitable.

In addition topoyoe, eukaryotic microbes such as filanientous fungi or yeast arc suitable cloning or expression hosts for PRO polypeptide-encoding vectors. Sacczaromyces cerevlslae is a cormmonly used lower ukaryotic host microorganism. Others include Schizosaceharomyces pombe (Beach and Nurse. Naur. 22Q: 140 (19811: EP 139,383 published 2 May 1985); Khtyvromycs hosts Patent No. 4,943,W2; Fleer et aL.

Biot~lrobiooy. 2: 968-975 (1991)) such as. K. kais (MW9S-8C, CBS693. CBS4574; Louvcourt raL. L Batril 737 [1983D. K. frugilis (ATCC 12,424), K. buigaricusr (ATCC 16,045). K. Wckeramui (ATCC 24.178), K. wuld (ATCC 56,500). K. drosop/ularum (ATCC 36,906;. Van den Berg dt at.. BiO/Technolory, 1: 135 (1990)).

K. daennotolerans. and K. mariiwm yarrod (EP 402.226), Pichiapasbons (EP 183,070. Sreekrishna et aL, L.

Basic Microil.. 21:265-278 1198]; Candda- Thchdenna reeuia (EP 244.234); Neurospora crassa (Case et aL., Proc. Natl- Acad- Sci- USA, 26: 5259-5263 (19179j); Schaanomyces such as Sdhwamiomyces occidenvalis (EP 394,538 published 31 October 1990); and filamentous fungi such as. Neurosrpora. Pemcilliwn. Tolypocladiwn (WO 91/00357 published 10 January 1991), and Asperg!Ilu hosts such as A. nIdulas (Ballance er al.. Biochern.

Biophys Res, ConInm, in-: 284-289([19831; T'ilburn et al.. 205-221 (1983]; Yelton et al., Emo...Nal *Acad. Sc* USA. RI: 1470-1474 11984D and A. niger (Kelly and Hynes. EMBO L, 4: 475-479 [1995D.

Methylotropic yeasts are suitable herein and include. but are not limited to. yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera. Pichia, Saccharozyce. Tondlopsis. and Rizodotondla. A list of specific speciet that are exemplary of this class of yeasts may be found in C. Anthony. 2L Biochemistry of Methylotrotnhs, 269 (1982).

Suitable host cells for the expression of glycosylatcd PRO polypeptides arc derived from multicelutlar organisms. Examples of invertebrate cells include insect ceils such as Drosophila S2 and Spodoptera as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COt cells.

More specific exmnples include monkey kidney CVl line transformed by SV40 (COS-7. ATCC CRL 1651): humnan embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al.. LjlG.YiRoL.

36"59 (1977)); Chinese hamster ovary cels/-DHFR (CHO. Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 22:4216 (1980)); mouse sctoli cells (M4. Mather. Biol. Repmd 2:243-251 (1980)); human lung cells (W138, ATCC CCL human liver cells (Hep 02, HB 8065); and mouse mammary umior (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.

Te 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 endonuclase 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 polypeptide of interest may be produced recombinantly not only directly, but also as a fusion polypepide with a hcterologous polypeptidc, 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 component 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.

S penicillinase, lpp, or heat-stable enterotoxin H leaders. For yeast secretion the signal sequence may be, the yeast invcrtase leader, alpha factor leader (including Saccharomyces and Kluyveronyces a-factor leaders, the latter described in U.S. Patent No. 5,010.182). or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In .i nmammalian cel expression. mamnalian signal sequenes may be used to direct secretion of the protein, such as signal sequences from secreted polypcptdes of the same or related species, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are wel 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 2 plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma. adcnoviius, 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 thymidinc kinase. 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. Scl. USA. 72:4216 (1980). A suitable selection gene for use in yeast Is the UPI gene pree in the yeast plasmid yRp7 (StincCtb ct al.. Nature. 2JZ:39 (1979); Kingmaet al.. &M 7:141 (19179); TschemPer cial., Q&i. IQ:157 (1980)1. Theatpi gene provides a selection mrarker for a muztant strain of yeast tacking the ability to grow in uyptphuf. for example, ATCC No. 44076 or PEP4- I (Jones, Genetcs, Al 2 (1977)].

Expreskon and cloning vectors usually contain a promoter operably linked to the PRO polypeptide nucleic: aci sapien to direct reRNA synthesis. Promoters recognized by a variety of potential host cells arewcU known.

Promoters suitable for use with prokaryotic hosts include the P-lactainas and lactose promoter systemns (Chang et al., N~aure. ZU-.6l5 (1978); Goeddel ct al.. Naurm, ZU.:544 (1979)). alkaline phosphatase. a tryptophan (urp) promoter system (Goeddel. Nujcic Acis Res. 1:4057 (1980); EP 36,7761, and hybrid promoters such as the tac promoter (deBocr ct al.. Proc- Nail Aad. Si. ISA, 10:21-25 (1983)1. Promoters for use in bacterial systems also [0 will contain a Shinc-Dalgarno sequence operably linked to the DNA encoding dhe desired PRO polypeptide.

*Examples of suitable promoting sequences for use with yeast hosts include the p romoters for 3mhshgyeae kinase (Hitznan et al..,I L CQb=. 2M-2073 (1980) or other glycolynic enzymes [Hess ct al..

Adv. Enmem Re: .:149 (1968); Holland, BjQ~bCMiLstl. 12:4900 (1979)). such as eniolase. glyceraldehyde-3phosphate dehydrogenase, hexokiome. pywaat decaboxytase. phosphoftuctoklaie. glucose.6-phosphate isomerase.

3-phosphoglyaculmse, pynvat Wiase. niosephos mt isomeae .hosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of trancription controlled by growth conditions. are the promoter regions for alcohol dehydrogenase 2, isocytochromie C. acid 0hosphatase, degradative enzymes associated with nitrogen mnetabolism metallodnionein. glycedyd-3phosphat and enzymes responsible for maltose anid galactose utilization. Suitable vectors and p romoters for use in yeast expression are further described in EP 73,657.

PRO polypeptide transcription from vectors in mamnmalian host cells is controlled, for example. by promoters obtained from the genomes of viruses such as polyona vlis. fowlpox virus (UK 2.211,504 published July 1989). adenovirus (such as Adenovirus bovine papiloma virus, avian sarcoma virus, cytomnegaloviflis. a ****retrovirus. hepatitis-B virus and Simian Virus 40 (SV40). from heterologous mnammalian promoters. the actin promoter or an immunoglabulin promoter. and from heat-shock promoters. provided such promoters are compatible with the host cell systems.

Ttrnscription of a DNA encoding the desired PRO potypeptide by higher eukaryoles may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-actiflg elements of DNA. usually about from to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, nc-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukasyotic cell vinis. Examples include the SV40 enhancr on the late side of the replication origin (bp 100270). the cytcnncgloviI's early promoter enhancer, the polyoma enhancer on the late si de 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 seqluence, but is preferably located at a site 5' from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animnal, humnan, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription anid for stabilizing the mRNA. Such sequa=C are coninanly available from the 5' and, occasionally untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenyLared fragments in the untranslated portion of the mRNA encoding PRO polypepddes.

Still other zncthods, vectors, and host cells suitable for adaptation to the synthesis of PRO polypeptides in recombinant vertebrate cell culture are described in Gething et al., Nature, 22:620-625 (1981); Mantej et al., Nature, 2&1:40-46 (1979); EP 117.060; and EP 117,058.

D. Detecting Gent AmpificationfMrnression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northernblouing to quantitate die transcription of mRNA Vlhomas, Proc. NatI Acad- Sci, USA, 22:5201-5205 (1980)). dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on t sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes. including DNA duplexes, RNA duiplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and die assay may be carried out where the duplex is bound to a surfitce, so that upon die formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Gene expression, almmratively, may be measured by immunological methods, such as imnmunohistocheniical 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* staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO polypeptde or against a synthetic peptde 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 of Polypetide Forms of PRO polypeptides3 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 means, such as freeze-thaw cycling. sonication, mechanical disruption, or celi lysing agents.

It may be desired to purify, PRO polypetie from recombinant cell proteins or polypeptdecs. The foMowing procdan-es are exemplary of suitable purification procedures: by fractionation on an ion-exchange columa; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exhange resin such as DEAE; chromatofocusing; SDS-PAOE; anmonium, sulfate precipitation; gel filtration using, for example, Sephadex protein A Sepharose columns to remove contaminants such as IgG; and metal chelatin columins to bind epitopetagged forms of Mie PRO polypeptide. Various methods of protein purification may be employed and such methods are known in the ant andI described for example in Deutscher, Methods in Emmlogy, 1B (19M). Scopes, Protein Purification: Princiles and Practice, Springer-Verlag, New York (1982). The purification step(s) -selected will depend, for example, en the nature of the production process used and the particular PRO polypeptidc produced.

S4. Uses for PRO Poirptides Nucleodde sequnce (or their complement) encodirig the PRO polypeptides Of the Present invention have various applications in the art of mnolecular biology. including uses as hybridization probes, in chromosome and gene mapping and in die generation of anti-seuse RNA and DNA. PRO polypeptide-cncoding nucleic acid will also be useful for thc preparation of PRO polypeptides by dhe recombinant techniques described herein.

The full-length native sequence PRO polypeptide-encoding nucleic acid or portions theroof, may be wedA as hybridization probes for a cDNA library to isolate the ful-lngth PRO polypeptdec gene or to isolate still other genes (for instance, those encoding naturally-occurring variants of the PRO polypeptdec or PRO polypeptides from other species) which have a desired sequence identity to the PRO polypeptide sucleic acid sequences. Optionally.

the length ofthe pobes wlbabout 2 to at50ses. 'Mehybridization probes may be derived from the nucleodd sequece of any of the DNA molecules disclosed herein or from genomic sequences including promoters, enthancer elermnt and introns of native sequee PRO polypeptide encoding DNA. By way of example. a screening e@:e *method will comprise isolating the coding region of die PRO polypeptide gene using the known DNA sequence to 0 synthesize a sedected probe of abourt 40 bases. Hybridization probes may be labeled by a variety of labels, inchling ".:00radionuceotides such as or"S S. or enzymatic labels such as alkaline phosphatase coupled to the probe via 0 15 avidintbjotin coupling systems. Labeled probes having a sequence complementary to that of the specific PRO ::09polypeptie gene of he present invention can be used to screen libraries of human eDNA, 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 5:20 disclosed herein.

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 0 *0 mapping the gene which encodes that PRO polypeptide and for the genetic analysis of individuals with genetic disorders. The nucleotide sequeCes Cprovided 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 0*000:markers, and hybridization screening with libraries.

0..0 sea* 0The PRO polypeptidc can be used in assays to identify its ligands. Similarly, inhibitors of the receptr/ligand binding inteation 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 compotunds 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. malking them particularly suitable for identify~ing small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. Mwe assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays. lmmimoassays and cell based assays.

which are well characterized in the art.

Nucleic acids which encode a PRO polypcptide or is modified forms ca also be used to generate either tazsgenic animals or *knock out* animals which, in barn. are useful in the developmeni and screening of therapcutically useful reagents. A trasgenic animal a mouse or rat) is an arnial having cells that contain a transgene. which transgece was introduced into dhe aknimal or an ancestor of the animal at a prenatal, an embryonic stage. A trnsgene is a DNA which is integrated into the genome ot a cell from which a tranagenic animal develops. In one embodiment. cDNA ecoding a PRO polypeptide of interest can be used to clone genomic, DNA ecowding the PRO polypeptide in accordanc with established techniques and the genomic sequences used to generate transgenic animnals that contain cells which express DNA encoding the PRO polypeptade. 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 tranisgene incorporation with tissue-pecific; enhancers. Traingenic animals that inctude a copy of a transgene encoding a PRO polypeptide introduced into the germ line of the animal at an embryonic stage can be used exminez the effec of increased expression of DNA encoding die PRO polypeptide. Such an1imals Can be Used as tester an-Ias for reagents thought go confer protection from, for example, pathological conditions associatd with its overexpression. In accordance with this fact of the invention, an animal is treated with the reagent and a reduced 15 incidence of the pathological condition. compared to untrated animals bearing the tmansgenc, 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 Imock out* animal which has a defective or altered gene encoding the PRO polypqtidc of interest as a result of homologous recombination between the endogenous gene encoding the PRO polypeptide and altered genomic DNA 20 encoding the PRO polypeptide introduced into an embryo&i cell of the animal. For example, cDNA encoding a PRO polypeptide can be used to clone geonic DNA encoding the PRO polypeptide in accordance with established *coo 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 mnonitor integration. Typically, several kilobases of unaltered flanking DNA (both at die 5' and 3' ends) are included in the vector [see Thomas and 25 Capeccii 51.503 (1987) for a description of homologous recomnbination vectors]. The vector is introduced into an embryonic stem cell line by electroporation) and cells in which the introduced DNA has honuologously* with the endogenous DNA are selected [see Li et al., "e fi2:915 (1992)]. The selected coils are then injectd into a blastocyst of an animal a mouse or rat) to form aggregation chimeras [see Bradley.

in Tercrocaranomas and Emnbryotuc Stan Cells: A Practical Approach. E. J. Robertson, ed. Oxford, 1987); p.113-152). A chimeric embryo can then be implanted into a suitable pscudopregnant fcmale foster animal and the embryo brought to term to crate 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 or the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defiend against certain palhological conditions and for their development at pathological conditions due to absence of the PRO polypeptide.

With regard to the PRO211 and PR0217 polypeptide, therapeutic indications include disorders associated with the preservaton and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions aimuocolitis. Zollinger-Eliison syndrome. gastrointestinal uleration and congenital microvillus atrophy), skin diseases associated with abnormal kcratinocyte differentiation- psoriasis, epithelia cancers such as lung squamous cell carcinoma, epidermoid carcinoma of the vulva and giornas.

Suxme the PR0232 polypeptide and nucleic acid encodirC it possess sequence homology to a cell surface stem cell antigen arnd its encodiag 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 PR0232 polypeptde way be employed as antagonists of membrane bound PR0232 activity both in vitro andS in Wvo. PR0232 polpptides may be employed in screening assays designed to identify agonsts or antagonists of the native PR0232 polypepde., wherein such assays may take the form of any conventional cell-type or biochemical binding assay. Moreover. the PR0232 polypeptidt: may serve as a molecular marker for the tissues in which the polypeptde is specifically expressed.

With regard to the PR0187 polypeptdes disclosed herein, FOP-S has been implicated in cellular differentiation and embryogenesis, including the pattering which appears during limb formation. FGP-8 a-ad 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.

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 PR0219 polypeptides of the present invention which play a regulatory role in the blood coagulation cascade may be employed in vivo for trapeutic purposes as well as for in Wfro purposes. Those of ordinary skill in the ail will well know how to employ PR0219 polypeptides for such uses.

The PR0246 polypeptides of the present invention which serve as cell surface receptors for one or more viruses will find other uses. For example, extracellular domains derived from these PR0246 polypeptides mnay be employed therapeuticall in Wvo for lessening the effects of viral infection. Those PR0246 polypeptides which serves as tumor specific antigens may be exploited as therapeutic targets for and-tumor drugs, and the like. Those of ordinary skill in the art will well know how to employ PR0246 polypeptides for such uses.

Assays in which connective growth factor andS other growth factors are usually used should be performed with PR0261. An assay to determine whether TOP beta induces PR0261. indicating a role in cancer is performed as known in dhe art. Wound repair and tissue growth assays ame also performed with PR0261.' The results are applied accordingly.

PR0228 polypeptides should be used in assays in which EMRI, CD97 and latrophilin would be used in to determine their relative activities. T'he resift can be applied accordingly. For example. a competitive binding assay with PR0228 and CD97 can be performed with the ligand for CD97. 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 100 level. DNA474 12. the EST from which PCR oigos were generated to isolate the full length DNA49435-1219. has be= observed to map to l Ip!5. Sequence homnology to the 11p]i5 locus would indicate that PR0533 may have utility in the treatment of Usher Syndrome or Atrophia areata.

As mestioned pmeviouly. fibroblast growth factoss can act upon cells in both a mitogenic sand non-snitogenic manner. These hctors are nitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, inducing grmnaosa cells. adrenal cortical cells. chrodrocytes. myoblasts. corneal and vascular endothelia cells (bovine or human), vascular smooth muscle cells. lens. retina and prostatic epithelial cells, oligodendrocytes.

astrocytes. chrondocytes, rnyoblasts and osteoblasts.

Non-mitogenic actions of fibroblast growth factors include promotion of cell migration into a wound area (cheimotaxis). initiation of new blood vessel farnulavion (angiogenesis). modulation of nerve regeneration and survival 1 (acurotrophism). modulation of endocrine functions, and stimulation or suppresion of specific cellular protein :~in escelarcW matrix production and cell survival. Baird, A. Bohlen. Handbook of Exp. Phumcol.

W 39-48 hes prperiesprovid a basis for using fibroblast growth factors In therapeutic approaches 0 to acceleratc wound hecaling, nerve: repair, collateral blood vessel formation. and the like. For example. fibroblast .0::Olgrowth factors. have been suggested to minimize myocardium damage in heart disease and surgery (U.S.P.

4.378.437).

Since the PRO245 polypeptide and mzck acid encoding it possess sequence homology to a transmcmbrane protein tyrosine kinase protein and its encoding nucleic acid, probes based upon the PR0245 nucleotide sequence may be employed to identify other novel tansuembrane tyrosine kinase proteins. Soluble forms of the PR0245 polypeptide may be employed as antagonists of anibrazw bound PRO245 activity both in Wtfv and in Wvo. PR0245 polypeptides ay be employed in screening assays designed to identify agonists or antagonists of the native PR0245 polypeptide. wherein such assays may 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. PR0220 and PR0221 have 025 homology so SLIT and leucine rich repeat protein. Therefore, these proteins are useful in assays described in the literature, vjM, wherein the SLIT and leucine rich repeat protein are used. Regarding the SLJT protein. PR0227 can be used in an assay to determinc the affect of PR0227 on neurodegenrative disease. Aditionally, PR0227 has homology to human glycoprotein V. In the case of PR0227. this polypeptide is used in an assay to determine: its affect on bleeding, clotting, tissue repair and scarring.

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

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

PR0287 polypepsides and portions thereof which effect the activity of bone mowrphogenic Protein 'BMPllprcolLagen C-proteinase (PCP) may also be useful for in vivo therapeutic purposes, as well as for various in vitro applications. In addition. PRO28 polypeptides and portion thereof may have thcrapeutic applications in wound healing and tisse repair. Peptides having homology to procollagen C-proteinase enhancer protein and its precursor may also be used to induce bowe and/or cartilage formation and ame therefore of particular interest to the scientific and mnedical communities.- Therapeutic indications for PR0214 polypeptides iclude disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions (ecg.. ezuerocolitis, Zollinger-Ellison syndrome. gastrointestinal ulceration and congenital aticrovilius atrophy), skin diseases associated with abnormal kerauinocyte differentiation psoriasis, epithelial cancers such as lung squamnous cell carcinoma.

cpidermoid carcinoma of die vulva and gliomas.

Studies on the genieration and analysis of mice deficient in inctmbers of the TGF- superfamily are reported in Matzuk, Trends in Endocrinol. and Metabol, fi: 120-127 (1995).

The PR0317 polypeptide, as well as PR0317-specific antibodies. inhibitors, agonists, receptors, or their analogs, herein ame useful in treating PRO3 17-associatcd disorders. Hence. for example, they may be employed in modulating endometrial bleeding angiogenesis, and may also have an effect on kldney tissue. Endometrial bleeding can occur in gynecological disease such as endounetrial cancer as abnormal bleeding. Thus, the compositions herein may find use in diagnosing and treating abnormal bleeding conditions in die endometniui, as by reducing or eliminating the need for a hysterectomy. The molecules herein may also find use in angiogenesis applications such as and-tumor indications for which the antibody against vascular endothelial growth factor is used, or, conversely.

ischemic indications for which vascular endothelial growth factor is employed.

*.:Bioactive comipositions comprising PR0317 or agonists or antagonists thereof may be administered in a suitable tierapeutic dose determined 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 complexed 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, bicactive composition may be delivered by intravenow infusion into the bloodstream or any other effective means which could be used for treating problems of the lddney, uterus, endometriumn, blood vessels, or related tissue, in the hemr 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 pharmacoiutics.

See. for example, Mordetui and Rescigoo.Pa c~-a-eer 2:17-25 (199); Morenti a aL. Phrmaceutical Reerh A:1351-1359 (1991); and Mordenti and Chiappell. -'Tbe use of interspecies scaling in toxicokinetics* in Toxicokinetics and New Drug Developmnent, Yacobi ar at. (eds) (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 route of administration, and the condition of the mammal.

Accordingly, it will be necessary for the therapist to titer the dosage and modify dhe route of admainistration as required to obtain the optimal therpeutic effect. A typical daily dosage might range from about 10 ngtkg to up to 100 mg/kg of the mammals body weigh or more per day, preferably about I pg/kg~day to 10 mg/Wgday. Typically, the clinician will administer PRO3 17, PR0317 agmiist. or PR0317 antagonist, until a dosage is reached that achieves the desired effect for treatment ofthde above mentioned disorders.

PR0317 or an PR0317 agonist or PR0317 antagonist may be administered alone or in combination wit 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 earrier medium preferably at a pH of about 5 to a. more preferably 6 to 8. although the pH may vary according to the chracteristics of the PROM,17 agonist, or antagonis t being formulated and die condition to be treated. Characteristics of the treatment compounds include solubility of the molecule, half-life, and antigenicity/imrnunogenicity; these and other characteristics may aid in defining an effective carrier.

PR0317 or PR0317 agonisus or PR0317 antagonists may be delivered by known routes of administration inldn bi noiitdt topical cram an ges wasu a spa and aerosol, Wasera patch and bandage; :irnjectable. intravenous, and lavage formulations; and orally administered liquids and pills, particularly formulated to resist stomach acid and enzymcs. The particular formulation, exact dosage, and route of administration will be deemie by die atedn physician and will vary according to each specific situation.

Such determinations of administration ame made by considering multiple variables such as the condition to be treated., the type of mammal tobe reated. the compound to be administered, dteanthepiric profieof 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, timne of administration, drug combination. reaction sensitivities.

and tolerance/response to therapy. Long-acting treatmentt compound formulations (such as liposomally encapsulated PR0317 or PEGylated PR0317 or PR0317 polymeric microspheres. such as polylactic acid-based snicrospheres) might beadmninistered every 3 to 4 days, every week, or once every two weeks depending on half-lif and clearance rate of the particular treatment compound.

Normal dosage anxoitm may vary from about 10 ngikg to up to 100 mg/kg of marmal body weight or more per day, preferably about I pig/kg/day to 10 mg/kg/day, depending upon die route of admintistration: Guidance as to particular dosages and methods of delivery is provided in the literature-. see, for example, U.S. Pat. Nos.

4,657,760; 5,206-W4; or 5,.212. It is anticipated that different formulations will be effective for diffecrent treatmen compounds and different disorders, that administration targeting the uterus, for example, may necessitate delivery in a manner different from that to another organ or tissue, such as cardiac tissue.

Where sustained-release administration of PR0317 is desired in a formaulation with release characteristics suitable for the treatment of any disease or disorder requiring administration of PROM. 17,icroecapsulation of PR0317 is contemplated. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH). interferon- (rhlFN- interleukin-2, and MN rgpl120. Johnson er aat.!Mod.. 2: 795-799 (1996); Yasuda. omed her- .22: 1221-1223 (1993); Ho etrat., i/Tcholgy 755-758 (1990); Clelani, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems.* in Vaocin Desiga.TeSintadAduanAmoc Powell and Newman, eds. (Plc- Pressi: New York, 1995), pp. 439-462; WO 97/03692. WO 96/40072. WO 96/07399; and U.S Pat. No. 5,654,010.

The sustaiced-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 bioactivc agents from lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.).

Biodegradable Polymers as Dnru Delivery Systems (Marcel Dckker: 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 S cardiac tissues may precpitate 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 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 PRO317 antagonist may be administered to a mammal with another biologically active agent, either separately or in the same formulation 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-I may be administered together with an antagonist to PR0317. such as an and-PR0317 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 Figure 44 with A33_HUMAN, an A33 antigen precursor. A33 antigen precursor, as explained in the Background is a tunm-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 arc inserted into the compared sequences. 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 imown 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 infction may be accordingly conceived. As mentioned in the Background, the expression of viral receptors is often associated with neoplastic tumors.

Tberapeutic uses for the PRO234 polypeptides of the invention includes treatments associated with leukocyte homing or the interaction between Icukocytes and the endotbelium during an inflammatory response. Examples Include, asthma rheumatoid arthritis, psoriasis and amlapic sclerosis.

Since the PR0231 polypeptide and nucleic ac" encoding it possess sequne homology to a putative acid phosphatase and its encoding zmcleic acid, probes based upon the PRO23 1 nucleotide sequence way be employed to identify other novel phospbatase proteins. Soluble forms of the PRO23 1 polypepode may be employed as antagonists of mebrr bound PRO231 activity bothin vitro and in vivo. PR0231 polypepdecs may be employed in screening assays designed so identf agoaists or antagonists of the native PR0231 polypeptide, wherein suchbssays may take.

the form of any onvenrional cell-ype or biochemical binding assay. Moreoivcr. the PR0231 polypeptide may serve as a molecular marker for the tissues in which the polypetdec is specifically expressed.

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

PR0238 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 cazilfdates which block, reduce or reverse its. reducing abilities. This is done by performing side by side assays where candidates are added in one assay having PR0238 and a substrate to reduce, and not added in another assay. being the sam but for the lack of the presec of the candidate.

PR0233 polypeptdes and portions thcreof which have homology to reductase may also be useful for in vivo therapeutic purposes, as well as for various other applications. MTh identification of novel reductase proteins and 20 related mnolcutles 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 toplay important roles in a number of disease processes, the identification of new reductase proteins and reductase-like molecules is of special importance in thor such proteins may serve as potential therapeutics for a variety of different human disorders. Such polypeptides may also play importan 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 PROW3.

The PR0223 polypeptides of the present invention which exhibit serine carboxypepridease activity may be employed in wv for therapeutic purposes as well as for in intro purposes. Those of ordinary skill in the art will well know how to employ PRO=2 polypeptides for such uses.

PR0235 polypeptides and portions thereof which many be involved in cell adhiesion are also useful for i vimo therapeutic purposes, as well as for various in v1&o applications In addition, PR0235 polypeptides and portions thereof may have therapeutic applications in disease states which involve cell adhesioa- Given the physiological importance of cell adhesion mechanisms in vivo. efforts are currently being undler taken to identify new, native proteins which are involved in cell adeion. 7Terefore, peptides having homology to plexin are of particular interest to. the scientific and medical communities.

Because the PR0236 and PR0262 polypeptides disclosed herein arc homologous to various known Pgalactosidase proteins, the PR0236 and PR0262 polypeptdes disclosed herein will find use in conjugates of monoclonal antibodies and the polypeptide for specific killing of aumor cells by generation of active drug from a galactosylated prodrug the generation of S1-fluorouidirai from d ie prodrug P-D-galactosyl.5-fluorouridine). The PR0236 and PR0262 polypcptddes disclosed herein may also find various uses both in vivo mnd in vitro, wherein those uses will be similar or identical to uses for which P-gahctosidase 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 usefxl for in vtvo therapeutic purfposes, as wdl as for various in Wimu applications. In addition, PR0239 polypetides and portions thereof may have therapeutc applications in disease states which involve synaptic mechanisms, regeneration or ell adhesion.

Given the physiological unportance of synaptic processes, regeneration and cell adhesion mechanisms in vivo. efforts arc cunritly being under talmn to identify new. native proteins which are involved in synaptic machinery and cell :.10 adhesion. Therefore. peptides having homology to densin are of particular interest to the scientific and medical communities.

The PRO260Opolypeptides described herein can be used in assays to determine the relation to fucosidase.

In particular. the PR0260 poly4Tides can be used in assays in determining their ability to remove fucose or other sugar residues from proteoglycans. The PR0260 polypeptides can be assayed to determine if they have any *15 functional or Jocational similarities as fucosidase. The PR0260 polypeptides can then be used to regulate the systems in which they are integral.

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.

PR0270 polypeptides; and portions thereof which effect reduction-oxidation (redox) state may also be useful for in vw 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 inlanimation. In addition, PR0270 polypeptides and portions; thereof may affect the expiression of a gene which have a role in apoptosi. Therefore, peptides having homology to thioredoxin are particularly desirable to the scientific and *44e25 mecalwcommuties.

PR0272 polypeptides and portions thereof which possess the ability to bind calcium may also have numerous in viva therapeutic uses, as well as various in virro applications. Therefore. peptides having homology to reticulocalbin are particularly desirable. Those with ordinary sill 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 viva therapeutic purposes, as well as for various other applications. The identification of novel collagens aix collage-like molecules may have relevance to a number of human disorders. Tus, the identification of new collagens and collage-likr molecules is of special imiportance in that such proteins ay 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 industrul applications. Given the large munbcr of uses for collagen, there is substantial interest in polypeptides with homology to the collagen molecule.

107 PR0295 polypeptides and portions thereof which have hmology 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 imnnune system.

Thus, the identification of new integrins and integrin-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 PR0295.

•As the PR0293 polypeptide is clearly a leucine rich repeat polypeptide h aologue, the peptide can be used in all applications that the known NUBR-1 and NLRR-2 polypeptides are used. The activity can be cropared 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 PRO247 relative to densin or these other proteins. The results can be used accordingly in diagnostics and/or therapeutic applications with PR0247.

20 PR0302, PRO303, PR0304, PR0307 and PR0343 polypeptides of the present invention which possess protease activity nay be erployed both in vivo for therapeutic purposes and in vitro. Those of ordinary skill in the art will well know how to employ the P0302, PR0303, PRO304, PR0307 and PRO343 polypeptides of the present invention for such purposes.

PR0328 polypeptides and portions thereof which have hcaology 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 number 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 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 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 PRO326 including uses in competitive assays with LIG-1, ALS and decorin to determine their relative activities.

The results can be used accordingly. PRO335, PR0331 or PRO326 can also be 107a used in assays where LIG-1 would be used to determine if the same effects are incurred.

PRO332 contains GAG repeat (GKX) at amino acid positions 625- 628 in Fig. 108 (SBQ ID 13:309). Slippage in such repeats can be associated with hunmran disease. Accordingly, PR0332 can use useful for the treatment of such disease conditions by gene therapy, i.e. by introduction of a gene containing the correct GKEK sequence nmtif.

Other uses of PR0334 include use in assays in which fibrillin or fibulin would be used to determine the relative activity of PRO0334 to fibrillin or fibulin. In particular, PR0334 can be used in assays which require the nechanisms inparted by epidernal growth factor repeats.

Native PRO346 (SEQ ID 1 :319) has a Blast score of 230, corresponding to 27% harology between amino acid residues 21 to 343 with residues 35 to 1040 C4M6_.HUMAN, a carcinoerbryonic antigen gm6 precursor.

This harology region includes nearly all but 2 N-terminal extracellular domain residues, including an imvnoglobuilin superfamily harology at residues 148 to 339 of PRO346 in addition to several transmanbrane.

reiues (340-343). Carcinbr/oic: antigen prcusoasexplie in the Back&run is a turnor-specific antigen.

and as such. is a rcognized markcr and therapeutic target for the diagnosis and treatment of colon cancer. 1he expresion of tumnor-specific antigens is often associated with die progression of neoplastic timse disorders. Native PR0346 (SEQ MD 40:320 and P WO6874. a human carciwewbyoic antigen CEA-d have a Blast score of 224 and homology of 28% between residues 2 to 343 and 67 to 342, respectively. Ibis homology- includes the entire extracefluiar domai residues of native PR0346. minus the initiator methionine (residues 2 to 18) al well as scveral transmembrane residues (340-343).

PR0268 polypeprides which have protein disulfidle isomerase activity will be useful for many applications where protein disulfide isomerase activity is desirable including. for example, for use in promotin proper disulfide bond formation in roconibinany produced proteins so as to increase the yield of correctly folded protein. Those of ordinary skill in the ant will readily know how to employ such PR0268 polypeptides for such purposes.

PR0330 polypeptddes 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 Polve~ide Antibodies The present invention further provides anti-PRO polypeptide antibodies. Exemplary antibodies include :polyclonal, monoclonal, humanized, bispecific. and becroconjugate antibodies.

A. Polvelonal Antibodies Il andi-PRO polypeptde antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. 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 wUi be injecte in the mnammal by multiple sucuaeous or intraperitoneal injections. The immunizing agent may include the PRO polypeptide or a fusion protein thereof. It my be useful to conjgate the immunizing agent to a protein known to be immunogenic in the mammal being irnmmized. Examples of such imamunogenic proteins mnclude but arm not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed inchidc Freund's complete adjuant and MPLPTDM adjuvant (monophosphoryl Lipid A. synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimmntation.

B. Morioclonal Antibodie Tie andi-PRO polypetide antdlies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, M:l495 (19175).

In a hybridoma. method, a mouse, hamster, or other appropriate host animnal, is typically immunized with an immunizing agent to elicit lymnphocytes diat produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes mnay be imm~unized in vitro.

The immunizing agent will typically include the PRO polypeptide of interest or a fuasion protein thereof.

Generally, citinr peripheral blood lymphiocytes (CPBLs') are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-humian mammalian sources arc desired. The lymphocytes are then fused with an inmortlized cell line using a witable fining agent, stih as polyethylene glycol. to form a hybridoma cell (Goding.

Mownclonal Anibodesi Principles and Practice. Academic Press, (1986) pp. 5-1031. Immortalized cell Lines are usually transformed mammralian cells, particularly mycloma cells of rodentr, bovine and human origin. Usually, rat or mtmne inycloma 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 unfitsed, immortalized cells.

For example, if the parenta] cells lack die enzyme hypoxanthin guaniine phosphoribosyl tranferase (HGPRT or HFRT). the culture medium for the hybridomas typically v/0l include hypoxanthine, aminopterin, and thymidine ('HAT mnedium*i). which substances preCvent the growth of HGPRT-deflcicnt Cells.

Preferred immortalized cell lines are those that fuse efficiently. support stable high evel expression of antibody by the seleced antibody-producing cells, and are sensitive: to a medium such as HAT medium. More preferred immortalized cell lines; are snurine myetom line, which can be obtained, for instance, from the Salk Institute Cell Distribution Center. San Diego, California and dhe American Type Culture Collection, Rockville, Maryland. Human myelomra and mouse-human heteronayeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, LImmwwl.. LU:3001 (1984); Brodeur el al.. Monoclonal Antibody Production Technques and Applications. Marce 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 bindin specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in viro birling assay, such as adiinmstacihassay (RIA) or eazymno-liniced imioabsorbent assay (ELISA). Such techniques andl assays acknown in the art. The binding affinity of the monoclonal antibody can, for example, be determined byteScatchard analysis of Munson and Pollard, Anat. Biorczem.. Jfl7:220 (1980).

After the desired hybridoma cells aic identified, the cloocs may be subcloned by limiting dilution procedures and grown by standard methods (Goding, aLv"W. Suitable culture media for this purpose include, for example, Duibecco's Modified Eagle's Medium and RPMI-1640 mediumn. Alternatively. the hybridonu cell may be grown in vo as ascites in a mammal.

The monoiclonal antibodies secreted by the subelones may be isolated or purified from the culture medium or ascites &luW by conventional immunoglobulin purification procedures such as, for example, protein A-Scpharose.

hydroxylapatite chromatography, gel eleetrophoresis, dialysis, or afiity chromatography.

The monoclonal antibodies may also be nade by recombinan DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures by using oligonucicotide probes that are capable of binding specifically to genes encoding the heavy andl fight chains of murie antibodies). 71c hybridoan cells; of the invention serve as a preferred source of such DNA. Owee isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells. Chinese hamster ovary (CHO) cells, or myclOrna cells that do not otherwise produce immumoglobulin protein, to obtain the synthesis of monoclonal antibodies in the 109 reconihinant 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 dhe homologous murie sequences (UiS. Patent No. 4.516,567; Morrison et al.. vZWa or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-irnnunoglobulin polypeptide. Such a non-imartmoglobulin 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 arc well known in thc art. For example, one method involves recombinant expession of immunoglobulin light chain and mnodified heavy chain. The heavy chain is trncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively. the relevant cysteine residues amn substituted with another amino acid residue or arc deleted soas w prevent cosslinking.

In vitro methods arc also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly. Fab fragments. can be acconplished using routine techniques known in the art.

Humanized Antibodies The and-PRO polypepuide antibodies of the invention may further comprise hunmized antibodies or human antibodies. Humanized forms of non-human murine) antibodies are chimeric immunoglobulins, imuolobulin chains or fragments thereof (such as Fv, Fab. Fab'. F(ab') 1 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient ame 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 somec ins tances. Fv framework residues of the human immunoglobrulin 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.

25 variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobutin and all or substantially all of the FR regions are those of a human imunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an inmmoglobulin constant region typically that of a human imenoglobulin (Jones er al.. Nature, M: 5227525 (1986); Riechmann et at., Nature. Ifl:323-329 (1958); and Presta, Ckir. Op. Struct. Bil., 2:593-596 (199)].

Methods for humanizing non-human antibodies ame well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import' residues. which are typically taken from an 'import" variable domain.

Humanizaton can be essentially performed following the method of Winter and co-workers [Jones et al.. Nature. 321: 522-525 (1986); Riechmana a al., Nature. Ml:323-327 (1988); Verhoeycn ef al.. Scee 2n2:1534-1536 (1988)].

by substituting rodent CDLs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "hu-nnzed" antibodies are chimeric antibodies Patent No. 4,816,567). wherein substantially less than an intacthuran variable domain has been substituted by the corresponding sequence from a non-hunman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues arc substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the ari, including phage display libraries Hogenboom aid Winter, J. Mot. Bio..2W:381 (1991); Marks et al.. J. Mo!.,Bio, nZ2:581 (1991)]. The techniques of Cole di al. and Boerner es al. arc also available for te preparation of human monoclonal antibodies (Cole et aL, mon owAmiboffes and aner TherAPY. Alan R. Liss. p. 77 (1985) and Boerner eci* J. ImmwtoL, 14=W:96-95 (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 specificities 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.

Methods for nukling bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chainflight-chain pairs, where 9:00:15 the two heavy chin have different specificities [Milstein and Cucllo, Nature. 2:537-539 (1983)]. Because of the random assortment of imnunoglobulint heavy and light chains, these hybridomas (quadronias) produce a potential mixture of ten differnt antibody molecules, of which only one has the correct bispecific structure. The purification .00. of dhe correct molecule is usually accomplishied by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 199, and in Traunecker et EMBO IQ:3655-3659 (1991).

off' 20 Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to initunoglobulia constant domain sequences. The fusion preferably is with an iinmunoglobulin heavy-chain cnt=n dcxnain comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy- *too. chain constars region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.

DNAs euicoding the itoimtnoglobulin heavy-chain futsions and, if desired, the imrutnoglobulin 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 ef al.. Mfeihodi in En~ynwlogy, M2:210 (1986).

E. Heteroconiugate-Antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example. -been proposed to target imuesystem cells to unwanted cells Patent No. 4,676,980], and for tatment of HIIV infection (WO 91/00360; WO 92/20073; EP 03089]. It is contemplated that the anibodies may be prepared in vitro using known methods in synthietic protein chemistry, including those involving crossliniting agents. For example, immunotOxins may be constiucted using a disulfide exchange reaction or by forming a thioeter bond. Examples of suitable reagents for this puirpose include irmnoiate and methyl4-mercaptobutyrididate and those disclosed, for example, ini U.S.- Patent No. 4.676,980.

S6. Uses for Anti-Pro PoljVoendedf Antibondies The anti-PRO polypeptide antibodies of the inven~tion 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 serum. Various diagnostic assay techniques known in the art may be used. such as competitive binding assays. direct or indirect sandwich assays and immunoprecipmation assays conducted in either heterogencous or homogeneous phases rZola, Monoclonal Antibodies- A Manual of Techniquies, tRC Press, Inc: (1987) pp. 1474158]. The antibodies used in dhe diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing. either directly or indireedy, a detectable signal. For example, the detectable moiety may be a radioisotope, such as C.,3 P.s S. ol" 1. a fluorescent or chemilurninsccmt compound, such as fluorescein isothiocyanate, rhodaniine, or luciferin. or an enzyme. such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method. known iti the sat for conjugating the antibody to die detectable moiety may be employed, including those methods described by Hunter et al., Nagure, 4:945 (1962).

David er at.. Biochemiry, 13:1014 (1974); Pain d aL. J. ImmwzaoL Met,. 40:219 (1981); and Nygren. J.

Histochem. and Cytochem., 3D:407 (1982).

.***Anti-PRO polypeptide antibodies also arc useful for the affinity purification of PRO polypeptide from recombinant cell culture or natural souces. In this process, the antibodies against the PRO polypeptide are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the PRO polypeptide to be purified, and thereafter the support is washed with a suitable solvent diat 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 anothcr suitable solvent that will release the PRO polypeptide from the antibody.

With regard to PRO211 and PR0217. therapeutic indications includc disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions aerocolmts. Zoliinger-Elisam 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 die vulva and gliomas.

With regard to anti-PR0187 antibodies, FGF4 has been implicated in cellular differentiation and emnbryogenesis. including the patterning which appears during limb formation. FGF-8 and die PRO 187 molecules of the invention therefore are likely to have potent effects on cell growth and development. Diseases which relate to cellular growth and differentiation arm therefore suitable targets for therapeutics based on functionality similar to FGP-S. For example, diseases related to growth or survival of nerve cells including Parkinson's disease, Alzlheimer's disease. ALS. neuropathies. Additionally, disease related to uncontrolled ell growth, cancer, would also be expected therapeutic targets.

Native PRO533 is a 216 amino acid polypeptide of which residues 1-22 are the signal sequenc. Residues 3 to 216 have a Blast score of 509, corresponding to 53% homology to fibroblast growth factor. At die macleotide level, DNA474 12, the EST from which PCR oligos were generated to isolate the full length DNA49435-12 19, has been observed tonp to llplS. Sequcocchoxuology to tin IlpiS locus would indicate that PR0533 may have utility.

in the treatment of Usher Syndrome or Atrophia areata.

112 As mtoned previously, fibroblast growth factors can actupon cells in both a mitogenic and non-mitogenic: normeT. These factors; are mitogenic for a wide variety of normal diploid mesodermi-derived and neural crest-derived cells, inducing gramilosa cells, adrenal cortical cells, chrosalrocytes. myobLasts, corneal and vascular enidothelial cells (bovine or human), vascular smooth muscle cells, lens, retina and prostatic: epitelial cells, oligodendrocytes, astrocytes. elirondocytes. myoblasts and osteoblasts. 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 (chernotaxis), initiation of new blood vessel formulaion (attgiogeniesis), modulation of nerve regeneration and survival (neurotrophism). modulation of endocrine functions, and stimulation or suppression of specific Cellular protein exres .n extroiclhlar matrix production and cell survival. Baird, A. Bohlen, Han dbook of Elp. Phrmacol.

21(l): 369-418 (1990). These properties provide a basis for using fibroblast growth factors in therapeutc approaches Io accelerate wound healing, nerve repair, collateral blood vessel formation, and the like. For example, fibroblast ::growth factors, have been suggested to minimize myocardium damage in hear 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 maintenance of gastrointestinal inucosa and the repair of acute and chronic inucosal lesions emterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulceration and congenital nmicrovillus atrophy). sldn diseases associated with abnrmal keratinocyte differentiation psoriasis, epithelial cancers such as lung squamous cell carcinoma.

epidermoid carcinoma of the vulva and gliozins.

Anti-PR0317 antibodies find use in anti-tunior indications if they are angiostatic, or in coronary ischemic indications if they are angiogenic.

Native PRO301 (SEQ WD NO:119) has a Blast score of 246 and 30% homology at residues 24 to 282 of Fig.

4 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 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 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-l 19) also has a Blast score of 165 and 29% homology at residues to 255 of Fig. 44 with l{S46KDA a human coxsackic 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 inifection and therapeutics for the prevention of such infction 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 PR0234 polypeptides of the invention includes treatments associated with leukocyte homing or the interaction between leukocytes and the crndothelium during an inflammatory restionse. 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 am useful in the diagnosis, prognosis and treatmnt of dhe disease.

Cancer specific mnonoclonal antibodies (uiAbs) which are specific to tumor antigens. Such niAbs. which can distinguish between normal and cancerous cells ame useful in the diagnosis. prognosis and treatment of the disease. Particulr anigens are known to be associated with neoplastic diseases. such as colorectat and breast cancr.

Since colon=c=cr is a widespread disease, early diagnosis and treatmen is an important medical goal. Diagnosis and treatment of cancer can be implemented wing monoclonal antibodies (mAbs) specific therefore having fluorescent. nuclear magnetic or radioactive tags. Radioactive genes. toxins sand/or drug tagged mAbs can be used for treatment in situ with minimal patient description.

The following examples are offered for illustrative purposes only. and are not intended to limit the scope of the pres ent invention in any way.

All patent and literature references cited in dhe present specification are hereby incorporated by reference their entirety.

EXAMPES~

Commercially available reagents referred to in dhe examples were used according to manufacture's instructions unless othewise indicated. The source of those cells identified in the following examples, and throughout the specification by ATCC accession numbers is the American Type Culture Collection. Rockville, Maryland.

EXAMPEL1: Extracellular Domain HomlogX ScreeiMj to ldcnift Novel Pobtyppdes and cDNA Encoding The extracelluar domain (ECD) sequences (including the secetion signal sequence, if any) from about 950 knwn serted protein fromz dhe Swiss-Prot public database were used to search EST databases. The EST databases included public databases Dayhoff. GenBank), and proprietary databases LIFESEQOh.- Incyte Pharnaceuticals. Palo Alto, CA). Thec search was performed using the computer program BLAST or BLAS2 (Altschl.l ar Gish, Methods in Enzmology 29: 460.80(1996); hfl :l/blast.wustlledu/blasaIREADME.bnnl) as a comnparison of the ECD protein sequences to a 6 frame tranlation of the EST sequences. Those comparisons with a Blast score of 70 (or in some cases 90) or greater dhat did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap' (Phil Green. University of Washington, Seattle, WA; (http:lboz~nt.washbigtoeduphrap.dosPhrapahl).

Using this extracellular domain homology screen, consensus DNA sequences were assembled relative to the other identified EST sequnes. 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 die consensus sequences obtained a described above, oligonaiclecaides were then synthesized and used to identify by PCR a eDNA library that contained the sequence: of interest and for use arprobes to isolate a clone of the full-length coding sequence for a PRO polypeptide. Forward (J1) and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequence are typically 40-55 bp in length. In sonie cases, additional oligonrucleotides are synthesized when the consensus sequence is greater than about 1-I .Skbp. In order to screen several libraries for a full-lengt clone. DNA from the libraries was screened by PCR amplification, as per Ausubel et aL. Currnt Protocol- in Molecular Biolngx, with the PCR primer pair. A positive library was then used to isolate clones encoding dhe gene of interest using the probe oligonucleoodde and one of the priner pairs.

Thec cDNA libraries wed to isolate the cDNA clones were constructed by standard mnethods using commnercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Nod site. linked with blunt to Sall hemaikinased adaptors. cleaved with Notd, sized appropriately by gel electroplioresis, and cloned in a defined orientaton into a suitable cloning vector (such as pRKB or pRKD; is a precursor of pRKSD that does not contain the SG1 site; see, Holmes et al.. Scene L:278-1280 (1991)) in the unique Xhol and Not! sites.

EXAMPEL2: Isolation of cDNA Clones Encodinp PRO211 and PRO217 Consensus DNA sequences were assembled as described in Example 1 above and were designated as DNA2973 and DNA28760. respectively. Based on these consensus sequences, oligonucleotides were synthesized and used to identify by PCR a eDNA library that contained the sequencs of interest and for use as probes to isolate a clone of the full-length coding sequence for the PRO211 and PR0217 polypeptides. The libraries used to isolate DNA32292-1131 and DNA33094-1131 were fetal lung libraries.

eDNA cloam were sequenced in their entirety. The entire nucleotide sequences of PRO21 1 (DNA32292- 1131; UNQ18S) and PR0217 (UNQI91; DNA33094-l 131) are shown in Figure I (SEQ ID NO: 1) and Figure 3 *:(SEQ ID) NO.3). respectively. The predicted polypeptides are 353 and 379 amnino acid in length, respectively, with .20 respective molecular weights of approximately 38.190 and 41.520 daltons.

The oligonucleotde sequences used in the above procedures were the following: 2 8730.p (OLI 516) (SEQ ID 5*AGACCGCGTTCGTTGC.GGTArGA3 28730.f (OU 517) (SEQ ID NO:6) s*-AGAGTGTATCTCTGGCTACGC-3' 29730.r (OU S.18) (SEQ ID NO:7) 5'-TAAGTCCGGCACATACAGGTC.3* 2876O.p (OLI 617) (SEQ ID NO:8) 5'CCCACGATGTATGATTGGACmGGTACTCCT-I-YJJCTGCTC3 29760.f (OLI 618) (SEQ ID NO:9) 5'-AAAGACOCATCTGCGAGTGTCC-3' 29760.r (OLI 619) (SEQ ID NO: 5,-TGCTGATITCACACTGCTCTCCC-3- EXAMPLE 3: Isolation of cDNA Clones ncoin~ Hmnn PR230 A consensus DNA sequence was assembled rclative to the other identified EST sequences as described in Example I above, wherein the consensus sequecec is designated herein as DNA30857. An EST proprietary to Gerimcch was employed in dhe consensus assembly. The EST is designated as DNA20088 and his the nuclcotide sequence shown in Figure 7 (SEQ ID NO: 13).

Based on the DNA308S7 consensus sequece-. oligora=Wtdes were synthesized to identify by PCR a cDNA library that contained the scquecec of interest and for use as probes to isolate a clone of the fufll-lngth coding sequence for PR0230.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR ad=me S'-TCGAGGCCTCTGAGAAGTGGCCC-3' (SEQ [D NO: 14) reverse PCR sirfiner S'-GGCGGTATCTCTCTGGCC*TCCC-3' (SEQID Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30857 sequence which had the following nucleotide sequence hybridization probe S-TTCTCCACAGCAGCTGT'GGCATCCGATCGTGTCrCAATCCAfl'CTCTGG.3- (SEQ ID NO:f6) In order to screen several libraries for a source of a full-length clone. DNA from the libraries was screened by PCR am~plification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0230 gene using the probe oligonucleotide and onr of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. DNAieqxrriz of the clones isolated as described above gave the full-lenth~ DNA sequence for PR0230 (herein designated as UNQ204 (DNA33223-1136)) and the derived protein sequence for PROM3.

The entire nucleotide sequence of UNQ204 (DNA33223-1136) is shown in Figure 5 (SEQ ID NO:11).

Clone UNQ2D4 (DNA33m2-l 136) contains a single open reading frame with an apparent translational initiation site *20 at nucleotide positions 100-103 and ending at the stop codon at nruclcotide positions 1501-1503 (Figure SEQ ID NO: 11). The predicted polypeptide precursor is 467 ammno acids long (Figure 6).

.E AMPLE 4: Isolationl of cDNA Clones lEncoding Human PRO232 A consensus DNA sequence was assembled relative to the other identified EST sequences as described in Example 1 above. wherein the consensus sequenc is designated herein as DNA30935. Based on the DNA30935 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 PROM3.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR 2riMer 5'-TGCTGTGCTACTCCrGCAMAGCCC-3' (SEQ ID NO:19) reverse PCR urimer 5'-TGCACAAGTCGMrTCACAGCACG-3' (SEQ ID Additionally, a synthetic oligonucleodde hybridization probe was constructed from the consensus DNA30935 sequence which had the following nceotde sequence 5S-AGCAACGAGGACTCCTGCAGGTGGAGAACGCACCCAGCTGGG-3 (SEQ ID NO:21) In order to sceena several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplificaton 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 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 sequece for PR0232 (herein designated as L1NQ206 (DNA34435-1140)J and the derived protein sequence for PROM3.

The entire nucleotdec sequence of UNQ206 (DNA34435-1140) is shown in Figure 8 (SEQ ID NO: 17).

Clone UNq206 (DNA34435-1 140) contains a single open reading frame with an apparent translational initiation site at isetide positions 17-19 and ending at dhe stop codon at nucleotde positions 359-361 (Fig. 8; SEQ MD NO: 17).

The predicted polypeptide precursor is 114 amino acids long (Fig. Clone UNQ2O6 (DNA34435-1140) has been deposited with ATCC on September 16. 1997 and is assigned ATCC deposit no. ATCC 209250.

Analysis of the amino acid sequece of the ful-length PR0232 suggests that it possesses 35% sequence identity with a stem ceUl surface antigen from Gallus gallus.

~EAMEPIS Isolation of cDNA Clones ncoding PRO1 87 A proprietary expressed sequmme tag OM~) DNA atbas 0UFESEQ 11 ncyte Pharmaceuticals, Palo Alto, CA) was searched and an EST 0843 193) was identified which showed homology to fibroblast growth factor (FGF-8) also known as androgceniced growth factor. inRNA was isolated from humnan fetal lung tissue using reagents and 15 protocols from Invitrogen, San Diego, CA (Fast Track The cDNA libraries used to isolate die cDNA clones were constructed by stand:,rd methods using commtercially available reagents lnvitrogen. San Diego, CA, Life Technologies. Gaithersburg. MD). The cDNA was primed with oligo dT containin a Notd site, linked with blunt to SaHl beniildnased adaptors, cleaved with Nod. 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 (Super Script Plasmid System). 'Me double-stranded cDNA was sized to greater than 1000 bp and the SailNodl tinkered eDNA was cloned into XboI/Nocl cleaved vector. pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sf1l restriction enzyme site preceding the XhoI/Not1 cDNA cloning sites.

.:Several libraries from various tissue sources were screened by PCR amplification with the following oligonucleotide probes: IN843193.f(0L1315).(SEQ ID NO:24) 5-CAGTACGT'GAGGGACCAGGtJCGCCATGA..3.

IN843193.r (Oil 317) (SEQ ID 5'-CCGGTGACCTGCACGTGCI-1GCA3 A positive library was then used to isolate clones encoding the PRO 187 gene using one of the above oligonueleotides and the following oligonucleotide probe: IN843193.p (OLI 316) (SEQ ID NO:26) 5SGCGGATCTGCCGCGCANG'GTCGAGCCC-3' A eDNA clone was sequenced in entirety. Thie entire nucleotide sequence of PR0187 (DNA27864-I 155) is shown in Figure 10 (SEQ ED NO:22). Clone DNA27864-1 155 contains a single open reading framne with an apparent translational Initiation site at nucdeotide position 1 (Figure 10; SEQ ID NO:22). The predicted polypeptide precursor is 205 amino acids long. Clowe DNA27864-1 155 has been deposited with the ATCC (designation: DNA27864-1155) and is assigned ATCC deposit no. ATCC 209375.

Based on a BLAST arid FastA squmee algmen analysis (using the- ALIGN computer program) of dhe full.

length sequence. the PRO197 polypptide shows 74% amino acid sequence identity (Blast score 310) to human fibroblast growth factor-S (androgen-induced growth factor).

EXAMEI.,E6: Isolation ofcDNA Clones EocodjnE PRY265 A consensus DNA scquence was assembled relative to other EST sequences as describcd in Example I above using phrap. This consensus sequence is herein designated DNA33679. Based on the DNA33679 consensus sequence, oligonucleotdes were synthesized: 1) to identify by PCR a cDNA library that contained ther sequence of interet, 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 primer A- S'-CGGTCTACCTGTATGGAACC-3' (SEQ ID NO:29); forward PCR Rrimer R: 5S-GCAGGACAACCAGATAAACCAC.3' (SEQ ID :reverse PQR gmimer 5'-ACGCAGAITGAGAAJGCrGTC.3* (SEQ ID NO:31) Additionally. a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA33679 sequence which had the following nucleotdec sequence hyridzation .pzge CCCA~rCTGAACTTGAGAC~riAC- r(SE M NO:32) In order to scr=e 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 wsed to isolate clones encoding the PR0265 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction ot the cDNA libraries was isolated from human a fetal brain library.

*DNA sequezicig of the clones isolated as described above gave the ful-length DNA sequence for PR0265 Pherein designated as UNQ232 (DNA36350-hl1581 (SEQ ID NO:27) and the derived protein sequence for PROM6.

The entire nucleotide sequence of UNQ232 (DNA36350-1 158) is shown in Figure 12 (SEQ MD NO:27).

.5...Clone UNQ232 (DNA36350-11S8) contains a single open reading fr-ame 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 Polypqntide precrsor is 660 amino acids long (Figure 13). Clone UNQ232 (DNA36350-1 158) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209378.

Analysis of the amino acid sequence of the full-length PRO265 polypeptide suggests that portions of it possess significant homology to the fibromodulin and the fibromodulin precursor, therby indicating that PROM~ may be a novel member of the leucine rich repeat family, particularly related to fibroenodulin.

EAMPLE 7: Isolation of cDNA Cln ECoding Human PR2 19 A consensus DNA sequence was assembled relative to other EST sequence using p2rap as described in Example 1 above. Tids consensus sequence is herein designated DNA28729. Based on the DNA2879 consensus sequence, oligomucleotides 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 PROM 1.

A pair of PCR primers (forward and reverse) were synthesized: frward PCR orirner S'-TGACCCTGG TTAATAcjtc-3- (SEQ ID revyrne PCR Sirirner 5'.ACAGCCATGGTCTATAGCTT-Iy.3. (SEtQ ID NO:36) Additionally. a synthetic oligonucloodde hybridization probe was constructed from the consensus DNA29729 scquence whiich had the following nucleotide sequence hybridization Rrob 3 (SEQ ID N 0:37) In order In screen several libraries for a source of a full-length clone, DNA from the libraries was scned by PCR amplification with the PCR Primer pair identified above. A positive library was then used to isolate cloncs encoding the PR0219 gene using the probe oigonuicleoodde and one of the PCR primers.

RNA for construction of die eDNA libraries was isolated from humnan fetal idney tissue.

DNA sequencig of the clones isolated as described above gave the full-length DAsequence for PR0219 therein designated as UNQ193 (DNA32290-1 164)1 (SEQ ID, 140:33) and the derived protein sequence for PR0219.

7be entire nuckotide sequence of UNQ193 (DNA32290-1164) is shown in Figures 14A-B (SEQ ID No033).

***.Clone UNQ 193 (DNA32290-1 164) coatains a single open reading firame with an apparent translationil initiation sice at micleotile 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 20984.

Analysis of the amino acid sequence of the MIl-length PR0219 polypeptide suggests that portions of it possess significant homology to tie mouse and human matrilii-2 precursor polypeptides.

EXAMPLEj8: Isolation of cD)NA Clones ncoding Human PR246 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example I above. This consensus sequence is herein designated DNA30955. Based on the DNA30955 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-lenigth coding sequence for PROM4.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR prer 5S-AGGGTCTCCAGGAGAAAGACTC-3' (SEQ ID reverse PCR n2riMer 5*-A7TGTGGGCC'rFGCAGACATAGAC..3. (SEQ ID NO:41) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA309S5 sequence which had the following inucleotide sequence TCTCGrC3(SEQ ID NO:42) In order to screen severa] libraies 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 use4 to isolate clones encoding the PR0246 gene using the probe otigor-leotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. DNAwT~rirg of the clones isolated as described above gave the MIl-length DNA sequence for PR0246 [herein designated as LJNQ=2 (DNA35639-1172)J (SEQ ID NO:38) and the derived protein sequence for PROM4.

The entire nucleotde sequence of UNQ220 (DNA35639-1 172) is shown in Figure 16 (SEQ ID NO;38).

Clone UJNQ220 (DNA3S639-1 lfl) contains a single open reading (iane with an apparent trnslational initiation site at nucleotide positions 126-128 and ending at the sto codon at aucleotide positions 1296 '-1298 (Figue 16). The predicted pojypeptide precursor is 390 amino acids long (F7igure 17). Clone UNQ220 (DNA35639-l 172) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209396. L Analysis of the amino acid sequence of the full-length PR0246 polypeptide suggests that it p ossess significant homology to the human cell surface protein HCAR, thereby indicating that PR0246 may be a novel cell surface virus receptor.

EXAMPIE 9: Isolation of clDNA Clones Encoding Human PR=22 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example I above. Ibis consensus sequence is herein designated DNA2S7SS. An EST proprietary to Genentech was *Ob* employed in the consensus assembly. This EST is shown in Figure 20 (SEQ ID NO:50) and is herein designated as DNA219S1.

Based on the DNA28758 consensus sequence, oligomicleotides 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 PrR grimer 5*-GGTAATGAGCTCCA'ITACAG.3' (SEQ ID NO:51) forward PcR primer 5'-GGAGTAGAAAGCGCATGG-3- (SEQ ID NO:52) forward PCR nRie 5'-CACCTGATACCATGAATGGCAG.3* (SEQ ID NO:S3) reverse PCR printer 5'-CGAGCTCGAA1TAATrCG-3' (SEQ ID NO:54) *reverse PCR Rrimer 5'-GGATCCCTGAGCTCAGG-.3' (SEQ ID N0:55) reverse PCR nrimer 5'-CCTAG7TGAGTGATCCTrGTAAG-3' (SEQ ID NO:S6) Additionally, a synthetic oigonucleotide hybridization probe w as constructed from the consensus DNA28758 S sequence which had the following nucleotide sequence bybridization orobe 5*AGGCCCCTAGCCGATAC)AAATTCAT3 (S E D NO:57) In order to screen. several libraries for a source of a fllI-lengths 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 dhe PR0228 gene using the probe oligozacleotide and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated from human fetal Winey tissue.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence; for PR0228 [hertin designated as UNQ202 (DNA33M9-1202)] (SEQ D1 NO:48) and the derived protein sequepce for PROM28 The entire nucleotide sequence of UNQ202 (DNA33D92-1202) is shown in Figure 18 (SEQ MD NO:48).

Clone UNQ202 (DNA33092.l202) contain a single open reading fre with an apparent translational initiation site at nucleodde postins 24-26 of SEQ MD NO:48 and ending at the stop codon after nucleotide position 2093 of SEQ ID NOAS. The predicted polypeptide precursor is 690 amino acids long (Figure 19). Clone U1NQ202 (DNA33092- 1202) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209420.

Analysis of the amino acid sequence of the full-length PR0228 polypeptide suggests that portions of it possess significant homology to the secrctmn-rclated proteins CD97 and EMRI as well as the secretin member, latrophilin, thereby indicating that PR0228 may be a new member of the secretin related proteins.

EXAMPLE 10: Isolation of cDMA Clones Encodinp Human PRO533 The EST sequence accession number AF007268. a murinc fibroblast growth factor (FGF-15) was used to searh various public EST databases GenBank. Dayhoff, etc.). The search was performed using the computer program BLAST or BLAST2 [Akidchul et al.. Methods in Enzymoloev, ZMAW0480 (1996).

hp:/fbst.wustl/edizilastEA M~btm as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. The search resulted in a hit with GenBanc EST AA220994. which has been identified as stratagcoc NT2 neuronal precursor 937230.

Based on the Genbanic EST AA220994 sequence, oligonuclotides 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 .15 coding sequence. Forward and reverse PCR primers may range from 20 to 30 nucleotides (typically about 24). and are 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 Azuubel et al. Cwrrent Protocols in Molecular Biology, with the PCR *:primner pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the PCR primers.

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 below. A positive library was then used to isolate clones encoding the PR0533 gene using the probe oligonucleotide and one of the PCR primers.

RNA far construction ofthe 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; Clontch, etc.) The cDNA was primed with oligo dT containing a Nod site, linked with blunt to Sall hemiltinased adaptors, cleaved with Nod, sized appropriately by gel electrophoresis. and cloned in a defined orientation into a suitable cloning vector (such as pRXB or pRKD; pRKSB is a precursor of pRK5D that does Nt contain the Sf1l site; see, Holmes et al., Science. M: 1278-1280 (1991)) in dhe unique Xhol and Nod sites.

A cDNA clonewas sequenced in its entirety. Ile full length nucleoodde sequence of PR0533 is shown in Figure 21 (SEQ ID NO:58). Clone DNA49435-1219 contains a single open reading frame with an apparent translational initiation site a nucleotide positions 459-461 (Figure 21; SEQ ID NO:S8). 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 PastA sequence alignmnt analysis of the full-length sequence. PR0533 shows amino acid sequence identity to fibroblast growth factor The oligonucleotide sequncs used in the above procedure were the following: FGFlS.forward: 5'-ATCCGCCCAGATGGCTACAATGTGTA.3 (SEQ ID FGFlS.probc: S'-GCCTCCCGGTCTCCCrGAGCAGTGCCAAMCAGCGGCAGTJTA..3 (SEQ ID NO:6 1); FGF1S.revcrse: S*-CCAGTCCGGTGACAAGCCCAA&3' (SEQ ID NO:62).

EXAMPLF 11: Isolation of cDNA Clones Eneodin Human PRO245 A consensus DNA sequence was assembled relative to die other identified EST sequences as described in Example 1 above. wherein the consensus sequence is designated herein as DNA309S4.

Based on the DNA309S4 consensus scq~ce. oligomwclotdecs wre synthesized to identify by PCR a cDNA library that contained the sequence of interest and for usc as probes to Isolate a clone of the full-length coding sequence for PROM4.

A pair of PCR primers (forward and reverse) were synthesized: :::forwad PCR primer S'-ATCGrGTGAAG7TATGCCCC.3* (SEQ ID reverse PCR primer 5'-ACCTGCGATATCCAACAGAArrG-3- (SEQ ID NO:66) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30954 sequence which had the following nucleotide sequence hbriddztinprbe 5'GAGGAAATATTGAGArGGCCACGTC3(SEQ ID NO:67) In order to screen several libraries for a source of a full-length clone. DNA from die libraries was screened 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. DNAw~iqtrg of the clones isolated as described above gave the full-length DNA sequence tbr PR0245 [herein designated as UNQ219 (DNA35638-1l41)] and the derived protein sequence for PROM4.

The entire nucleotide sequence of UNQ219 (DNA35638-l 141) is shown in Figure 23 (SEQ ID NO:63).

Clone UNQ219 (DNA3S63S-l 14 1) contains a single open reading f-rme with an apparent translational initiation site at nucleotide positions 89-91 and ending at the stop codon at nucleotide positions 1025-1027 (Fig. 23; SEQ ID NO:63). The predicted polyetder precursor is 312 amino acids long (Fig. 24). Clone UNQ219 (DNA3S638-l 141) has been deposited with ATCC on September 16. 1997 and is assigned ATCC deposit no. ATCC 20965.

Analysis of the amino acid sequence of the full-length PR0245 suggests that a portion of it possesses amino acid identity with the lamian c-myb protein and, therefore, may be a new member of the transmembrane protein receptor tyrosine kinase family.

EXAMPI.&12: Isolation of cDNA Clones Encoding Human PR0220. PR0221 and PR0227 PR22 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 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) wcrc synthesized: foyr PC rme 'TCACCTGGAGCCTArGGJ(-3, (SEQ ID NO:74) reverse PCRprimer S'-ATAC CAGCTATAACCAGGCTCG.3. (SEQ 11) NO:7S) Additionally. a synthetic oligonucleotidr hybridization probe was constructed from the consensus DNA28749 sequence which had the foWowing nucleotide sequence: hybdzatin rob 3 (SEQ MD NO:76).

In order lo screen several libraries toria source of a full-length clone. DNA from (he libraries was screened by PCR amplification with the PCR primer pair identified above. A positivc library was then used to isolate clones encoding die PR0220 gene; using dhe probe oligonmcleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from hum" fetal lung tisse. DNqxn*z~ of the clones isolated as descibed above gave the fui-length DNA sequence for PR0220 (herein designated as UNQ194 (DNA32298-1132) and the derived protein sequence for PR0220.

The entire nucleotide sequence of UNQ194 (DNA32298-1 132) is shown in Figure 25 (SEQ ED NO:68).

15 Clone UNQ194 (DNA32298-1132) 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 UNQ 194 (DNA32298-1 132) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209257.

Analysis of the amino acid sequence of the full-length PR0220 shows it has homology to member of the leticine rich repeat protein superlarnily. including the leucine rich repeat protein and the neuronal leucine-rich repeat protein 1.

PRO22 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 DNA28756. Based on the DNA28756 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 PR0221.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer 5S-CCATGTGTCTCCT'CCTACAAAG..3. (SEQ ID NO:77) reverse PCR primer 5'-GGGATAGATGTATCTJA-1GI-Jf3' (SEQ ID NO:78) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28756 sequence which had the following nucleotide sequence: CTCG3(SEQ ID NO:79) 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 die PR0221 gene wing the probe oligotnicleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. DNAzgqmfg of the clones isolated as described above gave the full-length DNA sequence for PRC1221 (herein designated as UNQ195 (DNA33089-l 132) and the derived protein sequevice for PR0221.

The entire aucleotide sequence of UNQ195 (DNA33089-1 132) is shown in Figure 27 (SEQ ID Clone UNQ195 (DNA33089-l 132) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 179-181 and ending at die stop codon at nuecotidc positions 956-958 (igure 27). The predicted polypeptide precuirsor is 259 amino acids tong (Figure 28). PR0221 is believed to have a tninsmembranc region at amin acids 206-225. Clone UJNQ195 (DNA33089-l 132) has been, deposited with ATCC and is assigned ATCC deposit no. ATCC 209262.

Analysis of the a2-; acid sequence of the full-length PR0221 shows it has homology to member of the leucine rich repeat protein superfamily, including the SUTr protein.

PRO227 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 DNA28740. Based on the DNA28740 consensus sequence, oligonucleotides were synthesized to identify by PCR a eDNA Library that contained the C:...:sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PR0227.

A pair of PCR primers (forward and reverse) were synthesized: forward PrCR Vr* S-AGCAACCGCCrGAAGCTCATCC-3' (SEQ ID NO:8O) :reverse PCR primer 5'-AAGGCGCGGTGAAAGATGTAGACG..3* (SEQ ID NO:81) Additionally. a synthetic oligonucleotdec hybridization probe was consmicted from the consensus DNA28740 sequence which had the fWlowing nucleotide sequence: ACTGAGG GGCJA.3' (SEQ ID NO: 92).

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 isolae clones encoding the PRO22 gene using the probe oligonucleotide and one of the PCR primers.

*..RNA for construction of the eDNA libraries was isolated from human fetal lung tissue. DNAWxig 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 sequenc for PR0227.

The entire wucleotide sequence of UNQ201 (DNA33786-1 132) is shown in Figure 29 (SEQ MD NO:72).

Cloac UNQ201 (DNA33786-l 132) contains a single open reading frame with an apparent translational initiation site at nucleotidc positions 117-119 and ending at the stop codon at nucleotide positions 1977-1979 (Figure 29). The predicted polypeptide precuirsor is 620 amino acids long (Figure 30). PR0227 is believed to have a transmenibrane region. Clone UNQ201 (DNA3378-1 132) has been deposited with ATCC and is assigned ATCC qposit no. ATCC 209253.

Analysis of the amino acid sequence of the full-length PR0221I shows it has homlogy to member of the lcucine rich repeat protein supecrfamily, including dhe platelet glycoprotein V precursor and the human glycoprotein

V.

EXAMPIY1: hsolatoon of cDNA Clonct Fnending Human PRO2 9 A consensus DNA sequence was assembled relative to other EST sequences using phrip as descuibed 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 che full-length coding sequence for PRO2SS.

PCR primers (forward and reverse) were synthesized: forard PCR prme 5'-GCTAGGAAITCCACAGAAGCCC-3' (SEQ ID ~reverse PCR primer 5T-AACCTGGAATGTCACCGAGCTG-3' (SEQ ID NO:86) reverse PCR primer 5'-CCTAGCACAGTGACGAGGGACTrGGC-3- (SEQ ID NO:M7 Additionally. synthetic oligotnicleotde hybridization probes were constructed from the consensus DNA29740 sequence which had the following nueleotide sequenc: WyridizinpiRbe 5.-AAGACACAGCCACCCTAAACTGTCAGTCTCTGGGAGCAAGCCTJCAGCC3* (SEQ ID NO:88) (SEQ ID NO:89) In order to sceen 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 oligonuceotde and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. DNAseqzcnkg of the clones isolated as described above gave the full-length DNA sequence for PR0258 (herein designated as UNQ225 (DNA359I8-1174)J (SEQ ID NO:83) and the derived protein sequence for PR0258.

The entire nuclotide sequence of UNQ225 (DNA3S918-1 174) is shown in Figure 31 (SEQ ED NO:83).

Clone UNQ225 (DNA3S9IS-1 174) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 147-149 of SEQ ID NO:S3 and ceding at the stop codon after nucleotide position 1340 of SEQ ID NO:S3 (Figure 3 The predicted polypeptide pre cursor is 398 amino acids long (Figure 32). Clone UNQ225 (DNA3S99-l 174) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 2094M2.

Analysis of the amino acid sequence of the full-length PR0258 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.

EXAMPLE14: Isolation of eDNA Clones Eiiiodine Human PRO266 An expressed sequence tag database was searched for ESTs having homology to SUT. resulting in the identification of a single EST sequence designated herein as T73996. Based on the T73996 EST sequence, oligonucleotidecs 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 done of the full-length coding sequence for PR0266.

A pair at PCR primers (forward aNd reverse) were syntheized: forward ECR Vrme SGTGATCGGCAACAATAAC-3y (SEQ ID NO:92) reverse PCR Vrme S-A1rTTGGCAGGCTGAGfTAAG-3- (SEQ ED t'O:93) Additionally. a synthetic oligoaucleouide hybridization probe was constructed which had the following nucleotide sequence 5SGGTGGCTATACATGGATAGCAATTACCTUGGACACG~lGTCCCGG(..3(SEQ ID NO:94) In order to sa~ce several libraries for a source of a fuil-length cdone, DNA fromn 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 PR0266 gene using the probe oligonucleotide and one of the PCR primers.

RNA for constrction of the eDNA libraries was isolated from human Jetal brain tissue. DNAseqpezrig of the clones isolated as described above gave the full-length DNA sequence for PR0266 therein designated as :9.9 UNQ233 (DNA371SO-1 17S)J (SEQ ID NO:90) and the derived protein sequence for PR0266.

71e entire nucleocde sequence of UNQ233 (DNA37150-1l78) is shown in Figure 33 (SEQ ID Clone UNQ233 (DNA37L50-1178) contains a single open reading frame with an apparent trainslational initiation site 99*999 at micleotide positions 167-169 and ending at the stop codon after nuclotide position 2254 of SEQ ID NO:90. The predicted polypeptide precuror is 696 amino acids long (Figure 34). Clone UNQ233 (DNA371SG-1 17S) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209401.

Analysis of dhe amino acid sequence of the full-length PR0266 polypeptide suggests that portions of it possess significnt homulogy to the SLIT protein, thereby indicating that PR0266 imy be a novel leucine rich repeat protein.

Isolation of cDNA Clones Encoding Human PRQ262 A consensus DNA sequence was assembled relative to other EST sequences using pbrap as described in Example 1 above. This consensus sequence is herein designated DNA35705. 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 clone: of the fulilens coding sequence for PR0269.

Forward and reverse PCR primers were synthesized: forward PCR primer S'-TGGAAGGAGATGCGATGCCACCTG -3' (SEQ ID NO:97) forward PCR primier 5'-TOACCAGTGGGGAAGGACAG-3' (SEQ ID NO:98) forward PCR primer 5'-ACAGAGCAGAGGGTGCCTTG-3' (SEQ ID NO:99) reverse PCR primer S*-TCAGGGACAAGTGGTCTCTCCC-3' (SEQ ID NO: 100) reverse PCR orimer 5'-TCAGGGAAGGAGTGTGCAGflCTG-3- (SEQ ID NO: 101) Additionally, a synthetic oligonuclcotidc hybridization probe was constructed ftrm the consensus DNA35705 sequence which had the following nucleotide sequence: '-ACAGCTCCCGATCTCAG1-rACITGCATCGCGGACGAA TCGGCGCTCGCr-3 (SEQ ID 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 libray was then used to isolate clones encoding the PR0269 geme using the probe oliganucleotide and one of' the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal Eidney tissue.

DNA sequencing of the clooms isolated as described above gave the full-length DNA sequence for OR0269 Pherin designated as UNQ236 (DNA38260-1 190)] (SEQ ID NO:95) and the derived protein sequence for PR0269.

The entire nucleodec sequecec of UNQ236 (DNA38260-1 190) is shown in Figure 35 (SEQ ID Clone UNQ236 (DNA38260-1 18D) contains a single open reading fre with an apparent translational initiation site at nucide positions 314-316 and ending at the stop codon at nucleotide positions 1784I786 (Fig. 35; SEQ ID Thc predicted polypeptide pmcursor is 490 ammno acids long (Fig. 36). Clone UNQ236 (DNA38260-1 180) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209397.

9.9.15 Analysis of the anom acid sequence of' the ful-length PR0269 suggests that portions of it possess significant homology to the human throruhomodulin proteins, thereby indicating that PR0269 may possess one or more thrombornodulin-like domnains.

5:EXAMPLE 16: Isolation of cDNA Clones Encoding Human PRO297 A consensn DNA sequence encoding PRO28 was assembled relative to the other identified EST sequences as described in Example 1 above. wherein the consensus sequenc is designated herein as DNA2872. Based on the DNA2M78 consensus sequene. oligonucleotides were synthesized to identify by PCR a cDNA library that contained 4 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 synthsized: forward PcR primer 5'.CCGATFCATAGACCTCGAGAGr-3- (SEQ ID NO:105) 9...*reverse PCR Sirinier 5'-GTCAAGGAGTCCTCCACAATAC-3* (SEQ ID NO: 106) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28728 scquece which had the following nucleodde sequence hybridization nvrobc 5 -GTGTACAATGGCCATGCCAGCCAGcGATrGGcCGCIJYGT-3- (SEQ ID NO: 107) [n order to screen several libraries for a source of a fuzll-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 PR0287 gene using the probe oligonucleotide 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 PRO287 (herin designated as UNQ250 (DNA39969-1 185). SEQ ID NO: 103] and the derived protein sequece for PROM.

127 The entdre nucleotide sequence of UNQ250 (DNA39969-1 185) is shown in Figure 37 (SEQ ID NO: 103).

Clone UNQ250 (DNA39969-1 18&5) cortains a single open reading frame with an apparent translational initiation site at nucleotide positions 30-309 andending at the stop codon at nuceotide positions 1552-1554 (Fig. 37; SEQ MD NO:103). The predicted polypeptide precursor is 415 amtino acids long (Fig. 38). Clone UNQ250 (DNA39969-1 185) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209400.

Analysis of the amnin acid sequence of the full-length PR0287 suggests that it may possess one or more procollagen C-proteinase enhancer protein precursor or procollagen C-proteinase enhancer protein-like domains.

Based on a BLAST and FastA sequence alignment analysis of the full-length sequence, PRO2W shows nucleic acid sequence identity to procoliagen C-proteinase enhancer protein precursor and procollagen C-proteuaase enhancer protein (47 and 54% respectively).

so:*EXA.MPLEi17: Isolation ofcN rloes n gn Human PRO214 .0 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. oligoniacleotides were sjalhesized: 1) to identify by PCR a cDNA library that contained the sequence of interest. and 2) for use as probes to isolate aclone of the ful-egth coding sequence.

In~ order to screca everal libraries for a source of a full-length clone, DNA from te libraries was screened by PCR amplificafton with the PCR primler pair identified below. A positive library was then used to isolate clones encoding the PR0214 gene using the probe oligonticleode: and one of the PCR primers.

.00. RNA for construction of the eDNA libraries was isolated from human fetal lung tissue. AcDNAcdone was sequenced in its entirety. The full length nucleotide sequence of DNA32286-1191 is shown in Figure 39 (SEQ ID NO: 108). DNA32286-1191 contains a single open reading frame with an apparent translational initiation site at nucleotide position 103 (Fig. 39; SEQ ID NO:108). The predicted polypeptide precursor is 420 ammno acids long (SEQ ID NO:109).

Based on a BLAST and FastA sequence alignment analysis of the full-length sequence, PR0214 polypeptide shows amino acid sequence identity to HT protein and/or Fibulin (49 and 38 respectively).

The oligonuicleotide sequences used in the above procedure were the following: 2844p..I55 5-CCGGCTATCAGCAGGTGGGCrCCAGTGTTCGATGTvGOAYAGr.TJA.3.(SEQ ID NO: 110) 28744.f (O11556) S'-ATCrGCGTGAACACGAGGGC.3* (SEQ ID NO: I111) 28744.r (0U557) 5'-ATCTGCTTGTAGCCCCGGCAC.3- (SEQ ID NO:112) EAMPE.IS: Isolation of ceDNA Clones Encodney HuMan PR0317 A consensus DNA sequence was assembled using phrap as described in Example 1 above, wherein the consensus sequence is herein designated as DNA28722. Based on this consensus sequence. oligonucleotides were syntheized: 1) to identify by PCR a cDNA library that eontained the sequence of interest, and 2) for use as probes to isolate a clone of the full4cogth coding sequence. 11W forward and reverse PCR primers, respectively. synthesized for this purpose were: S'-AGGACTGCCATTTCCG (0U489) (SEQ ID NO:l115) and -ATAGGAGTrGAAGCAGCGCTGC (0U490) (SEQ ID NO: 116).

The probe synthesized for this purpose was: 5S.TGTGTGGACATAGACGAGTGCCGC ACC ATGCAGCACCGC (0L1488) (SEQ ID NO: 117) niRNA for construction of the cDNA libraries was isolated from human fetal iddney tissue.

In order to scen several libraries for a source of a MIl-lengt clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et at.. Curret Protocols in Molecular Biology (1989). with the PCR primer pair identified above. A positive library was then used to isolate clones containing the PR0317 gene using the probe oligonucleotide identified above and one of the PCR primers.

A eDNA done was sequencd in its entirety. The entire nucleotide sequence of DNA33461-1 199 (encoding PR0317) is shown in Figure 41 (SEQ ID NO:113). Clone DNA33461-1199 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 69-70 (Fig. 41; SEQ ID NOAl 13). The predicted polypeptide precursor is 366 amino acids long. The pradicted 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 DNA33461-1199 has been deposited with ATCC and is assigned ATICC deposit no. ATCC 209367.

Based on BLAST~' and FastATm sequence alignment analysis (using the ALIGN'h computer program) of dhe full-length PRO3l7sequecec, PR0317 shows the most amino acid sequenice identity to EBAF-l The S.0: results also demonstrate a significant horology between lannan PR0317 and mouse LEFTY protein. The C-terminal end of dre PR0317 protein contains many conserved sequences consistent with the pattern expected of a member of the TUF- supierfamily.

ha situ expression analysis in humnan tissues performed as described below evidences that there is distinctly strong expression of the PRO3 17 polypeptide in pancreatic tissue.

EXAMPLE 19: Isolation of eDNA clones Encoding Human PRO301 A consensus DNA sequence designatedt herein as DNA35936 was assembled using phrap as described in 0**eeeExample I above. Based on this consensus sequence, oligorrucleoides 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.

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 PRO301 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal idney.

A eDNA clone was sequenced in its entirety. The full length nmcleotide sequence of native sequence PRO301 is shown in Figure 43 (SEQ ID3 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 molecular. weight of 32,583 daltons and p1 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 sequene alignment analysis of' the full-length sequence. pRO3OI shows aioacid sequence identity to A33 antigen precursor (30 and coxsacie and adenovis receptor protein (29 The oligonucleotide sequences used in the above procedure were the followinlg: 0112162 (35936.nl) 5S-TCGCGGAGCTGTG~rCTGTI-r CCC.31 (SEQ ID NO:120) 0112163 3

S

936 .pl) S'-TGATCGCGATGGGGACAGGCGCAIGCTCGAGAATGrJGGCCTJ. (SEQ MD NO: 121) 0112164 (35936.12) 5*-ACACCTGGflCAAAGATGGG-3 (SEQ ID NO:122) OLf5 (3S936.rl) S-TAGGAAGAG TGCTGAAGGCACGG-3- (SEQ ID NO:123) 0112166 (35936.03) s-TOCCrACTCAGGTGdTAC-3' (SEQ ID NO: 124) 15 0112167 (35936.r2) S-ACTCAGCAGTGGTAGGAAAG-3' (SEQ MD NO: 12) EXAMPfLE 2: Isolation of cDNA Clones Encodin Human PRO224 A consensus DNA sequence assembled relative to the other identified EST sequences as described in Exanple 1, wherein the consensus sequare is designated herein as DNA30845. Eased on the DNA30845 consensus sequence. oligoruicleotides 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 PR0224.

pair of PCR primers (forward and reverse) were synthesized: forward PCR primer S-AAG7TCCAGTGCCGCACCAGTGGC.3' (SEQ ID NO: 128) reverse PCR primer 5*-rrGGT7CCACAGCCGAGCTCGTCG-3 (SEQ ID NO: 129) Additionally. a synthetic oligonucleooide hybridization probe was constructed from the consensus DNA30945 sequence which had the following nucleotide sequence hybridization nrobe 5'GGAGGGAG7GGCTGACAAAGCAGCAC3 (SEQ WD NO:130) In order to screen several libraries for a source of a full-length clone. DNA from the libraries was screened 430 by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones; encoding the PR0224 gene using the probe oligomicleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. DNAacn of the clones isolated as described above gave the full-length DNA sequence for PR0224 (herein designated as UNQ198 (DNA33221-1l33)J and the derived protein sequence for PROM2.

The entire nucleotide sequence of UNQ199 (DNA33221-l 133) is shown in Figure 45 (SEQ ID NO: 126).

Clone UNQ198 (DNA33221-1133) conitains a single open reading framre, with an apparent translational initiation site at nucleotide positions 96-98 and ending at the stop codon at nucleotide positions 942-944 (Figur 45; SEQ IID NO: 126). The start of a transmeinbrane region begins at nuceotide position 777. The predIcted polypeptide precuror is 282 amino acid& long (Figure 46). Clone UNQ198 (DNA33221-l 133) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209263.

Analysis of the amino acid sequence of the full-length PR0224 suggests that it has homology to very low-' 130 density lipoprotrin receptors. apolipoprotein E receptor and chicken oocytc receptors P95. Based on a BLAST and FastA sequence alignment analysis of the full-length sequence. PRO224 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%.

EXAMPLE 21: Isolation of cDNA Clones Encodinp Human 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 DNA28771 consensus sequence, 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 PRO222.

A pair of PCR primers (forward and reverse) were synthesized: 10 forward PCR primer 5'-ATCTCCTATCGCTGCTTrCCCGG-3' (SEQ ID NO:133) *reverse PCR primer 5'AGCCAGGATCGCAGTAAAACTCC-3' (SEQ ID NO:134) Additionally, a synthetic oligomnuclcotide hybridization probe was constructed from the consensus DNA28771 sequence which had the following nuclcotide sequence: hybridization probe 15 5'-ATrFAAACTTGATGGGTCTGCGTATCTGOAGTGCTACAAAACCTTATCT-3' (SEQ ID NO:135) 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 PRO222 gene using the probe oligonucleotide 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 PRO222 [(herein designated as UNQ196 (DNA33107-1135)] and the derived protein sequence for PRO222.

The entire nuclcotide sequence of UNQ196 (DNA33107-1135) is shown in Figure 47 (SEQ ID NO:131).

Clone UNQI96 (DNA33107-1135) contains a single open reading frame with an apparent translational initiation site at nuclcotide positions 159-161 and ending at the stop codon at nucleotide positions 1629-1631 (Fig. 47; SEQ ID 25 NO:131). The predicted polypepid precursor is 490 amino acids long (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 full-length sequence, PRO222 shows amino acid sequence identity to mouse complement factor I precursor complement receptor mouse complement C3b receptor type 2 long form precursor (25-47%) and human hypothetical protein hkaaO247 EXAMPLE 22: Isolation of cDNA clones Encodine PRO234 A consensus DNA sequence was assembled (DNA30926) using phrap as described in Example 1 above.

Based on this consensus sequence, oligoauclcotides 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.

RNA for the construction of the cDNA libraries was isolated using standard isolation protocols. e.g., Ausubcl et al., Current Protocols in Molecular Biology, from tissue or cell line sources or it was purchased from commercial sources alontedh). The cDNA libraries used-to isolate the cDNA clones were constructed by standard methods Ausubel el at.) using commnercially available reagents Invitrogen). This library was derived from 22 week old fetal brain tissu.

A cDNA done was sequenced in its entirety. The entre nicleotide sequence of PR0234 is shown in Figure 49 (SEQ ID 140:136). The predicted polypeptide precursor is 382 amino acids long and has a calculated molecular weight of approximtely 43.1 Wa.

.The oigoaucleotide sequences used in the above procedure were the following: 3096.p (011826) (SEQ ID 40: 138): 5*-G CATrOAAAACCTCT1'GcCATCT GATGGTGACFFCTGGA7TGGGCTCA-3' 3096.f (0U827) (SEQ ID NO: 139): S'-AAGCCAAAGAAGCCTGCAGGAGGG-3' 10 3M96.r (011828) (SEQ ID 140:140): 5'-AGTCCAAGCATAAAGGTCCTGGC-3' EXME ~23- Isolation of eDNA Clones Encodin, Humnan PRO231 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, oligomicleotides 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 PR023 1.

Three PCR primers (tw~o forward and one reverie) were synthesized: *forward PCR primar 1 5'-CCAA4CTACCAAAGCTGCTGGAGCC-3' (SEQ ID NO: 143) forward PCR pmiter 2 S'-GCAGCTCTATTACCACGGGAAGGA3' (SEQ ID NO: 144) reverse PCR primer 5'-TCCflCCCGTGGTAATAGAriCTGC-3' (SEQ ID NO:145) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30933 sequenc which had the following nucicoide sequence hybriizaion probe 5-GC1CAGAGAACCAGAGGCCGGAGGAGACTGCCTCTITACAGCCAGG-3 (SEQ ID 140:146) 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 PR0231 genec using the probe oligonudeotide and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated from human feral liver tissue. DNAsequeadzv of the clones isolated as described above gave the full-length DNA sequence for PRO231 [herein designated as UNQ205 (DNA34434-l 139)J and the derived protein sequence for PR0231.

The entire nuclootdec sequenice of UNQ205 (DNA34434-1 139) is shown in Figure 51 (SEQ ID NO: 14 1).

Clone UJNQ205 (DNA34434-1 139) conains a single open reading frame with an apparent translational initiation site at nucleotide positions 173-175 and ending at the stop codon at nucleotide positions 1457-1459 (Fig. 51; SEQ ID NO: 141). The predicted polypepde pmrursor is 428 amino acids long (Fig. 52). Clone UNQ205 (DNA34434-l 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 acdd phosphatase precursor proteins, respectively.

132 EXAMPLE14: Isolation of cDNA Clones Fjicodinp Hum;In pR0229 A consensus DNA sequence was assembled relative 10 other EST sequences Using phrap as described in Example I above. This consensus sequence is herein designated DNA28762. Based on the DNA29762 consensus Sequence. oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that cont .ained the sequence of interest. and 2) for use as probes to isolate a clone of the full-length coding sequenice for PR0229.

A pair of PCR primers (forward and reverse) were synthesized: frad C pie 5'-TTCAGCTCATCACrTCACC-GCC-3. (SEQ ID NO: 149) reverse PCR mimer 5'-WGCCATACAAATACCACAGJ(S.3. (SEQ Additionally. a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28762 sequience which had the following ntuceotide sequence CCAC~GGACGGGGAGG;AAAA CAT3 (SEQ ID NO: 151) In Order to san= several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR printer pair identified above. A positive library was then used to isolate clones encoding the PR0229 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from 1humain fetal liver tissue. DNAseqmdne Of the clones isolated as described above gave the fulfl-length DNA sequence for PR0229 [herein designated as UNQ203 (DNA33100-1 159)] (SEQ ID NO: 147) and the derived Protein sequence for PROM2.

entire nudleodde sequence of UNQ203 (DNA331OO-1 159) is shown in Figure 53 (SEQ ID NO: 147)*.

Clone UNQ203 (DNA33 IOO-1 L59) contains a single open reading frame with an apparent translational initiation site :at nucleotide positions 98-100 and ending at the stop codon at nudeootide positions 1139-I1141 (Figure 53). The Predicted polypeptide precursor is 347 amino acids long (Figure 54). Clone UNQ203 (DNA33100-l 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 Enicoding Human pR023g A consensus DNA seaue= was assembled relative to other EST sequences using phrap as described above in Example 1 This consensus sequenc is herein designated DNA30908. Based on the DNA30908 consensus seqce,= oligonueleoticles were synthesized: 1) to identify by PCR a CDNA library that contained the sequence of interest, and 2) for use as Probes to isolatesa clone Of the full-length coding sequence for PROM3.

PCR primers (forward and reverse) were synthesized: forward PCR nrirncr I 5'-GGTGCrAAACTGGTGCTCTGTGGC.3 (SEQ ID NO: 154) forward PCR primer 2 5'-CAGGG;CAAGATGAGCATTrCC.3 (SEQ ID NO: 155) reverse PCR nride S'-TCATACrGrFCCATCTCGGCACGC-3. (SEQ ID NO: 156) Additionally, a synthetic oligomuxleotide hybridization probe was constructed from the consensus DNA30908 sequence which had the following smelootide sequence hybridization probe CATC3 (SEQ ID NO: 157) In order to screen several librauies for a source of a full-length clone, DNA from die libraries was screened by PCR amplification wMi the PCR primner pair identified above. A positive library was then used to isolate clones encoding die PR0238 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of dhe cDNA libraries was isolated from human fetal liver tissue. Dt4Asqrirg of the clones 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 DNA35600-1162 contains a single open reading frame with an apparent translational initiation site at nucleotide :::.positions 134-136 and ending prior to the stop codon at nmclodde positions 1064.1066 (Figure 55). The predicted polypeptide prwmoris 310amino acids long (Figure 56). Clone DNA356O-1162 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 15 possess significant homology to redwitase, particularly oxidoreductase, thereby indicating that PR0239 may be a novel reductase.

EXA.MPL. 26: Isolation of cDNA Clones Encoding Human PRO233 The extraceilular domain (ECD) sequececs (including die 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)l :databases. The EST database included public EST databases GenBarik) and a proprietary EST DNA database

(UFESEQ

Tm Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAS12 (Altshul et al., M bosi am zy2M6:460-480 (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 of 70 (or in some eases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program lphrap (Phil Green, University of Washington. Seattle. Washington; huip:/Ibozeian.mbt.washington.edipp.does/phraphm) An expressed sequencie tag (EST) was identified by the EST database search and a consensus DNA sequence was assembled relative to other EST sequences using phrap. Ibis consensus sequence is herein designated DNA30945. Based on the DNA30945 consensus sequenc, 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 PR0233.

Forward and reverse PCR primers were synthesized: forward PCR 'rn~ 5S-GGTGAAGGCAGAAAI7GGAGATY-3, (SEQ ID NO:160) reverse PCR primer 5'-ATCCCATGCATCAGCCTG1TACC-3. (SEQ ID NO: 161) Additionally, a synthetic oligonucleotide hybridization probe was constructed from dhe consensus DNA30945 sequence which had the following aucleotide sequence hrdizationjlzrb 5-GCTGGTGTAGTCTATACATCAGATTGTrGrCTACACAAGATCCTCAG-3* (SEQ ID NO: 162) In order to screert several libraries for a source of a full-length clone, DNA from the librarie s was screened by PCR amplification with t PCR priner pair identified above. A positive library was then used to isolate clonss encoding the PR0233 gene using the piobc oligonucleodde.

RNA for construction of the cDNA libraries was isolated from human fetal brain tissue. DNAzqcozg of the clones isolated as described above gave the full-lenigth DNA sequence for PR0233 (herein designated as UNQ207 (DNA34436-1238)] (SEQ ID NO:158) and the derived protein sequence for PR0233. The entire nucieotde sequence of UNQ2O7 (DNA34436-1238) is shown in Figure 57 (SEQ ID NO:158).' Glone UNQ207 (DNA34436-128) contains a single open reading frame with an apparent tnanslationial initiation site at nucleotide positions 101-103 and ending at the stop codon at ideotide positions 1001-100 (Figure 57). The predicted polypeptide precursor is 300 amino acids long (Figure 58). The fulli-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 UNQ2O7 15 (DNA34436-1238) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209523.

Analysis of the amno acid sequence of the fUl-lcngth PR0233 polypeptide suggests that portions of it possess significant homology to reduetase proteins, thereby indicating that PR0233 may be a novel reductase.

EAMPLE 2 Isolation of cDNA Clones Encodino Human PRO223 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Examplel1above. This consensu~s sequceis herin designated DNA30836. Based on the DNA30836 consensus sequence, oligonucleotides were synteized: 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-length coding sequece= for PR0223.

PCR primer pairs (one forward and two reverse) were synthesized: 25 forward PCR primer 5*-TCCATGCCACCTAAGGGAGACTC-3' (SEQ ID NO:165) reverse PCR nrimerI S'-TGGATGAGGTGTGCAATGGCTGGC-3' (SEQ ID NO: 166) reverse PCR p~rimer 2 5*-AGCTCTC-AGAGGCTGGTCATAGGG-3' (SEQ ID NO:167) Additionally, a synthetic oligowuclotide hybridization probe was constructed from the consensus.- DNA30836 sequence which had the following nucleotide sequence 5'GTCGGCCCMrCCCAGGACTGACATAAGAGrATGCCCCCC7AC.3' (SEQ ID NO:168) In order to scren 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 PRO22 gene using the probe ollgonucleotide and one of the PCR primers.- RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. DNAsequar*g of the clones isolated as described above gave the full-length DNA sequence for PR0223 (herein designated as UNQI 97 (DNA33206-1 165)] (SEQ ID NO: 163) and the derived protein sequence for PR0223.

The entire nucleotidce sequence of UNQI97 (DNA33206-1 165) is shown in Figure 59 (SEQ ID NO:163).

Clone UNQ197 (DNA33206-1165) contains a single open reading frame with an apparent tanslational initiation site at nucleotide positions 97-99 and ending at the stop codon at nucleotide positions 1525-1527 (Figure 59). The predicted polypcptid precursor is 476 amino acids long (Figure 60). Clone UNQI97 (DNA33206-l 165) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209372.

Analysis of the amino acid sequence of dithe full-length PRO223 polypeptide suggests that it possesses significant homology to various scrine carboxypeptidase proteins, thereby indicating that PRO223 may be a novel serine carboxypcpidase.

EXAMPLE 28: Isolation of cDNA Clnes Encodin Human PR0235 10 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Examnple 1 above. Thi 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 clone of the full-length coding sequence for A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer S'-TGGAATACCGCCTCCTGCAG-3' (SEQ ID NO:171) reverse PCR primer S'-CITCTGCCCTrTGGAGAAGA TGGC-3' (SEQ ID NO: 172) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30927 *sequence which had the following nucleotide sequence hvbridization probe 5s-GGACFCACTGGCCCAGGCCTCAATATCACCAGCCAGGACGAT-3' (SEQ ID NO:173) In order to screeii several libraries for a source of a full-length clone, DNA from the libraries was screened *by PCR amplification with the PCR primer pair idendfied above. A positive libray was then used to isolate clones encoding the PR0235 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. DNAqumcirg 25 of the clones isolated as described above gave the full-length DNA sequence for PRO235 [herein designated as UNQ209 (DNA35558-1167)] (SEQ ID NO:169) and the derived protein sequcce for PRO23S.

The entire nucleotide sequence of UNQ209 (DNA35558-1167) is shown in Figure 61 (SEQ ID NO:169).

Clone UNQ209 (DNA35558-1167) 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 61). The predicted polypeptide 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 PRO235 polypeptide suggests that portions of it possess significant homology to the human, mouse and Xenopus plexin protein, thereby indicating that PRO235 may be a novel plexin protein.

EX&MeE 2: Isolation of eDNA Clones Eindong Human pR0236 and Hu=a PRO262 Consensus DNA sequences were assembled relative to other EST seqces using Phrap, as described in Example I above. These consensus sequenes are herein designated DNA30901 and DNA30947. Eased on the DNA3090i and DNA30847 coneum sequel=e, ofigoamcleotide3 were synthesized: 1) to identify by PCR a cDNA library bhat-contained the sequence of interest, and 2) for use as probes to isolate a clone of the ful-length coding Sequence for PR0236 and PR0262, respectively. 7 Based upon the DNA30901 consensus sequence. a pair of PCR primers (forward and reverse) were synthesized: forward PCR primer S-TGGCTACTCCAAGACCCTGGATG-3. (SEQ ID NO:l78) reverse PCR primer S.-TGGACAATCCCMGCTCAGCC(-3' (SEQ ID NO: 179) :.10 Additionally, a synthetic oligonueleotide bybridizazon probe was constructed from the consenus DNA30901 **sequenc which had the following nucleotide: sequec bybtdino mob ACAGAGGACrA TACA~GTTCGG3(SEQ ID NO: 180) Based upon the DNA30847 consenss sequence, a pair of PCR primers (forward and reverse) were 15 synthesized: forward PCR primner 5'-CCAGCTATGACTATGATGCACC..3* (SEQ ID NO:181) reverse PCR primer S'-TGGCACCCAGAATGCJTGTTGGCT)C.3' (SEQ ID NO: 182) Additionally, a synthetic oligonucleotde hybridization probe was constructed from the consensus DNA30847 sequence which had the following nucleotate sequence tryriiaiprobe SCGAGATGTCATCAGCAAGT CCAGG ajMGACC7J A Cl-l (SEQ ID NO:183) In order to screen several libraries for a source of full-length clones, DNA from the libraries was screened by PCR amnplification with the PCR primer pairs identified above. Positive libraries were then used to isolate clones encoding the PR0236 and PR0262 genes using the probe oligonuclotdes and one of the PCR primers.- RNA for construction of the eDNA libraries was isolated from human fetal lung tissue for PR0236 and human fetal Eiver tissue for PR0262.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0236 therein designated as UNQ210 (DNA35599-1168)J (SEQ MD NO:174), the derived protein sequence for PR01236, the fil-length DNA sequaee for PRO262 Iherein designated as UNQ229 (DNA36992-1168)] (SEQ MD 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 (DNA35599-1168) contains a tingle open reading frame with an apparent translational initiation site at nucloodde positions 69-71 and ending at the stop codon at nucleotdec positions 1977-1979 (Figure 63). The predicted polypeptide precursor is 636 amino acids long (Figure 64). Clone UNQ21O (DNA3SS9Q-1168) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209373.

The entire niucleotide sequence of UNQ229 (DNA36992-1168) is shown in Figure 65 (SEQ ID NO: 176).

Clone UNQ229 (DNA36992-1168) contains a single open reading frame with an apparent translational initiation site at nucleocide positions 240-242 and ending at the stop codon as nuclcotide positions 2202-2204 (Figure 65). The predicted polypeptide precursor is 654 amino adids 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 PR0262 polypeptides suggests that potons of diosc pobvypdpr possess significant homology to A-Salactosidase proteins derived from various sources.

thereby indicating that PR0236 and PR0262 may be novel P-galacttmidase homologs.

EXA?4PELE..I Isolation of eD)NA Clones Encoding Human PRO239 A consensus DNA sequence was assembled relative to odher EST sequences using phrap as described in Example I above. This consensus sequence is herein designated DNA30909. Based an the DNA3099 consensus 10 sequence. oigonaeleotdes 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 PROM3.

A pair of PCR primers (forward and reverse) were synthtesized: *forward PCR nrimer S'-CCTCCCTCTATrACCCATGTC.3' (SEQ ID NO:186) reverse PCR primer 5'-GACCAACTrrCTCTGGcjAcTGAGG.3* (SEQ ID NO: 187)- Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus 'DNA30909 sequence which had the following nucleotide sequence hbrdiation probe 5SGTCACFAmCTCTAACACAAGCATCCACAGrJ(CA.3' (SEQ ID NO: 188) 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 PR0239 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated from husman fetal lung tissue. DNAseqzmiz of the clones isolated as described above gave the full-length DNA sequence for PR0239 [herein designated as UNQ213 (DNA34407-1 169)] (SEQ ID NO:184) and the derived protein sequence for PROM3.

*The entire nucleotide sequence of UNQ213 (DNA34407-1169) is shonin Figure 67 (SEQ ID NO: 184)'.

Clone UNQ213 ODNA34407-1169) contains a single open reading frame with an apparent translational initiation site at szjcleotide positions 72-74 and ending at the stop codon at micleoodde positions 1575-1577 (Figure 67). The predicted polypeptdec precursor is 501 amin acids long (Figure 68). Cone UNQ213 (DNA34407.l 169) has been deposited with ATCC and is assigned ATCC deposit no.ATCC 209383.

Analysis of the amino acid sequence of the ful-length PR0239 polypeptide suggests that portions of it possess significant homology to the densin protein, thereby indicating that PR0239 may be a novel molecule in the densin family.

EXAMPLE.3.1: Isolation of cDNA Clones Encoding uin PRO257 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example I above. This consensus sequence is herein designated DNA2873 1. Based on the DNA28731 consensus sequence. oligonuclooddes were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of intercst. and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO2S7.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR 12der S-TCTCTATCCAAACTGTGGCGc-r.31 (SEQ ID NO:191) reverse POR primer 5'-MGrATGACGA-rCGAAGGTGG.3' (SEQ ID NO: 192) Additionally. a synthetic oligonuclcotidc hybridization probe was constructed from the consensus DNA28731 sequence which had the following nucleotide sequence 5'GAGACICCCGCCArCCAACGACTAC3 (SEQ ID NO:193), 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 identi ed above. A positive library was then used to isolate clones encoding dhe PR0257 gen using the probe oligomicleotide and one of tie PCR primers.

RNA for contucton 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 PR0257 (bertindesignazted as UNQ224 (DNA35841-1173) (SEQ ID NO:189) and the derived protein sequence for PR0257.

.The entire nuicleotide sequence of UNQ224 (DNA3594I-l 173) is shown in Figure 69 (SEQ ID N6:189).

Clone UNQ224 (DNA35841-l 173) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 964-966 and ending at the stop codon at nucleotide positions 2785-2787 (Figure 69). The predicted polypeptide precrsor is 607 amino acids long (Figure 70). Clone UNQ224 (DNA35S4 1-1 173) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 20943.

Analysis Of the amin acid sequence of the ful-lngth PR0257 polypeptide suggests that portions of it popsessiagnificant homology to the ebnerin protein, therby indicating that PR0257 my be a novel protein member reltedto heebnerin protein.

EXAMPLE 32: Isolation of cDNA Clones Encoding Human PR026Q *25 A consensus DNA sequence was assembled relative to other EST sequences using phrap, as described in E~xample I aove. Thi consensus seuec is herein- designated DNA30834. Based on the DNA30834 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 PR0260.

PCR primers (forward and two reverse) were synthesized: forward PCR D cR 5'-TGGTTGACCAGGCCAAGTrCGG-.3' (SEQ ID NO: 196); reverse PCR nrimer A 5'-GATTCATCCTCAAGGAAGAGCYG.3' (SEQ ID NO:197); an reverse PCR Strimrer Be 5AACTrGCAGCATCAGCCACTCrvC.3* (SEQ IM NO: 198) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30834 sequence which had the following nucotide sequence: 5S-1FCCGTGCCCAGCTrCGGTAGCGAGTGGnJ~rGTJGTATTGG1CA-3' (SEQ ID NOJ19) In order to scree seveual libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with t PCR primer pair identi fied above. A positive librazry was then used to isolate clones encoding the PR0260 gene using the probe oligonucleotide and one of fte PCR primers.

RNA for construction of dhe cDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave the full-lngth DNA sequence for PR0260 (herein designated as LJNQ2Z7 (DNA33470-1 175)] (SEQ ID NO: 194) and the derived protein sequenc for PR0260.

The entire nucleotide sequence of UNQ227 (DNA33470-l 175) is shown in Figure 71 (SEQ MD NO: 194).

Clone UNQ227 (D5NA33470-1 175) cotntains a single open reading frm 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 p oris 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 the amino acid sequence of the fullcngth MR0260 polypptde suggests that portions- of it possess significant homoalogy to the alpha-l-fucosidase precursor, thereby indicating that PR0260 may be a novel EXA1EX33: Isolation of cDNA Clones Emneodne umran PR0263 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, 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 PROM6.

PCR primers (tow forward and one reverse) were: synthesized: forward PCR urimer is 5S-GAGCFICCATCCAGGTGTCATGC-3' (SEQ ID NO:202); forward PCR primer 2 5'-GrCAGTGACAGTACCTACTCGG-3' (SEQ ID NO:203);mrverse PCR ndrime 5'-TGGAGCAGGAGGAGTAGTAGTAGG-3' (SEQ ID NO:204) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensu DNA30914 sequence which had the following nucleotide sequence: 5'-AGGAGGCCTGTAGGCrGCTGGGACTAAG1TGGCCGGCAAGGACCAAGTr-3' (SEQ ID NO:205) In order to screen several briis for a source of a fuill-length clone, DNA Erom 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 oligonucleotde and one of the PCR primers.

RNA for constrction of the eDNA libraries was isolated from human fetal liver tissue. DNA0*=cing of the clones, isolated as described above gave the full-length DNA sequence for PR0263 (herein designated as UNQ230 (DNA3443 1-1177)1 (SEQ ID NO:200) and the derived protein sequence for PROM6.

The entire tuclotide sequence of UNQ230 (DNA34431-1 177) is shown in Figure 73 (SEQ ID NO:200).

Clone UNQ230 (DNA3443 1-117T7) contains a single open reading fre with an apparent tranislational initiation site at nucleotde positions 16D-162 of SEQ ID N0200 andl ening 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 (DNA34431-ll77h has been de-posited with ATCC and is assigned ATCC deposit no. ATCC 209399.

Analysis of the amino acid sequence of the full-lengthi PR0263 polypeptide suggests that portions of it possess stgoiflcanr homology to CD44 antigen, thereby indicating diat PR0263 may be a novel cell surface adhesion molecule.

EXAMPJL 3: Isolation of cDNA Clones Etncoding Human PR07 A consensus DNA sequence was assembled relative to dhe other identifed EST sequences as described in Exampl 1 above. wherei die consensus sequence was designated herein as DNA35712 Based on the DNA35712 consensus sequence. oligozmcleotides 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 dhe full-length coding sequee for PROM7.

Forward and reverse: PCR primers were synthesized: forwar PCR V 5'C= GTTCCAGGCA- (SEQ ID O28 forar PR rier(12) S'-TGGAGACAATATOCCGAIC.3' (SEQ ID NO:209) reverse PCR primer (A)l 5'-AACAGTTGGCCACAGCATGGCAGG-3' (SEQ IDNO:210) Additionally, a synthetic oigonuceotide hybridization probe was constructed from the consensus DNA35712 Sequence which had the following nucleotide sequence (SEQ ID N:21 1) *n -norder to screen several libraries for a source of a full-length clone, DNA from the libraries was scened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones :encoding the PR0270 gene using'the probe oligonicleodde and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. DNA eqxring of the clones isolated as described above gave the full-legth DNA sequence for PR0270 Dierein designated as UNQ237. DNA39510-1 181] (SEQ ID N0:206) and the derived protein sequence for PROM7.

25 The entire nucleotide sequence of UNQ237. DNA39510-1181 is shown in Figure 75 (SEQ ID NO:206).

Clone UNiQ237 (DNA39510-1 181) contains a single open reading fre with an apparent translational initiation site at miloridc positions 3-5 and ending at the stop codon at nucleodde positions 891-893 (Fig. 75; SEQ ID N0.206).

The predicted polypepude prsor is 296 amino acids long (Fig. 76). Clone UNQ237 (DNA39S 10.111) has been deposited with ATCC and is assigned ATCC deposit no.. ATCC 209392.

Analysis of the amino acid sequence of the full-ength PRO770 suggests that portions Of it possess Significant homology to the thioredoxin-protein, thereby indcating that the PR0270 protein may be a novel member of the thioredoxin family.

EXME 35: Isolation of eDNA Clones ncoding Human 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 designated DNA3S737. Based on the DNA35737 consensus sequence. oligonuecotides were synthesized: 1) to identify by PCR a cDNA library that contained dhe sequence of interest. and 2) for use as probes to isolae a clone of the fll-length coding sequence for PR0r271.

Forward and rcverse PCR primers were synthesiz;.ed: forward PCR Rrirner I S'-TGCITCGCTACTGCCCI'C-3, (SEQ ID NO:214) forward PCR nrimer 7 5*-TTCCCrTGTGGGTTGGAG-3' (SEQ ID 140215).

forward PCR nrirner 3 5S-AGGGCTGGAAGCCAGTrC.3' (S EQ ID NO:216) reverse PCR prirner I S'-AGCCAGTGAGGAAATGCG-3,. (SEQ ID NO:217) reverse PCR n~rimer 2 5'-TGTCCAAAGTACACACACCFOAGGC3' (SEQ ID NO:21 8) Additionally, a synthetic oligoucleodde hybridization probe was Constructed from die consensus DNA35737 sequence which had the following nucleotide sequence 5-GATGCCACGATCGCCAAGGTGGGACAGCTC rrCCCMJ 4 &AA3 (SE.Q ID NH0219) In order to screen seveal 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 encoding the PR0271 gene using die probe oligonuceotde and one of the PCR primers.

RNA for construction of the eDNA libraries' was isolated from human frtal brain tissue. DNAeqmixg .15 of the clones isolated as described above gave the full-lengt DNA sequence for PR0271 (herein designated as UNQ238 (DNA39423.l 182)1 (SEQ ID NO:212) and the derived protein sequence for PR0271.

The entire zarcicotide sequence of UNQ238 (DNA39423,-1 182) is shown in Figure 77 (SEQ IM NO:212).

Clone UNQ238 (D)NA39423-l 182) contains a single open reading frame with an apparent translational initiation site at nuclcotide 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 (DNA39423-1182) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209387.

Analysis of the amino acid sequence of the flil-length PR0271 polypeptide suggests that it possess significant homology to the protoglcan link pruttin. theteby indicatig that PR0271 may be a link protein hornolog.

EAMPE a36: 1kolatinn of eDNA Clones Encoding Human PR07 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Examiple 1 above. This consensus sequence is herein designated DNA3 6460. B~ased on the DNA36460 consensus sece=. oligonucleotides were synthesized: 1) to identify by PCR a cD)NA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full1-length coding sequence for PROM7.

Forward and reverse PCR primers were synthesized: forward PCR primer S'-CGCAGGCCCTCATGGCCAGG..3' (SEQ HD NO:222) forward PCR Vrimr 5*-GAAATCCTGGGTAArroc-3' (SEQ ID NO:223) reverse PCR riner 5'-GTGCGCCGGCTCACAGCTCATC..3* (SEQ ID NO:224) Additionaltly, a synthetic oligomacleotide hybridization probe was constructed from the consensus DNA36460 sequence which had the folowing nucleotide sequence CCC G r-3(SEQ ID NO:225) In order to seree several libraries for a source of a full-kugtb clone. DNA from the libraries was screened by PCR amplification with the PCR primer pairs ideatified above. A positive library was then used to isolate clones encoding the PR0272 gene using the probe oligoraiceotidec and one of the PCR primers.

RNA for construction of the cOHA libraries was isolated from human fetal lung tissue. DNAsnqmnkg of the clones isolated as described above gave the full-length DNA sequence for PR0272 therein designated as UNQ239 (DI4A40620-1l183)J (SEQ IM NO:220) and the derived protein sequence for PROM7.

The entire nucleotide sequence of UJNQ239 (DNA40620-l 183) is shown in Figure 79 (SEQ ID NO:220).

Glone UNQ239 (DNA40620-l 183) contains a single open reading fr-ame with an apparent translational initiation site at micleoide positions 35-37 and ending at the stop codon at nucleotide positions 1019-1021 (Figure 79). The predicted polypeptide precursor is 328 amino acids long (Figur 80). Glone UNQ239 (DNA40620-11S3) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209388.

Analysis of the amino acid seuneof the Mul-length PRO272 polypptde suggests that portions of it possess significant homology to the human and mouse retiaxiocalbin proteins, respectively, thereby indicating that PR0272 may be a novel reticulocalbin protein.

EXAMPLE..37: Isolation of eDNA Clones Encoding Human PRO294 A conscnsus DNA sequenice was assembled relative; to other; EST sequences using phrap as described In Example I above. This consen sequence is herein designated DNA35731. Based on the DNA35731 consensus sequez=, oligonucleotides were syntheized: 1) to identify by PCR a eDNA library that contained the sequence of interest, and 2) for use as probes to isolate a done of the full-length coding sequence for PROM9.

Forward and reverse PCR primers wer synthesized: *.:forward PCR primer (fl) 5'-TGGTCTCGCACACCGATC-3' (SEQ ID N0:228) forward PCR mmr(12) 5'-CTGCTGTCCACAGOGGAG-3' (SEQ ID NO:229) forwar PCR lidmer (13) 5'Cr AGAATCr!3 (SEQ ID NO:230) forward PCR nrimer (1f4) S-GAGATAGCAATrTCCGCC-3' (SEQ ID NO:23 1) rvre C Rd=ni (rI) Y-TICCTCAAGAGGGCAGCC-3' (SEQ ID NQ-132) m-ese CRRrier 5'-CrrGGCACCAATGTCCGAGATrr-3* (SEQ ID NO:233) Additionally, a synthetic oligonucleotide hybridization proibe was constructed from the consensus DNA35731 sequence which had the following nucleotide sequence bybhidxztion prob 5'-GCrCTGAGGAAGGTGACGCGCGGGGCCTCCGAACCCrrGGCCInG.3' (SEQ ID NO:234) In order to screen several libraries for a sourcc of a full-length clone, DNA from the libraries was screened by PCR amplification with die PCR printr pairs Identified above. A positive library was then used to isolate clones encoding the PR0294 gene using the probe oligonrucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal brain tissue. DN&MUEqmaing of the clones isolated as described above gave the full-length DNA sequence for PR0294 (herein designated as 144 UNQ257 (EM40604-1187) (SDE ID NO:226) and the derive protein sequence for PR0294.

The entire nucleotide sequence of UNQ257 (INN40604-1187) is shown in Figure 81 (SEQ ID N3:226) Clone UQ257 (EIA40604-1187) contains a single open reading frane with an apparent translational- initiation site at nucleotide positions 396-398 and ending at the stop codon'at nucleotide positions 2046-2048 (Figure 81). 'IThe predicted polypeptide precursor is 550 amino acids long (Figure 82). Clone U.N257 (EM40604-1187) has been deposited with AItC and is assigned ATC= deposit no. 209394.

Analysis of the amino acid sequence of the full-length PRO294 polypeptide suggests that portions of it possess significant hMndogy to portions of various collagen proteins, thereby indicating that PRO294 riay ~be collagen-like molecule.

EWIPLE 38: Isolation of cE MCanes Eacoding muan PRO295 A consensus [M sequence was asseabled relative to other EST sequences using phrap as described in Ecanple 1 above. This consensus sequence is herein designated oNA35814. Based on the ENA35814 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cEA 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 prirners were synthesized: forward PCR primer(.fl) 5'-AGAGX1GAICOGGCIG (SEQ ID ND:237) forward PC primer(.f2) 5' -CCAGCA7GWAC'ICtAG-3' (SEQ ID N3:238) 25 forward PCR prier(.f3) 5'-T1GCAGCITCAIAGG-3' (SED ID N:239) forward PR primer(.f4) 5' -Or333CAAAAAAA C-3' (SED ID ND:240) reverse PCR priner(.rl) 5' CltIXA lrC-3' (SEQ ID NO:241) Additionally, a synthetic oligonucleotide hybridization probe was constructed fromn the consensus ENA35814 sequence which had the following nucleotide sequence.

hybridization probe 3IAG (SEX ID NO:242) In order to screen several libraries for a source of a fulllength clone, rA fran the libraries vas screened by ICR arplification 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.

RM for construction of the cIA libraries was isolated frnom human fetal lung tissue. IEA sequencing of the clones isolated as 145 described above gave the full-length EM sequence for PRO295 (herein designated as UNQ258 (ENA38268-1188) (SEQ ID N0:235) and the derived protein sequence for PRO295.

The entire nucleotide sequence of LUV258 (O438268-1188) is shown in Figure 83 (SED2 ID W:235). Clone UNQ258 (I=A38268-1188) contains a single open reading frane with an apparent translational initiation site at nucleotide positions 153-155 and ending at the stop codan at nucleotide positions 1202-1204 (Figure 83). The predicted polypeptide precursor is 350 amino acids long (Figure 84). Clone UNQ258 (MA38268-1188) has been deposited with AIC and is assigned AIC deposit no. 209421.

j -Analysis of the amino acid sequence of the full-length PRO295 polypeptide suggests that portions of it possess significant hcanology to ~the integrin proteins, thereby indicating that PRO295 nmay be a novel integrin.

EXAMPLE 39: Isolation of c!l A Clones Emooding Sman PR293 The extracellular danain (ECD) sequences (including the secretion signal, if any) of fran about 950 known secreted proteins franm the Swiss-Prot public protein database were used to search expressed 20 sequence tag (EST) databases. The EST databases included public EST databases GenBank) and a proprietary EST E database (LIFESEDP, Incyte Phararrceuticals, Pal Alto, CA). The search was perforred using the catruter program BLAST or BLAS2 (Altshul et al., Methods in Enzynology 266:460-480 (1996)) as a caparisan of the ECD protein sequences to a 6 frane translation of the EST sequence. Those caiparisons resulting in a BLAST score of 70 (or in sare cases 90) or greater that did not encode known proteins were clustered and asserbled into consensus I sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington; http: //bozenan.nt .washington.edu/phrap.docs/phrap.html) Based on an expression tag sequence designated herein as T08294 identified in the above analysis, oligonucleotides ware synthesized: 1) to identify by PCR a cmA 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 prinmers (forward and reverse) were synthesized: forward PC prirer 5 -AACAAGGrAAGAOCATCCG-3' (SEQ ID ND:245) reverse PRpriner 51 -AAA rvwAu -3 (SEQ ID 3NO:246) Additionally, a synthetic oligonucleotide hybridization probe was constructed fran the expression sequence tag which had the following 146 nucleotide sequence.

hybridization probe TCA CCIAGACATGACAACCAGC-3' (SBQ ID ND:247) In order to screen several libraries for a source of a fulllength clone, UA frcrn the libraries was screened by PCR .aplification 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 primers.

RNA for construction of the cEIA libraries was isolated fromn hurman fetal brain tissue. EA sequencing of the clones isolated as described above gave the full-length MNA sequence for PRO293 (herein .designated as UNQ256 (tA37151-1193)] (SEQ ID N):243) and the derived protein sequence for PRO293.

The entire nucleotide sequence of UQ2256 (ENA37151-1193) is S 15 shown in Figures 85A-B (SEQ ID NO: 243). Clone U42256 ([NA37151-1193) contains a single open reading frane with an apparent translational initiation site at nucleotide positions 881-883 and ending at the stop codon after nucleotide position 3019 of SEQ ID N3:243, Figures 85A-B). The predicted polypeptide precursor is 713 amino acids long (Figure 86). Clone UQ256 (INA37151-1193) has been deposited with ATCC and is assigned A'ITC deposit no. A'ICC 209393.

Analysis of the amino acid sequence of the full-length PRO293 polypeptide suggests that portions of it possess significant haology to the NIUR proteins, thereby indicating that PI293 may be a novel NLRR protein.

E**MPE 40: Isolation of cma Clones Encoding Eamn PIRO247 A consensus MA sequence was assembled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus sequence is herein designated ENA33480. Based on the EA33480 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cEIA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO247.

A pair of PCR prinmers (forward and reverse) were synthesized: forward PCR prirrer 5' -CAACAAIGAGGCCACAAGC-3' (SE ID NO:250) reverse PCR primer 5' -3A3 (SEQ ID NO:251) Additionally, a synthetic oligaonucleotide hybridization probe was constructed from the NA33480 expression sequence tag which had the following nucleotide sequence.

147 hybridization probe (SED ID NJ:252) In order to screen several libraries for a source of a fulllength clone, MA frma the libraries was screened by PCR anplification with the ICR 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 prinmers.

RNA for construction of the clMA libraries was isolated fran human fetal brain tissue. IA sequencing of the clones isolated as described above gave the full-length NA sequence for PRO247 (herein designated as IUN221 (ENA35673-1201)) (SEQ ID NO:248) and the derived protein sequence for PRO247.

The entire nucleotide sequence of tNQ221 (INA35673-1201) is shown in Figures 89A-B (SEQ ID NO:248). Clone UNQ221 (NA35673-1201) **15 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 80-82 of SEQ ID N3:248 and ending at the stop codon after nucleotide position 1717 of SE ID NO:249 (Figures 89A-B). The predicted polypeptide precursor is 546 amino acids long (Figure 88). Clone UNQ221 (IEA35673-1201) has been deposited with ACC and is assigned A'ICC deposit no. 209418.

Analysis of the amino acid sequence of the full-length PR0247 polypeptide suggests that portions of it possess significant honology to the densin molecule and KIAA0231, thereby indicating that PRO247 may be a novel leucine rich repeat protein.

Ej MPLE 41: Isolation of c!nA Clones Ebrcodvig Hman PR302, PMO303, PR0304, PIo307 and PRO343 Consensus EA sequences were assenrbled relative to other FES sequences using phrap as described in Exanple 1 above. These consensus sequences are herein designated ENA35953, rEA35955, ENA35958, INA37160 and [IA30895. Based on the I35953 consensus sequence, oligonucleotides were synthesized: 1) to identify by ICR a ca-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 PRO302.

PCR primers (forward and reverse) were synthesized: forward PCR priner 1 5' -G-CCA A'CTA'I 3' (SEQ ID NO:263) forward ECR primer 2 5' -GCAGAGGIIC'AAG=I'-3' (SED ID NO:264) reverse PCR priner 5' -AGCICTAGACCAAGOCAGCIOC-3' (SEQ ID ND:265) Also, a synthetic oligacniucleotide hybridization probe was constructed froman 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, IEA 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 PRO302 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cEl libraries was isolated from human fetal kidney tissue (LIB228) IA. sequencing of the clones isolated as described above gave the full-length [NA sequence for PR0302 (herein designated as UQ265 S(INA40370-1217)] (SEe ID N:253) and the derived protein sequence for ^PR0302.

The entire nucleotide sequence of UNQ265 (ENA40370-1217) is shown in Figure 89 (SEQ ID ND:253). Clone UN265 (ENA40370-1217) contains a single open reading frane 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 UIM265 (ENA40370-1217) has been deposited with the ATCC on Novenber 21, 1997 and is assigned ATCC deposit no. ATCC 209485.

Based on the ENA35955 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cl1A library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0303.

A pair of PCR priners (forward and reverse) were synthesized: forward PCR primer 5' -G331AIACTGA'IACATIOC-3' (SEQ ID ND:267) reverse PR primer 5' -AG XA A 3' (SEQ ID NO:268) Additionally, a synthetic oligonucleotide hybridization probe was constructed fran the consensus IIA35955 sequence which had the following nucleotide sequence: hybridization probe 5' C-3'ACAAAA t AGCtt (SEQ ID ND:269) In order to screen several libraries for a source of a fulllength clone, IMA from the libraries was screened by PCR anplification with the PCR prirer pairs identified above. A positive library was then used to isolate clones encoding the PR0303 gene using the probe oligonucleotide and 149 one of the PCR priners.

RNA for construction of the cEA libraries was isolated frcn human fetal lung tissue DA sequencing of the clones isolated as described above gave the full-length EA sequence for PR0303 (herein designated as UM266 (EMA42551-1217) (SEQ ID ND:255) and the derived protein sequence for PR0303.

The entire nucleotide sequence of W2266 (=A42551-1217) is shown in Figure 91 (SED ID NO:255). Clne UNQ266 (I4255i-1217) contains a single open reading franre 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 tQ266 (EI42551-1217) has been deposited on November 21, 1997 with the A'IC and is assigned AWtC deposit no. A'ITC 209483.

Based on the IDAN35958 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 clane of the full-length coding sequence for PR0304.

Pairs of PCR primers (forward and reverse) were synthesized: Sforward PCR priner 1 -GOGAAGIAGAA IGGCAMCTAAG-3' (SED ID NO:270) forward PCR prinrer 2 5'-CAGCCCIGCAAI-3' (SEQ ID NO):271) forward PCR priner 3 5*-TAC10GGGIWvCAGCANC-3' (SEQ ID N:272) 25 reverse PCR priner 5 -0 GlZ.AGAAQAOG'IGMXXC-3' (SEQ ID N:273) Additionally, a synthetic olignucleotide hybridization probe was constructed fron the consensus NA35958 sequence which had the following nucleotide sequence hybridization probe 5 'GCCi'cICc.IcAAAACAGCCGAGOCGAC-3' (SE ID N3:274) In order to screen several libraries for a source of a fulllength clone, INA fromn the libraries was screened by PCR anplification with the PCR 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 priners.

RNA for construction of the cNA libraries was isolated frnm 22 week human fetal brain tissue (LIB153).

IA sequencing of the clones isolated as described above gave the full-length NA sequence for PR3-4 therein designated as UNQ267 150 (E139520-1217) (SEQ ID NO:257) and the derived protein sequence for PR0304.

The entire nucleotide sequence of UN267 (IEA39520-1217) is shown in Figure 93 (SEQ2 ID NO:257). Clone UM267 (ENA39520-1217) contains a single open reading frane 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 (IR39520-1217) has been deposited with A'ICC on Novenber 21, 1997 and is assigned A'IOC deposit no.

AICC 209482..

.Based on the IIA37160 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a clIA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO307.

Pairs of PCR priners (forward and reverse) were synthesized: forward PCR prinr 1 5' -GOCAGOGAkTICAGGCIO (SEQ ID N:275) forward PCR priner 2 5' -Or3CIGACAGCAG33 IC-3' (SE ID N:276) Sforward PCR.priner 3 5' -IGACAAGACOGAOCAGG-3' (SEQ ID NO:277) reverse PCR priner 5' -rArIoCATIGCv=GAGAGCAAG-3' (SEQ ID NO:278) Additionally, a synthetic olignucleotide hybridization probe was .constructed from the consensus IN37160 sequence inhich 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 fran the libraries was screened by PCR anplification with the PCR pairs identified above. A positive library was then used to isolate clones encoding the PRO307 gene using the probe olignucleotide and one of the PCR priners.

RNA for construction of the cEA libraries was isolated fran humnan fetal liver tissue (LIB229).

LNA sequencing of the clones isolated as described above gave the full-length IA sequence for PRO307 (herein designated as UNQ270 (IA41225-1217) (SEQ ID NO: 259) and the derived protein sequence for PR0307.

The entire nucleotide sequence of UNQ270 (I41225-121 7 is shown in Figure 95 (SED2 ID ND:259). Clone U2270 (EWa41225-1217) contains a single open reading framre 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 UN2270 (IRA41225-1217) has been deposited with ATICC on November 21, 1997 and is assigned A'TQC deposit no.

ATCC 209491.

Based on the ENA30895 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 PRO343.

A pair of PCR prinmers (forward and reverse) ware synthesized: forward PCR primer IGAG OCATAC TI'CCA-3' (SEQ ID N0:280) reverse Cprier CTCA1='GCCAGGIGA-3' (SEQ ID ND:281) Additionally, a synthetic olignucleotide hybridization probe was constructed fromn the consensus DNA30895 sequence which had the following nucleotide sequence hybridization probe G IGCTGCACT-3' (SEQ ID NO:282) 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 PRO343 gene using the probe olignucleotide and one of the PCR primers.

RNA for construction of the clEA libraries was isolated frcm human fetal lung tissue (LIB26).

CNA sequencing of the clones isolated as described above gave the full-length NA sequence for EO343 [herein designated as UNQ302 (EMA43318-1217) is shown in Figure 97 (SEQ ID N0:261) and derived protein sequence for PRO343.

The entire nucleotide sequence of UNQ302 (ENA43318-1217) is shown in Figure 97 (SEQ ID N0:261). Clone UN302 (ENA43318-1217) 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 UN1302 (INA43318-1217) has been 152 deposited with ATCC an Noverber 21, 1997 and is assigned A'ITC deposit no.

AT'C 209481.

EXAMPLE 42: Isolation of cEM Clanes Encoding man P1RO328 A consensus IUJA sequence was asserbled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus sequence is herein designated rzA35615. Based on the 1M 35615 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cEMA 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 priners were synthesized: forward PCR prier 5*-TICCI'UAGTITXIGIO3C-3'I (SEQ ID NO:285) reverse P*J prier 5'-CTCATAToC A _GrAATICG-3' (SEQ ID NO:286) Additionally, a synthetic olignucleotide hybridization probe was 15 constructed fran the consensus EINA35615 sequence which had the following nucleotide sequence.

hybridization probe -AAG AA IA (SEQ ID NO:287) In order to screen several libraries for a source of a full- 20 length clone, IMA fran the libraries was screened by PCR arrplification with the ICR prinxer pair identified above. A positive library was then used to isolate clones encoding the PRO0328 gene using the probe olignucleotide and one of the PCR priners.

mNA for construction of the dl1A libraries was isolated fran htman fetal kidney tissue.

DA sequencing of the clones isolated as described above gave the full-length IWA sequence for PR0328 (herein designated as IM289 (ID40587-12131)] (SEQ ID ND:284) and the derived protein sequence for PRO328.

The entire nucleotide sequence of tJl289 (EiA40587-1231) is shwn in Figure 99 (SEQ ID ND:283). Clone UTQ289 (IUr40587-1231) contains a single open reading frane with an apparent translational initiation site at nucleotide positions 15-17 and ending at the stop codan at nucleotide positions 1404-1406 (Figure 99). The predicted polypeptide precursor is 463 amino acids long (Figure 100). Clone UNQ289 (IlA40587-1231) has been deposited with ATCC and is assigned AC deposit no. A'TC 209438.

Analysis of the amino acid sequence of the full-length PR0328 polypeptide suggests that portions of it possess significantly honology to the human glioblastca protein and to the cysteine rich secretory protein 153 thereby indicating that PR0328 my be a navel glioblastcan protein or cysteine rich secretory protein.

EUM'LE 43: Isolation of cM Clon Encoding Ebkrn PRO35, PR3331 or PR326 A consensus ENA sequence was assoibled relative to other EST sequences using phrap as described in Eaanple 1 above. This consensus sequence is herein designated IN3 6685. Eased on the E36685 consensus sequence, and Incyte EST sequence no. 2228990, oligonucleotides were synthesized: 1) to identify by PCR a cEM library that contained the seqence of interest, and 2) -for use as probes to isolate a clone of the full-length coding sequence for PRO335, PRO331 or PR326.

Forward and reverse FCR prirers were synthesized for the determination of PR0335: forward PJ. prinrr 5'1 -OAACUC.IIkTC Nr1'A-3' (SE2 ID IS:294) forward pR 5' -CTAAACIkACGA CfAOA-3' (SE2 ID NO:295) forward PCR primer 5' -X'ITGGAGACflCAA a-3' (SEQ ID NO:296) forward PCR priner 5' -A-CIO3ZACAGCIAGAAA 3' (SE) ID NO:297) reverse PCR priner 5' -CATTCCAATAAAAT'1C-3' (SE2 ID ND:298) reverse 1C priner 5' -GOGICT7UMM (SB2 ID N):299) reverse PCR priner 5' 3 (SEa ID NO:300) Additionally, a synthetic oligonucleotide hybridization probe was constructed for the determination of PRO335 which had the following rncleotide sequence.

hybridization probe 51 Wx (SE2 ID N):301) Forward and reverse PCR priners; were synthesized for the determination of PR0331: forward PJ. pCriaur 51 3 (SE ID N3:302) reverse PUC priner 5' -A30dA CICTICXC-3' (SE2 ID ND:303) Additionally, a synthetic oligonucleotide hybrization probe was constructed for the determination of PR0331 wich had the following nucleotide sequence hybridization probe -T3I XI(A91AOTC tC1A. (sDE ID lfl:304) Forward and reverse PCR primers were synthesized for the determination of PRD326: forward priner 5' CI3AFPAAtOXA A'ItOTIC-3' (SE2 ID 305) reverse PJ. priner 5' -3 ID ND:306) Additionally, a synthetic olignucleotide hybrization probe was constructed 154 *5* S...rc v.0** a. i* S~~ S CS tSOO* 0000 *0

S

SI...

S

555 S ~r

SS

S

@0 0@ 0550 0~ for the determination of PRO0331 which had the following nucleotide sequence hybridization probe (SEQ ID NO:307) In order to screen several libraries for a source of a fulllength clone, IEM fran the libraries was screenedby PCR anplification with the PC priner pairs identified above. A positive library was then used to isolate clones encoding the PRO335, PR0331 or PR0326 gene using the probe olignucleotide and one of the PCR priners.

NA for construction of the cEIM libraries was isolated fran human fetal kidney tissue (PR0335 and PR0326) and human fetal brain (PR0331).

M1A sequencing of the clones isolated as described above gave the full-length IM sequence for PROD335, PRO331 or PRO326 (herein designated as SEQ ID 1NS:288, 290 and 292, respectively; see Figure 103A-B, 105 and 107, respectively), and the derived protein sequence for PRO335, PRO331 or PRO326 (see figures 104, 106 and 108, respectively; and the derived protein sequence for PRO0335, PRO331 or PRO326 (see Figures 104, 106 and 108, respectively; SEY ID NDS:289, 291 and 293, respectively).

The entire nucleotide sequences are shown in Figures 103A-B, 105 and 107, deposited with the AtC on June 2, 1998, November 7, 1997 and Novenber 21, 1997, respectively.

Analysis of the amino acid sequence of the full-length PRO335, PR0331 or PRO326 polypeptide suggests that portions of it possess significant harology to the LIG-1 protein, thereby indicating that PRO335, 25 PR331 and PRO326 may be a novel LIG-1-related protein.

EZAMMIE 44: Isolation of CA Clanes Mooding Rmian PRD332 Based upon an ED harology search performed as described in Exanple 1 above, a consensus IEA seuence designated herein as INA36688 was 30 asserbled. Based on the INA36688 consensus sequence, oligonucleotides were synthesized to identify by PCR a cML 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 prinmers (forward and reverse) were synthesized: 5'-MATPGGOOGOGAGACtrlCC-3 (SED ID NO:310) -GCDCocCoGIC'liGGMA=-3' (SEp ID NO:311) A probe was also synthesized: 3 (SE ID NO:312) In order to screen several libraries for a source of a full- 155 length clone, DNA fran the libraries sas screened by PCR arrplification with the PCR prirer 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 priners.

RNA for construction of the cMNA libraries was isolated fran a human fetal liver library (LIB229).

NA sequencing of the clones isolated as described above gave the full-length [A sequence for [A40982-1235 and the derived protein sequence for PR0332.

The entire nucleotide sequence of IA40982-1235 is shown in ~Figures 109A-B (SEX ID NO:308). Clone EMA40982-1235 contains a single open reading frane (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 nolecular weight of 72,067 (pl:6.60). Clone EA40982-1235 has been deposited with A'ITC and is assigned AICC deposit no. A=C 209433.

Based on a BLAST and FastA sequence aligrment analysis of the full-length sequence, PR0332 shows about 30-40% amino acid sequence Sidentity with a series of know proteoglycan sequences, including, for exanple, fibrcmanodulin and fibrcmodulin precursor sequence of various species (EDBOVIN, RFD_IMCK, F4DM_RAT, FOD_.MOUSE, FUDHUMAN, PJ36773), ostearnedulin sequences (AB000114_1, AB007848_1), decorin sequences (CE831411, OCU03394_1, [LR42266, P R42267, P R42260, PR89439), keratan sulfate proteoglycans (BU48360_1, AF022890_1l), corneal proteoglycan (AF022256_1), and bone/cartilage proteoglycans and proteoglycane precursors (IS1BOVIN, PGS2MUSE, PGS2JiUMAN).

EXAMPIE 45: Isolation of cmA Clones Ebcoding Dxmn PRO334 A consensus IIA sequence was assenbled relative to other EST sequences using phrap as described in Ebcanple 1 above. Based on the consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cMNA 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 priners were synthesized for the determination of PRO334: forward PR priner 5' -GA PTC A t-3' (SE3 ID NO:315) reverse CR priner 5' -TICrAClGTAPdACcCACGrAOG-3 (SEQ 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 -CnAIO GA AGAGTIGAT-3' (SBQ ID NO:317) In order to screen several libraries for a source of a fulllength clone, DJA 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 PR0334 gene using the probe oligonucleotide and one of the PCR priners.

Human fetal kidney clNA libraries used to isolate the cIDA clones were constructed by standard methods using comercially available reagents such as those from Invitrogen, San Diego, CA.

ONA sequencing of the clones isolated as described above gave the full-length NA sequence for PR0334 [herein designated as ENA41379- 1236] (SBQ ID N0:313) and the derived protein sequence for PRO334.

The entire nucleotide sequence of INA41379-1236 (also referred to as UNQ295) is shown in Figure 109 (SEQ ID N0:313). Clone EIA41379-1236 contains a single open reading frame 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 A'TC and is assigned AT~CC deposit no.

ATT 209488.

Analysis of the amino acid sequence of the full-length PR0334 polypeptide suggests that portions of it possess significant honology to 25 the fibulin and fibrillin proteins, thereby indicating that PR0334 may be a novel renber of the EGF protein family.

EXAMPLE 46: Isolation of cEM Clones Ecoding Hanan PRO346 A consensus INA sequence was identified using phrap as described in Example 1 above. Specifically, this consensus sequence is herein designated ENA38240. Based on the ENA38240 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a ctNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length PRO346 coding sequence.

RNA for construction of the cMA libraries was isolated frmn human fetal liver. The cENA libraries used to isolate the cEA clones were constructed by standard methods using cmonercially available reagents Invirtogen, San Diego, CA; Clontech, etc). The cEIl was primed with oligo dr containing a NotI site, linked with blunt to Sall henikinased 157 adaptors, cleaved with NotI, sized appropriately by gel electrophroresis, 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, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

A cIMA clone was sequenced in entirety. The entire nucleotide sequence of ENA44167-1243 is shown in Figure 111 (SED ID NO:318). Clone SA44167-1243 contains a single open reading frate with an apparent translational initiation site-at nucleotide positions 64-66 (Fig. 113; SEQ ID ND:318). The predicted polypeptide precursor is 450 amino acids long.

Clone ENA44167-1243 has been deposited with AMC and is assigned A'IC deposit no. AICC 209434 (designation EA44167-1243).

Based on a BLASr, BALS2 and FastA sequence aligrment analysis (using the ALIC 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: 0LI2691 (38240.f1) -GA-3' (SEQ ID NO:320) OLI2693 (38240.rl) -CAC'AA3 CIXCCL 3' (SED ID NO:321) OLI2692 (38240.p1) 51 (8 0.'1 AC'TG-3' (SEQ ID N3:322) EMPLE 47: Isolation of cmA Clones Ecoding Munan PRO268 A consensus MA sequence was asserbled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus sequence is herein designated NA35698. Based on the UNA35698 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cU1A 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 primers were synthesized: forward PCR priner 1 5' -'IAG7IO3CAACDOGAAA'IG-3' (SEQ ID NO:325) forward PCR primer 2 5' -TA'IGIGATCAPGACMICX-3' (SEQ ID N:326) forward PCR priner 3 5' -Tc;CN 'rie AGATIG-3' (S3EQ ID NO:327) reverse PCR priner 5' -T Q IC G C (SBC ID NO:328) Additionally, a synthetic olignucleotide hybridization probe was constructed frcmn the consensus EINA35698 sequence which had the following nucleotide sequence 157a *Oe, C C

C.

C

**C

S S.

C.

S.C tO CC. 00CC

C

S C 0 0 C. 9*

C

C

C

hybridization probe 3' (SEQI mD :329) In order to screen several libraries for a source of a fulllength clone, EINA fran the libraries uas screened by PCR anplification with the PCR priner pair identified above. A positive library was then used to isolate clones encoding the [1)268 gene using the probe olignucleotide and one of the PCR prirers.

RNA for construction of the cNA libraries was isolated froan hnan fetal lung tissue. UI4 sequencing of the clones isolated as 10 described above gave the full-length IA sequence for PR0268 [herein designated as U=235 (LEA39427-1179)] (SEQ ID N3:323) and the derived protein sequence for PRO268.

The entire nucleotide sequence of lN235 (U1A39427-1179) is shown in Figure 113 (SE) ID N:323). Clone tUN235 (ENA39427-1179) contains 15 a single open reading frate 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 (139427-1179) has been deposited with A'TCC and is assigned ATCC deposit no. ATCC 209395.

20 Analysis of the amino acid sequence of the full-length PRO268 polypeptide suggests that it possess significant hIomlogy to protein disulfide iscmerase, thereby indicating that PRO268 many be a novel protein disulfide isanerase.

EAMPE 48: Isolation of cKM Clones Encoding rman PI330 A consensus IM sequence was assaxbled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus sequence is herein designated IEA35730. Based on the LNR35730 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 PRO330.

Forward and reverse PCR priners were synthesized: forward PCR priner 1 5'-CCAGCACAA rCltAG-3' (SE2 ID M:332) forward PCR priner 2 5'-GACrIr* 3' (SD ID m :333) forward PCR priner 3 5'-GICICAAGAACICC7IGC-3' (SE ID N:334) reverse PCR priner 5' -ACACICAGCAIGCTIGACIG-3' (SEQ ID M:335) Additionally, a synthetic olignucleotide hybridization probe was constructed fran the consensus which had the following nucleotide sequence 157b hybridization probe -3CAMAICAI (SBQ ID ND: 336) In order to screen several libraries for a source of a fulllength clone, IDL 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 prinmers.

RA for construction of the cINA libraries was isolated fron human fetal lung tissue. MA sequencing of the clones isolated as described above gave the full-length ENA sequence for PRO330 (herein designated as tUNQ290 (Ii40603-1232) (SEQ ID N1:330) and the derived protein sequence for PRO330.

IThe entire nucleotide sequence of iWl290 (M40603-1232) is shown in Figure 115 (SED ID N:330). Clone UNQ290 (EW40603-1232) contains 15 a single open reading frare with an apparent translational initiation site

S..

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 (IDN40603-1232) has been deposited with ACC and is assigned ATCC deposit no. M'CC 209486 on 20 Novenber 21, 1997.

Analysis of the amino acid sequence of the full-length PRO0330 polypeptide suggests that portions of it possess significant honology to nouse prolyl 4-hydroxylase alpha subunit protein, thereby indicating that PR0330 nay be a novel prolyl 4-hydroxylase alpha subunit polypeptide.

25 EAMMPE 49: Isolation of cENA Clcnes Encxoding Buan PRO310 A consensus I1A sequence was asserbled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus sequence is herein designated M40553. Based on the NA40553 consensus sequence, olignucleotides were sythesized: 1) to identify by PCR a cELIA 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 PC. prinrer 1 5*-'IC CAAGO=CCGCAAOCI T-3' (SEQ ID ND:341) forward PCR primer 2 5' -CIGGTICTICIOCIDCA3 (SfEQ ID NO: 342) reverse PR priner 5' AACC TAAMOL3TATACTATACCC-3' (S3EQ ID O3:343) Additionally, a synthetic olignucleotide hybridization probe was constructed fran the consensus which had the following nucleotide sequence 157c hybridization probe Am I rlGiCA G I CACTA'ItCtAGGAC-3' (SED ID ND:344) 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 PR0310 gene using the probe olignucleotide and one of the PCR priners.

RM for construction of the crMA libraries was isolated fromn human fetal liver tissue. IA sequencing of the clones isolated as described above gave the full-length EIA sequence for PRO310 (herein designated as (ENA43046-1225)] (SEQ ID NO:339) and the derived protein sequence for PRO310 (SEQ ID 1:340).

The entire nucleotide sequence of (D43046-1225) is shown in ~Figure 119 (SED ID ND:339). Clone (IEM43046-1225) contains a single open *15 reading franme 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 molecular weight of approxdimately 36,382 daltons. Clone (ENA43046-1225) has been deposited with ATC and is :20 assigned ATIOt deposit no. A'ITCC 209484.

Analysis of the amino acid sequence of the full-length PR0310 polypeptide suggests that portions of it possess harology to C. elegans proteins and to fringe, thereby indicating that PRO310 may be involved in deelopment.

EXRMPIE 50: Isolation of cENA clones Erlcodig aman PRO339 An expressed sequence tag (ESr) IM database (LIFESE2, Incyte Pharmaceuticals, Palo Alto, CA) was searched and ESrs were identified. An assaebly of Incyte clones and a consensus sequence was forned using phrap as described in Ecanple 1 above.

Forward and revers PCR priners were synthesized based upon the asseanbly-created consensus sequence: forward PCR prinmer 1 5' -GOCATRXAomG31 ItIUAG3GG-3' (SEQ ID 1NO:345) forward PCR prinmer 2 5' -ClCCTXTrTAOcGO (SEQ ID NO:346) forward PCR prinIer 3 S' -G7*IGACACKOGI (SE) ID NO:43) forward PCR prier 4 5' -GACCCA PICIG (SEQ ID ND:44) reverse PCR priner 1 5' 3 (SEQ ID reverse PCR priner 2 5' -IX133 CP't-3' (SEQ ID NO:46) Additionally, a synthetic olignucleotide hybridization probe was KU:%3gUtK..\Upecia3178I-9S2.c ISOI100 157d constructed from the consensus sequence which had the following nucleotide sequence.

hybridization probe aCTACGAIGTICATIOCAGGGAC A -3 (SEQ ID N3:47) In order to screen several libraries for a source of a fulllength clone, IA fron the libraries was screened by PCR anplification with the PCR primer 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 primers.

RNA for constructing of the cKNA libraries was isolated from human fetal liver tissue. A cEM clone was sequenced in entirety. The "..entire nucleotide sequence of DNA43466-1225 is shown in Figure 117 (SEQ ID N:337). Clone EIA43466-1225 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; SBQ ID N):337). The predicted polypeptide precursor is 772 amino acids long and has a calculated molecular weight of approximately 86,226 daltons. Clone ENA43466-1225 has been deposited with ATCC and is assigned ATICC deposit no. AITC 209490.

Based on a BLAST and FastA sequence alignment analysis (using the ALIGN carputer program) of the full-length sequence, PR0339 has haology to C. elegans proteins and collagen-like polymer sequences as well S" as to fringe, thereby indicating that PR0339 may be involved in development Sor tissue growth.

EXAMPLE 51: Isolation of DNA Clones Encoding Human PR0244 A consensus IEA sequence was asserbled 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 PR0244.

A pair of PCR primers (forward and reverse) were synthesized: 5'-TICAGCT1G3G AGGG-3' (30923.fl) (SEQ ID NO:377) -TATTCCOACCTIC rACAAATCOG-3' (30923.rl) (SEQ ID ND:378) A probe was also synthesized: TI-O-3' (30923.pl) (SEQ ID NO:379) In order to screen several libraries for a source of a full-length clone, ENA fran the libraries was screened by PCR anplification with the PCR 157e priner 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 cEA libraries was isolated fromn human fetal kidney library. CNA sequencing of the clones isolated as described above gave the full-length rA sequence and the derived protein sequence for PR0244.

The entire nucleotide sequence of PR0244 is shown in Figure 121 (SEQ ID N1:375). Clone INA35668-1171 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 ATOC (designated as NA35663-1171) and is assigned ATC deposit no. A'CC 209371. The protein has a cytoplasmic donain (aa 1-20), a transmenbrane domain (aa 21-46), and 15 an extracellular damain (aa 47-219), with a C-lectin domain at aa 55-206.

Based an a BLAST and FastA sequence aftigncm analysis of the full-length sequence, PR0244 shows notable amn add sequence identity to hepatic etin gallus pillus HIC hpl2O-binding C-type lectin macrophage lecin 2 (HUMHML2-1, 41 and sequecec PR32189 (44 EXAMiPUE 52: Use of PRO Pob ~etide-Encodine Nucleic Acid asq Hybidzatjon Probes The following rsehod &escribes use of a nucleotide sequnc 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 emuployed 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 Inman tissue cDNA libraries or human tissue genomic libraries.

Hybridization and 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 Denhardtsg solution, and 10% dextran sulfate at 42C for 20 hours. Washing of the filter is performed in an aqueous solution of 0.lIx SSC and 0. 1% SDS at 42 0

C.

DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO :polypeptide can then be identified using standard techniques iown in the art.

20 EXAMPLE 53: Expression of PRO Polvoeptides in E. coli This example illustrates preparation of an unlycosylaWe form of a desired PRO polypeptide by recombinant expression in E. coil.

The DNA sequence encoding the desired PRO polypeptide is initially amplified using selected PCR primers.

The primners should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected 25 expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coil; see Bolivar et al.. 2:95 (1977)) which contains genes for anipicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephasphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistac gene, a tip promoter. a polyhis leader (including the first six STUI codons, polyhis sequence, and enteroltinase cleavage site), the specific PRO polypeptide coding region, Lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coi strain using the methods described in Sanmbrook et al., m= Transformants arc identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolatd and confirmned by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.

After culturing the cells for scveral more hours. t cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art. and the solubilized PRO polypeptide c= then be purified using a metal chelatin column under conditions that allow tight binding of the protein.

PROMS, PR0317. PRO3Ol, PR0224 and PR0238 were successfully expressed in E. coil in a poly-His tagged form, using the following procedure. The DNA encoding PROW8, PROM.7 PRO3OI. PR05224 or PR0238* was initially amplified using selected PCR primers. 'The pimners contained restriction enzyme sites which corresponid 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 meta chelation column, and proteolytic removal with enterokinase. The PCR-amplified. poly-His tagged sequences were then ligated into an expression vector, which 10 was used to transform an E. coil host based an strain 52 (W31 10 fuhAftonA) Ion galE rpo~tsohrpRts) clpP(laclq).

Transformants were first grown in LB containing 50 mg/mI carbenicilun at 30'C with shaking until an 0.D.600 of' was reached. Cultures were the diluted 50-100 (old into CRLAP median (prepared by mixing 3.S7 g (NH~zSO, 0.71 g sodium citrate*21120, 1.07 g KCI, 5.36 g Difco yeast extract. 5.36 g Sheffield hycase SF in 500 mL. water, as well as 110 mM MPOS, pH 7.3, 0.55 (wtv) glucose and 7 mM MgSO 4 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 cells. 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 :guanidine, 20 mM Tris. pH 8 buffer. Solid sodium sulfite- and sodium tetrathionate is added to make final concentrations of 0.IM and 0.02 M. respectively, and the solution was stirred overnight at VC. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution was centrifuged at 40.000 rpm in a Beckman Ultracerifuge for 30 mini. The supernatant was diluted with 3-5 volumes of metal chelate colum '.:buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. Depending the clarified extract was loaded onto a 5 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 (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with buffer cornaining 250 mM imida zole. Fractions containitig the desired protein were pooled and stored at 41C. Protein concentration was estimated by its absorbance at 280 am using the calculated extinction coefficient based on its amino acid sequence.

lic 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 volurnes were chosen so thai the final protein concentration was between 50 to 100 mnicrograms/mI. The refolding solution was sdtird gently at 4*C for 12-36 hours. The refolding reaction was quenched by the addition oftTFA to a final concentration of 0.4% (pH of approximately Befoire further purification of the protein, the solution was filtered througha 0.22 micron filter and acetonitrile was added to 2-10% final concentration. The refolded protein was chronatographed on a Pores RUIH reversed phase columin using a mobile buffer of 0.1 TFAwith elution with a gradient of acetoniurilc 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 eluted at the lowest concentrations; of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetomitrile coreenzrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also remaves aedotoxin from the samples.- Fractions containing the desired folded PR0187. PR0317. PRO3O1. PR0224 and PR0238 proteins.

respectively, were pooled and the acetoaitrile removed using a gentle stream of nitrogen dircted a the solution.

Protin were formulated Into 20 aiM Hepes. pH 6.8 with 0.14 M sodium chloride and 4% mannitiol by dialysis or by gcl filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.

EXML 4 Ewerssion of PRO PohMpenides in Mammalian Cells This example illustrates preparation of a glycosylated form of a desired PRO polypeptide by recombinant expression in nammalin cells.

Thec vector. pRKS (see EP 307.247. published March 15. 1989). is employed as the expression vector.

Optionally. the PRO polypeptide-cnicoding DNA is ligted into pRKS with selected restriction enzymes to allow insertion of the PRO polypeptide DNA using ligation methods such as described in Sambrook etal., The resulting vector is called pRKS-PR0 polypeptide.

In one embodiment. the selected host cells may be 293 cells. Humn 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in mediurr such as DMEM supplemented with fetal -calf serum and optionally, nutrient components and/or antibiotics. About 10 p~g pRKS-PRO polypeptide DNA is mixed with about :I pg DNA encoding the VA RNA gne [Thimmappaya et :543 (1982)) and dissolved inSO 501i of ImM mrs-Ha. 0.1 mM EDTA, 0.227 M CaCl,. To this nmixire is added. dropwise, 500 pl ofS50 mM HEPES (pH 7.35), 280 mM NaCI, 1.5 mM NaPO 4 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 37TC. The culture medium is aplizeoff 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 #LCi/mI MS-cysteine and 200 #pCi/mI "S-methionine. After a 12 *...hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15 SDS gel.

Ihe 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 trasfected cells my 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 tranisiently using the dextra sulfate method described by Soasparyrac et al.. PEn. ad. Acad. Sci-, 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700jpg pRK5-PRO polypeptide: DNA is added. The cells are first concentrated from die spinner flask by centrifugation, and washed with PBS. The DNA-dcxtra precipitate is incubated on the cell pellet for four bours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue cultur miediumi, and re-introduced into the spinner flask containing tissue culture medium, 5 p~g/ml bovine insulin and 0.1 pg/mI bovine transferyn. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PRO potypeptide can then be concentrated and purified by any selected method, such as dialysis and/or columnromaography.

In another embodiment. PRO polypeptides can be expressed in CHO cells. The pRKS-PRO polyppde can be tansfactcd into CHO cells using known reagents such as CaPO, or DEAE-dextran. As described above, die cell cultures can be incubated, and the medium replaced with culture medium (alone) or mnediumn containing a radiolabel such as 'IS-methionine. After determining the presence of PRO polypeptide. the culture medium may be replaced with sertm free niedbwm Preferably, the cultures are incubated for about 6 days, and dien tbe conditioned* medium is harvestd The mediumt containing the expressed PRO polypptde can then be concentrated and purified by any selected method.

Eitope-tagged PRO polypeptide may also be expressed in host CHO cells. The PRO polypeptide may be subcloxmd out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovinus expression vector. The: poly-his tagged PRO polypeptide insert can then be subcloned into a SV40 driven vector containing a selection masker such as DHFR for selection of stable clones.

Finally. die CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be perfomed, as described above. to verify expression. The culture mediumn containing the expressed poly-His tagged PRO polypeptide can then be concentrated and purified by any selected method, such as by Ni'-chebtce affinity chromatograhy.

PRO2il. PRO2I7. PR0230. PR0219. PR0245. PRO2i. PR0258, PRO3OI, PR0224, PR0222, PR0234.

PR0229, PR0223. PR0328 and PR0332 were successfully expressed in CHO cells by both a transient and a stable expesion procedur. In addition, PR0232. PR0265, PR0246. PR0228, PR0227, PR0220, PR0266. PR0269.

PR0287. PR0214, PRO231. PR0233. PR0238, PR0244, PR0235. PR0236, PR0262. PR0239. PR02S7, 20 PR0260, PR0263, PR0270, PR0271, PR027, PR0294, PR0295. PR0293, PR0247. PR0303 and PR0268 were successfully transiently expressed in CHO cells.

Stable expression in CHO cells was performed using t following procedure. The proteins were expressed as an IgG construct (immnoadhesin), in which the coding sequences for the soluble forms extracellutlar domains) of the respective proteins were fused to an IgGI constant region sequence containing the hinge, CH2 and 9:.:25 CH2 domainsand/or is apoly-His agged form.

Following PCR amplification. the respective DNAs were subcloned in a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology. Unit 3.16, John Wiley and Sons (1997). CHO expession vectors arc constructed to have compatible restriction sites 5S and 3' of the DNA of interet to allow the convenient shuttling of cDNA's. 11e vector used expression in CHO cells is as describedin Lucas et al.. Nuc!. Acds Rea. 2A: 9 (1774-1779 (1996). and uses the SV40 early promoter/enhancer 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 mirograms of the desired plasmid DNA were intoduced into approximately 10 million CHO cells wing conmmercially available transfecton reagents Superfbcs (Quisgen). Dosper or Fugene' (Boehuinger Manohun).

Thc cells were grown and described in Lucas at supra. Approximately 3 x 10lls are frozen in an amipule for further growth and production as described below.

The ampules containing the plasmid DNA were thawed by placement into water bath and mixed by vorsexizg. The contents were pipcttod into a centrifuge tube containin 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. Ile supernatant was aspirated and the cells were resuspended in 10 niL of selective media (0.2 jum filtered PS20 with 5% 0.2 pmt diafiltered fetal bovine serum). The cells were then aliquotod into a 100 niL spinner containing 90 niL of selectivc media. After 1-2 days. the cells were transferred into a 250 nml; spinner filled with 150 mL selectve growth medium and incubated at 370C. After mnother 2-3.days a 250 adL, 500 niL and 2000 niL spinners were secded with 3 x IV~ cclls~ml.. Ile cell media was exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media my be employed, a production mnedium describe in US Patent No. 5.122,469. issued June 16. 1992 was actually used. 3L production spinner is seeded at 1.2 x 106 ceis/mL On day 0. the cell zaimber pH were determined. On day 1. the spinner was sampled and spurging with filtered air was comnced. On day 2. the spinner was sampled, the temperatur shifted to 339C, and.

mLof500 g/Lglucoie ad 0.6 mLof10% antioam 35% polydixnethylsiloxaneemulsion, Dow Corming 365 Medical Grade Emulsion). Throughout the production. pH was adjusted as necesary to keep at around 7.2.

After 10 days, or until viability dropped below 70%, die cell culnure was harvested by centrifugtion and filtering through a 0.22 pmi filter. T'he filtrate was either stored at 4*C or immediately loaded onto columins for purification.

For the poly-His tagged constructs. the proteins were purified using a Ni-NTA column (Qiagen). Before purification. imidazole was -added to the conditioned media to a conceration of 5 mM. The conditioned media was pumped onto a 6 nml Ni-NTA cohinm equilibrated in 20 mM Hepes. PH 7.4. buffer containing 0.3 M NaCI and 5 mM imidazole at a flow rate of 4-5 mlimin. at VC. After loading. the cofunm was washed with additional equilibration buffer and the protein eluted wkh equilibration buffer containing 0.25 M ianidazole. The highly purified protein was ooo..' 20 subsequntly desalted into astorage buffer containing 10 tnM Hepes. 0.14 M NaCI and 4% mannitol, PH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at Inmunoadhesin (Fc containing) constucts of were purified from the conditioned media as follows. The conditioned miedium was pumped onto a 5 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 elution with 100 mM citric acid, pH 3.5. T11c eluted protein was immediately neutralized by collecting 1.1l fractions into tubes containing 275 1 tL of 1 M Tris buffer, PH 9. The highly purified protein was subsequenitly deialted into storage buffer as described abovc for the poly-His tagged proteins. The homogeneity was assessed by SDS polyacrylaniide gels and by N-erminal amino acid sequencing by Edman degradation.

PRO211. PR0217. PROM3. PR0232. PROIS7. PROM6, PR0219, PROW4, PR0228, PROM,3 PROM4.

PR0221, PR0227. PR0220. PR0258. PR0266, PR0269. PROW9. PR0214, PR0317. PROMO, PR0224.

PR0222. PR0234, PR0231, PRO229. PROM3, PROM.8 MR0223. PROM3. PROM3. PR0262, PR0239.

PR0257. PR0260, PROW,3 PROM7. PR0271, PROM7. PR0294, PROM,5 PR0293, PR0247, PR.0304.

PROM0, PR0307, PROM,3 PROW,3 PR0328. PR0326, PR0331, PR0332. PR0334, PR0346, PROM26, PROM,0 PRO310 and PR0339 were also succssfully transiently expressed in COS cells.

Expression of PRO Polyneddet in YeaSt The following method describes recombinant expression of a desired PRO polypeptide in yeast.

First. yeast expression vectors ame constructed for intracellular production or secretion of PRO polypeptides from die ADH2/GAPDH pro moter. DNA encoding a desired PRO polypeptide. a selected signal peptide and the promoter isinserted into suitable restriction curyme sites in the selected plasmid to direct intracelluiar expression of the PRO polypeptide. For secretion, DNA encoding dhe PRO polypeptide can be cloned into the iclected plasmid, together with DNA encoding dhe ADH2JGAPDH promoter. the yeast alpha-factor secretory signal/lader sequence.

and linker sequences (if needed) for expression of dhe PRO polypeptide.

Yeast cells, such as yeast strain ABIO. can then be transformed. with the expression plasmids 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 fcanentation mediuom by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing the PRO polypeptide may further be purified using selected column chromatography resins.

EXAMPE 56: Exorssion of PRO Polvpeptdes in Haculovinis-Infected Insect -Cells The following method describes recombinant expression of PRO polypeptides in Baculovirus-infected insect cels. lm desired PRO polypeptide is fused upstream of an epitope tag contained with a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed. including plasmids derived from commercially available plasnids such as pVL1393 (Novagen). Briefly. the PRO polypeptide or the desired portion of the PRO polypeptide (such as the sequence encoding the extracclhilar domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subeloned into the expression vector.

Recombinant baculovinis is generated by co-trasfecting the above plasniid and BaculoGold"4 virus DNA (Pharmingen) into Spodopterafrugiperda CSI9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28C, the released viruses are harvested and used for further amplifications. Viral infe on 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-his tagged PRO polypeptide can then be purified. for example, by Ni 2 "-chelate affinity chromatograhy as follows. Eixtracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert er al.. Nature. MI: 175-179 (1993). Briefly. Sf9 cells arc washed, resuspended in sonication buffer (25 niL Hepes, pH 7.9; 12.5 mM MgCl,; 0. 1 mM EDTA; 10% Glycerol; 0. 1% NP-40; 0.4 M KCl). and sonicAted twice for seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer mM phosphate, 300 ruM NaCI. 10% Glycerol, pH 7.8) and filtered through a 0.45jurm filter. A Ni 24

-NTA

agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL. washed with 25 ML of water and equilibrated with 25 ml of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minut. llmn column is washed to baseline Aso with loading buffer, at which point fraction collection is started.

Next. the column is washed with a secondary wash buffer (50 niM phosphate; 300 mM NaCI. 10% Glycerol, pH which elutes nonspecifically bouri protein. After reaching A,,baseline again,. the column is developed with a 0 to 500 suM Irnidazoie gradiettin the secondary wash buffer. One nt fractions are collected and analyzed by SDS-PAGE and silver staining or western blot with Ni'*-NTA-conjuiated to alkaline phospliase (Qiagen).' Fractions containing the eluted Hisw-tagged PRO polypeptide amu pooled and dialyzed against loading buffer.

Nlternvcly. purification ofthe IgG tagged (or Fe tagged) PRO polypeptide can be performed using known chromatography techniques, including for instance. Protein A or protein G column chromatography.

PRO211. PROM1, PROM.0 PRO197. MR0265. PR0246. PR0228. PROM3. PROW4. PR0221. PR0220, PRO2.58. PR0266, PR0269. PR0287. PROM1. PROMO, PR0224, PR0222, PR0234. PR0231, PR0229, PROM3. PROM3. PR0257. PROM7. PROM9. PR0295. PR0328. PROM2, PROM.1 PR0334. PR0346 and PR03 10 were successf"l expressed in baculovints infected SM or high5 isect cells. While the cxpression was actually performed in a 0.5-2 L scale, it can be readily sicaled up for larger 8 L) preparations. The proteins were expressed as an IgG construct (nmaunoadiesin). in which die protein extracellular region was fused to an IgGI constant region sequence containing the hinge. CH2 and CH3 domnains arnd/or in poly-His tagged forms.

Following PCR amplification. dhe respective coding sequences were subeloned into a baculovirus expression vector (pb.PHlgG for IgG fusions and pb.PH.His.c for poly-Hlis tagged proteins), and the vector and Baculogold* :baculovirus DNA (Pharagen) were co-tramfcited into 105 Spodoptrmfrugiperda cells (ATCC CRL 1711). using Upofectin (Gibco BRL). pb.PH.lgG and pb.PH.His are modifications of the cornmereially available baculovinis expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences. The cells were grow in Hink's TNM-FH mediumn supplemented with 10% FES (Hyclone). Cells were inaiubatod for 5 days at289C. The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hunk's TNM-FH medtium supplemented with 10% FBS at an approximate multiplicity of infection (MQI) of 10. Cells were incubated for 3 days at 28T. The supernatant was harvested and the expression of dhe constructs in the baculovinas expression vector was determined by batch binding of 1 ml of supernatant to mL of Ni-NTA beads (OIAGEN) ro itdn agged proteins or Protein-A Sepharose CL-4B beads (Pharanacia) for lgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Cooinassie blue staining.

he first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 Cells grown in ESF-921 medium (Expression Systems LW)Q at an approximate MOI of 0.1. Cell were incubated for 3 days at 280C. The superniatam was harvested and filtered. Batch binding and SDS-PAGE analysis was repeated, as necessary. until expression of the spinner culture was confirmed.

The conditioned mediumn 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 die poly-H is tagged constructs, the protein construct were purified using a lNh-NTA column (Qiagen). Before purifcation. imidazoie was added to the conditioned mnedia to a concntration oif S mM. The conditioned media were purnped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM inidazole at a flow rate of 4-5 mI/mmn. at VC. After loading. dhe column was washed with additional equilibration buffer and the protein cluted with equilibration buffer containing 0.25 M iniidazolc. The highly purified protein was subsequently desalted into a storage buffer containn mM Hepes, 0. 14 M NaCI and 4% mauikol, pH 6.8. with a 25 ad G2.5 Superfine (Pharmacia) column and stored at Immnunoadhesin (Ec comainizig constructs of proteins were purified from dhe conditioned medi as follows.

The conditioned media were pumped onto a 5 ml Protein A column (Pharmiacia) which had been eillibrated in mM Na phosphate buffer, pH 6.8. After loading, the ohutm was washed extensively with equilibration buffer before ehition with 100 mM citric acid. pH 3.5. flIr chimed protein was immediately neutralized by collecting-I ml fractions into tubes containing 275 mL of 1 M Tris buffer. pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the proteins Was verified by SDS polyacrylamnide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.

EAMP~LE57: PEiiaration of Antibodies that Bind to PRO Polvpepides *This example Uistrates preparation of monoclonal antibodies which can specifically bind, to a PRO polypeptide.

S. 15 Techniques for producing the monoclonal antibodies arc known in the art and are described, for instance.

in Goding. m=x. Imnim ogens that may be employed include purified PRO polypeptde, fusion proteins- containing the PRO polypetide. and cells expressing recombinant PRO polypeptide on the cell surface. Selection of the immunogen can be nade by the skilled artisan without undue experimentation.

Mice, such as Balb/c, are iminizod with the PRO polypdec immnunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitceally in an amount from 1-100 micrograms. Alternatively, the inrnmogen is emulsified in MPL-DM adjuvant (libi Iinntnochemical. Research, Hamilton, MT) and-injected into the animalshind foot pads. The immunized mice are then boosted10 to 12 days later with additional inmmnogen emulsified in the selected adjuvant. Thereafter. for several weeks, the mice may also be boosted with additional imminization injections. Serum samples may be periodically obtained from die mice by retro-orbital bleeding for testing in EUSA assays to detect anti-PRO polypeptide antibodies.

After a suitable antibody titer has been detected, the animals 'positive* for antibodies can be injected with a final intravenous injeccion of PRO polypeptid. Three to fouir day later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35 polyethylene glycol) to a selected rnurine mycloma ccli line such as P3X63AgU. 1. available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (yoxanihin, aminopterin, and thymidine) mediumn to inhibit proliferation of non-fused cells, myclomna hybrids, and spleen CCUi hybrids.

The bybridoma cells will be screened in an El-SA for reactivity against the PRO polypeptide.

Determination of "positive' hyWdoma cells serting the desired monoclonal antibodies against the PRO polypeptide is within the skill in the art.

The positive hybridtxn cells can be injecte inraperitoneally into syngeneic Balb/c mice to produce ascites cotining the andi-PRO polypeptide mionoclonal antibodies. Alternatively, the hybridorna cells can be grown in tissue culture flasks or roller bottles. purification of tie monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by get exclusion chromatography. Alternatively, affinity chromatography bascd upon binding of antibody to protein A or protein G can be employed.

AML.Sit: Cimneric PRO Polynentdes PRO polypcptdces may be expressed as chimeric proteins with one or more additional jiolypeptide domains added to facilitate protein purification. Such purification facilitating domkiiu include, but are notf united to, metal checlating peptides such as histidine-tzytophan modules that allow purification on immobilized metals, protein A domains that allow purification on imnmobilized inixtunoglobizlin. and the domain utilized in the FLAGSTm extensioafnity purification system (lImmuncx Corp.. Seattle Wash.). The inclusion of a cleavable linker sequence such as Factor XA or cnierokinase,(lnvitrogen, San Diego Calif.) between die purification domain and the PRO polypeptide sequence may be useful to facilitate expression of DNA encoding the PRO polypeptide.

EAMPELP.11: Purification of PRO Pollnetides Using Sucdfic Antibodies Native or recombinant PRO polypeptides may be purified by a variety of standard iechniques in the art of protein purification. For example. pro-PRO polypeptide. mature PRO polypeptide, or pre-PRO polypeptide is ~:15 pified by ininoamfnity chromatography using antibodies specific for the PRO polypeptide of interest. In gencetal.

an iminunoaffinity column is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin.

Polyclonal imnmomglobulins arm prepared from immniae Bern either by precipitation with ammonium, sulfate or by purification on immobilized *Protein A (Pharinacia LKB Biotechnology, Piscataway. Likewise.

mnoclonal antibodies amt prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography en immobilized Protein A. Partially purified immumoglobulin is covalendty attached to a chromatographic resin such 9: as CriBr-activated SEPHAROSE' (Pharmacia LKB Biotechnology). The anbody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the marnufacturer's instructions.

Sucha i n c is utilized in the purification of PRO polypeptide by preparing a frtion from cells containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcdilular fraction obtained via differential centrifurgation by the addition of detergent or by other fl~tlids well known in the art. Alternatively, soluble PRO polypeptdde containing a signal sequence may be secreted in use ful quntzity into dhe mediumn in which the cells are grown.

A soluble PRO polypeptide-containting preparation is passed over die iinmunoaffinity columnr, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide high ionic strength buffers in the presnc of detergent). Then, the column is cluted under conditions that disrupt annbody/PRO polypeptide bindling a low pH buffer such as apprximately pH 2-3. or a highi concentration of a cliaotrope such as urea or thiocyanate ion), and PRO polypeptide is collected.

EXAMPLE 60: DrpSrenn 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 sobation, affixed to 2 solid support, borne on a cell surface. or located intraceliularly.

One method of drug screening utilizes eukaryotic or prokaryotic: host cells which are stably transtormed with r ecombi- m icleic acids expressing the PRO polypptdd or fragnmcnt. Drugs are screced 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 example, the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively, one can examine the dimizzation in complex'formation between the PRO polypeptide' and its target cell or targcE receptors caused by the agent being tested.

Thus, die present invcntion provides methods of screening for drugs or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comnprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying for the presecec of a complex between the agent and the PRO poiypeptid. or fragmnt, or (ui) for the presnc of a complex between- the PRO polypeptide or fragment and dic cel.

96 by methods well known in the art In such competitive binding assays, the PRO polypeptide or fragment is typically 010.labeled. Ahmc suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, .:go and the amount of free or uncomplexed label hi a measure of dhe ability of the particular agent to bind to PRO ::..polypeptide or to interfere with the PRO polypeptidecell complex.

@9:15 Another technique for drug screening provides high throughput screening for compound having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564. published on September 13, 1984.

Briefly stated, large mumibers of different small peptide test comnpounds 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 polypeptide and washed. Bound PRO polypeptide is detected by mtethods well known in the art. Purified PRO polypeptide can also be coated directly onto plates for use in the aforementioned drug screening tecliniques. 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 polypeptde specifically compete with a test compound for binding to PRO pnlynentide or fragnsci therrof. In this manner, the antihodii can he used to detect die nresence of any nentide which shares one or more antigenic determinants with PRO pohypeptide.

EXAMPLE 61: Rational Druz Desigm he goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest a PRO polypeptide) 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 polypepidc in vivo (cf. Hodgson. Bio~MchnlgY. 9: 19-21 (1991)).

In one approach, the three-dimensional structre of tie PRO polypeptide, or of an PRO polypeptide-inhibitor comnplex, is determined by x-ray crystallography, by computer modeLing or, most typically, by a comnbination of the two approaches. Both the shape and charges of the PRO polypeptide must be ascertained to elucidate the structure and to dctermine active site(s) of the molecule. Less often. useful informiation regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, 'relevant structual infornation is used to design analogous PRO polypepdde-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. Riochzmizu. UI:7796-7901 (199) or which act as inhibitors, agonists. or antagonists of native peptides as shown by Athauda el at., 1.BIodw.. JML:742-746 (1993).

It.is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. Tbis approach. i principle, yields a-pharmacorc upbon which-subsequemt drug design can be based. it is possible to bypass protein crysallograpb altogether by generating mnti-idiocypic antibodics *(anti-ids) to a functional. pharmacologically active antibody. As a mirro image of a mirror image, tie binding site *of the andi-ds would be expected to be an analog of die original receptor. 'The anti-id could theni be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmactire.

By virtue of the present invention. sufficient amounts of the PRO polypeptide may be made available to perform stich analytical sti~es as X-M rayystallography. In additon, 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.

EXAM1PLE Diaynosncr Test Using PRO317 Egtvpdde4Snefic Antibodies Particular anti-PR0317 polypeptide antibodies arc useful for the diagnosis of prepathologic conditions, and chronic or acute diseases such as gynecological disease or ischentic diseases which arc characterized by differences in dhe amourit or distinion of PR0317. PR0317 has been found to be expressed in humn kidney and is thus likely io 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 endometnium and other genital tissues. Further, due to library sources of certain 0 *O ESTs. it appears that Pi0317 may be involved as well in forming blood vessels and hence to be a modulator of see* angiogenesis.

Diagnostic tests for PR0317 include methods utilizing the antibody and a label to detect PR0317 in human body fluids, tissues, or extracts of such tisses. 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 sees covalently or noncovalently, with a substance which provides for a detectable signal. A wide variety of labels and conjugation techniqlues are known and have been reported extensively in both the scientific and patent Literature.

Suitable labels include radiorsiclidecs, cmymes, su-bstrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles, and the like. Patents teaching the use of such labels includ U.S. Pat. Nos. 3.817.837; 3.M50752. 3.939,350; 3,996,345; 4,277437; 4,275,149; and 4,366.241. Also, recombinant inmmoglobulins may be produced as shown in U.S. Pat. No. 4,616.567.

A variety of protocols for measuring soluble or membrane-bound PRo3 17, using either polyclOnal or monoclonal antibodies specific for that PROM 1, are kown in the art. Examples include enzyme-linked imniunosorbent assay (ELISA), radioimmunassay radioreceptor assay and fluorescent activated cell sorting (FACS). A two-site monoclonal-based inmmunoassay utilizing monoclonal antibodies reactive to two mon-interfering epitopes on PR0317 is preferred. but a competitive binding assay may be employed. These assays arc described. among other places. in Maddox ea a. I Ex -Me- 1L 121 1 (1983).

EXAMPLjE 63: Identification of PR03 17 Receptors Purified PR0317 is useflul fo r characterization and purification of specific cell surface receptors and other bindling moolocules. Cells which respond to PR0317 by metabolic changes or other specific responses are likely to express a receptor for PR03 17. Such receptors include. but arc niot limited to, receptors assdciatcd with and activated by tyrosine and serine/dueonine kinases. See Kolodziejczyc and Hall, hupra, for a review on known receptors for dft TGF- superfamily. Candidate receptors for this superfamily bai into two primary groups, termed typ I and type UI receptors. Both types art sericAhnronine kinases. Upon activation by the appropriate ligand, type I and type 1I receptors physically intrct to form hetcro-oligomers and subsequently activate intracellular signaling cascades, ultimately regulating gene transcription and expression. In addition, TGP- binds to a third receptor class, type III. a membrane-anchored protooglycan lackting die kinasc activity typical of signal transducing molecules.

PR0317 receptors or other PRO3 17-binding molecules may be identified by interaction with radiolabeled PRO3 17. Radioactive labels may be incorporated into PR0317 by various methods known in the art. A preferred emodientz is the labeling of primary amino groups in PRO3 17 with '23 Bolton-Hunter reagent (Bolton and Hunter, Bicen 1:529 (1973)), which has been used to label other polypeptdecs without concomitant loss of-biological activity (Hebert ar LioL-Chm_. 2M:18989 (1991);. McColl a JImnol,.. M:45504555 (1993)).

Receptor-bearing cells are incubated with Labeled PRO3 17. The cells arc then washed to removed unbound PRO3 17.

and receptor-bound PR0317 is quantified. The data obtained using different concentrations of PR0317 are used to *:calculate values for the number and affinity of receptors.

LAbed PRO317 is ueful asa reagent for puiication of isspecific receptor. In oneecmbodiment of affnity purification, PRO3 17 is covalently coupled to a chromaitography column. Receptor-bearing cells arc extracted, and :the extract is passed over the column. The receptor binds to the column by virtue of its biological affinity for PROW 1. Thne receptor is recovered from the cohznm anid sujected to N-termninal protein sequencing. This amino acid sequence is theni used to design degenerate oligonucleoude probes for cloning the receptor gene.

In an alternative method, mRINA is obtained from receptor-bearing cells and made into a cDNA library. The library is transbuced into a population of cells, and those cells expressing the receptor are selected using fluorescently labeled PRO3 17. The receptor is identified by recovering and sequecming recombinant DNA from highly labeled Cells.

In another alternative methoid, antibodies are raised against fth surface of receptor bearing cells, specifically monoclonal antibodies. The monoclonal antibodies an screened to identify those which inhibit the binding of labeled PRO3 17. 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 thant 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 unbvwn.

EAMLE 64: Deterjiaion of PRO3 17-Induced- Cellular Rrete The biological activity of PR0317 is measured. for example by binding Of an PR0317 of the invention to an PR0317 recptor. A test coqnipd is screened as an antagonist for its ability to block binding of PRO317 to the receptor. A test compound is screened as an agonist of the PROM1 for its ability to bind an PR0317 receptor and hnfuece diame physiological events as PR0317 using. for example. the KIRA-EUSA assay described by Sadick e at.. Analytical Biochemistry 2U.:207-214 (1996) in which activation of a receptor tyrosine kiae hs monitored .by Jimmwio-cature of the activated receptor and quantitation of the evel of ligand-induced phosphorylation. The -assay may be adapted to MOnitor PR03174ndticed reepor activation through the use of an PR0317 receptor-specific antibody to capture the activated receptor. These techntiques are also applicable to Other PRO polypeptides described herein.

EXAMPLR 65: Use of PRO224 far Sreeninc Comeoands PR0224 is expressed in a cell stripped of membrane proteins and capable of expressing PR0224. Low density liporoteina having a detectable label are added to the cells and incubated for a sufficient time for endocytosis.

The cells ame washed. The cells are then analysed for label bound to the membrane and within the cell after cell lysis.

Detection of the low denisity lipoproteins within the cell determines that PRO224 is within the family of low density li'poprotein Meceptr proteins. M s bifond wizhin (is family are then used for scrtening compounds which affect these receptors, and particularly the uptake of cholesterol via these receptors.

EXAMP2L 6: Ability of PRO Poyneoddes to Inhibit Vasuar Ezvlothelial Growth Factor (VEGFR Stimulated.

Proliferation of Endothelial Cell Growth The ability Of various PRO polypeptdecs to inhibit VEGF stimulated proliferation of endothelial cells was tested. Specifically, bovine adrena cotical capillary endothelial (ACIE) Cells (from primary culture, maximumn 12-14 Passages) were plated on 96-well mnicrotiter plates (Amersham Ufc Science) at a density of 500 cellstweU per 100 stL in low glucose DMEM, 10% calf serum, 2 suMA glutamine, lX pen/strept and fungizone. supplemented with 3 aghnL VEGF. Controls were Plated the same way but some did not include VEOF. A test sample of the PRO POlypeptie Of interest was added in a 100 gl volume for a 200 jil final volume. Cellswr iubad fo 67 dys at 37*C. The media was aspirated and the cells washed Ix with PBS. An acid phosphatase reaction mixture (100 F4, 0.l1M sodium acetate. pH 5.5. 0.1 Triton-100, 10 mm pnitrophen.yl phophate) was added. After incubation for 2 hours a37C.the rection wasstopped by addition of 10pl IN NaOH. OD was measured on microtiter plate Meader at 405 ma. Controls weno cells, cells aloecell FGF (5ng/mL). cells VEGF (3 ng/mL), cells VEGF (3 ag/ml) TGF-p (I ag/mI). and cells VEGF (3ng/mL) UF (S ng/mL). (TGF-P at a 1 ng/ml concentration is known to block 70-90% of VEGF stimulated cell proliferation.) Ile results were assessed by calculating the percentage inhibition of VEOF (3 nglml) stimula~ted cells proliferation, determined by measuing acid phosphatase activity at 0D405 ma,, relative to cells without stimulation, and relative to the reference TGF-P inhibition of VEGF stimulated activity. The results, as shown in Table 2 below, are indicative of the utility of the PRO polypeptides in cancer therapy and specifically in inhibiting tumor angiognesis. The numerical values (relative inhibition) shown in Table 2 are determined by calculating the pecal inhhirdo of VEGF stimulated prolifertion by the PRO polypcpdde relatve to cells without simuution and tln dividing diat peentage into the pcnt inhibition obtained by TGF-p at I ng/ml which is Iowa to block of VEGF stimulated cclU proliftradion.

0...0 .0 .0 00..

00 0 0 0 0 0.0.0 PRO Nmc PRO211 PR0211 PRO211 PRO211 PRO21I PRO211 PR0217 PR0217 PR0217 PR0217 PR0217 PR0217 PRO187 PRO197 20 PR1S7 PR0219 PR0219 PR0219 PR0246 25 PR0246 PR0246 PR0228 PR0228 PR0228 PR0228 PR0228 PR0228 PR0228 PR0228 PR0228 PR0245 PR0245 PR0245 PR0245 PR0245 PRO245 PR0221 PR0221 PR0221 PR0221 PR0221 PR0221 PR0258 PR0258 PR0258 PR0258 PR0258 PR0258 PRO Conmntrntion 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% 0.01% 0.1% 1.0% 2.5 uM 25nM 250 nM 0.01% 0.1% 1.0% 5.7 gM 57nM 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% Rebtive JnhbidQo 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 PRO Nm PRO301 PRO301 PRO301 PR0301 PRO301 PRO301 PR0224 PR0224 PR0224 PR0272 PR0272 PR0272 PR0328 PR0328 PR0328 PR033 1 PR0331 20 PR0331 PRO Cnncerntrtion 7.0 jAM 70,uM 700 pM 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% elaiv nbid2 1.02 0.88 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 EXAMPLE 67: Retinal Neuron Surviva This example demonstrates that PR0220 polypeptides have efficacy in enhancing the survival of retinal neurn cells.

Sprague Dawlcy rat pups at postnatal day 7 (mixed population: glia and retinal neuronal typs) are killed by decapitation following CO, anesthecsia and the eyes are removed under sterile 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 Cai*, M g-re PBS. The retina are incubated at 37*C for 7-10 mi'nutes after which the trypsin is inactivated by adding 1 ml soybean trypsin inhibitor. The cells are plated at 100,000 cells per well in 96 30 well plates in DMEM/F12 supplemented with N2 and with or without the specific test PRO polypeptide. Cells for all experiments are grown at 37'C in a water saturated atmosphere of 5% CO,. After 2-3 days in culture, cells are stabled with calcin AM then fixed usin 4% paraformaldehyde and stained with DAPI for determination of total cell count The total cells (fluorcsccni) are quantified at 20X objective magnification using CCD camera and NIH image software for Macintosh. Fields in the well arm chosen at random.

The effect of various concentration of PRO220 polyppids are reported in Table 3 below where percent survival is calculated by dividling the total nmber of ealcein 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.

TabIl ERQJNarmc PR0220 PR0220 PR0220 PR0220 PR0220 PRO220 PRO Concentration 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% 2.4% 4.1% 3.1% 72.4% 42.1%e EXAMPILL 6: Rod Photorrceptor Survival This example d~cionstrates that PR0220 polypcptidcs have efficacy In enhancing the survival of rod photoreceptor cells.

Sprigue Dawley rat pups at 7 day postnatal (mixed popultion: glia and retinal neuonal cell types) are killed by decapitation following CO% anesthesis and the eyes arc removed under sterile conditions. The neural retina is dissected away form dt pigmnt cpittliumn and other ocular tissue and then dissociated into a singlec cell suspension using 0.25% trypsin in Ca 2 mg 2 -free PBS. The retinas are incubated at 37*C tar 7-10 minues after which the eypsin is inactivated by adding 1 m! soybean trypsia inhibitor. Tbe cells are plated at 100.000 cells per well in 96 well plates in DMEM/F12 supplemented with N2 and with or without the specific test PRO polypeptide. Cells for all experimets are grown at 376C in a water saturated atmoshere of 5% C02. After 2-3 days in culture. cells are fixed using 4% paraformaldehyde. and then stained using Cellfracker Green CMFDA. Rho 4D2 (ascites or IgG 1:100). a ntenclonal antbody dirce towardc h visual pigment rhodopsin is used to detect rod photorecepocel by indirect immunofluoresec. The results are reported as survival: total cnmber of caleinirceIlTracker rhodopsin positive cells at 2-3 days in cultin. divided by dte total number of rhodopsin positive cells at time 2-3 days in culture. The total cells (fluorescent) are quantified at 20x objec magnification using a CCD camera and NIH image software for Macintosh. Fields in the well are chosen at random.

*The effect of various concentraion of PRO220 polypeptides are reported in Table 4 below. Anything above survival is considered positive..

EBg.oiNm PRO Concentration Penturial PR0220 0.01% 0.0% PR0220 0.1% 0.0% :PR0220 2.0% 0.0% PR0220 10% 0.0% PR0220 20% 66.9% PR0220 1.0% 56.9% EXAMPLE 69: Induction of Endothetial Cell Anoptosis The ability of PR0228 polypeptides to induce apOPtosis in endothelia cells was tested in humian venous umbilical vein endotclial cells (HUVEC. Cell Systems), using a 96-well format. in 0 serum media supplemented with 100 ng~ml VEOF. (As HUVEC cells are easily dislodged from the plating surface, all pipetting in the wells must be done as gently as practicable.) The mediawas aspired andthe cells washied once with PBS. mnloflIx trypsin was added to the cells in a T-175 fask. and the cells were allowed to stand until they were released from the plate (about 5-10 mnutes).

Trypsininio was utopped by adding 5.m1 of growth media. The cells were spun at 1000 tpm for 5 minutes at 4'C.

The media was aspirated and the cells were resuispended in 10 ml of 10% serum complemented mediumi (Cell Systems), I x penstrep.

The cells were plated on 96-well nicrotiter plates (Amershamn Life Science, cytostar-T scintillating m-icroplate. RPNQ16O, sterile, tissue-culture treated, individually wrapped), in 10% serum (CSG-tnedium. Cell System). ata density of 2x I'cel sper wllin aotlvolume of 100Id. he PRO29polyide wasadded i trilicateatddtoos; of 0.33% and 0.11%. Wells without cellawere used asablank and wells with cells only as a negative control. As a positive cotrol 1:3 serial dilutions of 50 #Ld of a 3x stock of utaurosporne were wed.

Iic ability of the PRO228 polypeptide to ieduce apaptosis was determined using Annexin V, a member of the calcium and phospholipid binding proteins, to detect apoptosis.

0.2 mlAnnexin V -Biotinstock solution (100 pg/ml) wered d j n4.6 nl 2 xCa?* bimling buffer aid BSA (1:2 dilution). 50 phl of the diluted Annexin V Bioti solution were added to each well (except controls) to a final concentration of 1.0 pg/mI. 'Me samples were incubated for 10-15 minutes with Annexiih-Biotin prior to direct addition of 3 5 S-Streptavidin. "4 trptavidin. was diluted in 2x Ca* binding buffcr. 2.5% BSA and was added to all wells at a final concaerrauionof 3 x 10 cpm/well. The plates were then sealed, centrifuged at 1000 rpm for 15 nuirrues and placed on orbital shelter for 2 hours. The analysis was perforrmed on 1450 Microbeta, Trilux (Waflac). The results ame down in Table 5 below whre percet above background represents the pcrcetage amo0unt counts per minute above the negative eorrols. Percents greater than or equal to 30% above background are considered positive.

PRO Name POCnetainPercnt Above Backound PR0228 0.11% 0.7% PR0228 0.11% 47.6% PR0228 0.33% 92.2% PR0228 0.33% 123.7% PRO228 1.0% 51.4% .PR0278 1.0% 95.3% 2 EXAMPLE2 PDR12 Cell Inibition This example demeristes that various PRO polypeptides have efficacy in inhibiting protein production by PDB12 pancreatic ductal cells.

PDBI2 pancreatic ductal cells arc plaWe on fibronectin coated 96 well plates at l.SxlO' cells per well in 100 grL/180 puL of growth media. 100 p1.. of growth media with the PRO polypeptide test sample or negative control lacking the PRO polypeptide is then added to well. for a final volume of 200 pL Controls contain growth medium containing a protein shown to be iactive in this assay. Cells are incubated for 4 days at 3VC. 20 pL of Alamar Blue Dye (AD) 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 ore excitation and 590 am emission. Toe standard employed is cells without Bovine Pituitary Extract (BPE) and with various conicentrations of BPE. Buffer or CM controls from unknowns are run 2 times on each 96 well plate.

The results from thee assays are shown in Table 6 below wherein percent decrease in protein production is calculated by comparing the Ala=a Blue Dye calculated protein concentration produced by the PRO polypeptidetreated cells with the Alamar Blue Dye calculated protein concentration produced by the negative control cells. A percent decrease in protein production of greater tha or equal to 25 as compared to thc negative control cells is considered positive.

Table PR am PRO211 PRO211I PRO211 PR0287 PRO28 PRO29 PRO2W PR0287 PROW9 PRO301 PRO301 PRO301 PR0293 PR0293 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 Decrease in Protein Prod-cio 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% EXAMPLE 71: Stimulation of Adult Heart flv~errophy This assay is designed to measure the ability of various PRO polypeptides to stimlat hypertrophy of adult heart.

Ventricular myocytes freshly isolated from adult (250g) Sprague Dawley rats are plated at 2000 cell/well in 180 ju1 volume. Cells are isolated and plated on day 1. the PRO polypeptide-comining test samples or growth medium only (negative control) (20 IL1 volume) is added on day 2 and the cells are then fixed and stained on day After staining, cell size is visualfized wherein cells showing no growth enhancement as compared to control cells are 25 given 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 enhanceement as compared to the negative control cells is considered positive for the assay.

Thec results are shown in Table 7 below.

-IT be: PRO Nam PR0287 PR0287 PRO301 PRO301 PRO293 PRO293 PR0303 PR0303 PR ocentrsio 20% 20% 20% 20% 20% 20% 20% 20% Growth Enhancement Score EXAMPLE 22: PDB12 Cell Proliferation This example demostates that various PRO polypeptides have efficacy in inducing proliferation of PDB12 pancreatic ductal cells.

PDB12 pancreatic ductal cells ame plated on fibrariectin coated 96 well plates at I .5x10' cells per well in 100 gIJI 80 iL of growth media. 100 iL of growth media with the PRO polypeptide test sample or negative control laciting the PRO polypeptide is then added to well. for a final volutne of 200,uL. Controls contain growth medium containizig a protein shown to be inactive in this assay. Cells art incubated for 4 days at 371C. 20 puL of Alamar Blue Dye (AB) is then added to each well and the flourescent reading is measured at 4 hours post addition of AD. on a microtiter plate reader at 530 am excitation and 590 nm emission. The standard employed is cells withou Bovine Pituitary Extract (BPE) a&d with various concentrations of BPE. Buffer or growth medium only controls from uninowns are run 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 polypeptidetreted cells with the A1a2-a Blue Dye calculated protein concentration produced by the negative control cells. A perent increase in protein production of greater than or equal to 25 as compared to the negative control cells is considered positive.

:PRO3Nam PRO Concentration Percent Increase in Protein Produiction PR312.0% 44.0% PR0301 10% 67.4% PRO301 -50% 185.8% PR0303 2.0% 27.9% PR0303 10% 174.9% PR0303 50% 193.1% ~EXAMPLE173: FEnhancent of Heart NeorimaW H=neSruhy Induced by PRO224 This assay is desigred to measure the ability of PR0224 polypeptides to stimualate hypertrophy of neonatal heart.

25 Cardiac snyocyte from W~ay old Harlan Spragute Dawley rats were obtained. Cells (180 p1l at 735 x iO 4 /i, 25 serum freshly isolated) are added on day 1 to 96-wcl plates previously coated with DMEMIF12 4% PCS.

Test samples containing the test PRO224 polypeptide or growth medium only (hegative control) (20 u1/well) are added directly to the wells on day 1. PGF (20 id/wel) is then added on day 2 at final concentration of 104 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 showing-a large increase an size as compared to negative controls are scored 2.0. The results are shown in Table 9 below.

Table 9 PRO Name POCnetainGrowyth Enhancement Score PR0224 0.01% 0.0 PR0224 0.1% 0.0 PR0224 1.0% EAMP.LE74: In situ Hybrdization In situ hybridizaton is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression.

mnalyw the issue dismriuron oftranscription. identify and localize. viral infection. follow changes in specific mRLNA synthesis and aid in chromosome mapping.

In sit hybridization was performed following an optimized version of the protocol by Lu and Gillett, C&U XYign 1:169-176 (1994). using PCR-generated '"P-labeled riboprobes. Briefly. formalin-tixed. paraffin-embedded human tissues were: sectioned. depanaffnized. deproteinared in proicinasc K (20 g/inl) for 15 minutes at 37'C. and.

further processed for In situ hybridization as described by Lu and Gillen;. jqira. A [33-P] UTP-labeled anisense niboprobe 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.

21 PMboltrobe synthesis Id (125 mCi) of 3"P-UT? (Amersham BF 1002. SA <2000 Cilmmnol) were speed vac dried. To each tube comntnn dried 'P-tflT. the following ingredients were added: 2.0 ul 5x transcription buffer #1 DTF (100 mM) INTP mix (2.SM :l10 I; each of10 mM GTP, CTP &A7P 10 IdHO) Al UTP (50 AM) p±1 DNA template (lpg)

ILHO

Id RNA polymerae (for PCR products 73 AS. 7 S. usually) The tubes were incubated at 37*C for one hour. 1.0 p1 RQ1 DNase were added, followed by incubation at 37 c for 15 minutes. 90 A1 TE (10 mM Tris pH 7.61mM EDTA pH 8.0) were added, and the mixture: ;ws pipetted oam DEBI paper. The remaining solution was loaded in a Microcon-SO ultrafiltration unit, and spun using program 10(6 mhaites). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes).

Aftertdiefinal recovery spin. 100 iTE were added. 1 Aloftthe final product was pipeted on DEBI paper and counted in 6 ml of Biofluor 11.

The probe wasrnnon a ThEurea gel. 1-3 Idof theprobe or 5pl ofRNA MrklIIIwere added to 3 plof loading buffer. After heating on a 95*C beat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, the sample loaded, and rn at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR filmn with'an intensifying screen in -70*C feezer one hour to overnight.

A. Pretreatment of frozen sections The slides were rmomved from the freezer, placed on aluminium trays and thawed at room temperature for minutes. The tray were placed in 55*C incubator for five minute to reduce condensation. The slides were fxed for 10 minutes in 4% paraormaldehyde on icc in the fume hood, andi washed in 0.5 x SSC for 5 minutes, at room temperatur (25 mld 20 x SSC 975 ml SQ 1120). After deproteination in 0.5 I~g/ml proteinass K for 10 minutes at 37*C (12.5 Id of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%. 95%. 100% ethanol, 2 minutes each.

178 B. Prtreatmn of parffin-aedded sections The slides were deparaffinized, place in SQ H 2 0, and rinsed twice in 2 x S9C at room tenperature, for 5 minutes each time. The sections were deproteinated in 20 r/mrl proteinase K (500 pl of 10 mg/ml in 250 ml RNase-free RNase buffer; 37 0 C, 15 minutes) human embryo, or 8 x proteinase K (100 l in 250 ml RNase buffer, 37 0 C, 30 minutes) formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.

C. Prebbridization The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% fornamide) 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 were added, the tissue was vortexed well, and incubated at 42 0 C for 1-4 hours.

D. I4bridization x 10' qn probe and 1.0 pl 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 20 hybridization buffer were added per slide. After vortexing, 50l 33 P mix were added to 50 pl prehybridization an slide. The slides were incubated overnight at E. Washes Washing was done 2 x 10 minutes with 2 x SSC, EDrA at roan 25 temperature (400 ml 20 x SSC 16 ml 0.25M EDTA, Vf=4L), followed by RNaseA treatnment at 37 0 C for 30 minutes (500 pl of 10 rg/ml in 250 ml RNase buffer 20 IThe slides ware washed 2 x 10 minutes with 2 x SSC, EDVA at roan terperature. The stringency wash conditions were as follows: 2 hours at 55 0 C, 0.1 x SSC, EDEA (20 ml 20-x SSC 16 ml EDrA, Vf=4L).

F. Oligmnucleotides In situ analysis was perforned on a variety of ENA sequences disclosed herein. The oligonucleotides aeployed for these analyses are as follows: MA33094-1131 (PRO217) p 5 -OCTIARGACACI3 (SEQ2 ID NO: 347) p2 5' -crA3AATAACOCICACPAAQGUCltICITt AACTIc-3 (SEQ ID NO: 348) a...r I33223-1136 (PI230) p1 5' -TI ATA CAC AT A-3' (SE ID N:349) 179 p2 5' 3' (SE2 ID NO: 350) ENA34435-1140 (PRO232) p1 5# -3 (SE2 ID NO: 351) p 2 5'3-(E ID ND: 352) *0.

(6) p1 p2 (7) 20 p 1 p2 (8) p1 p 2 ENA35639-1172 (P1R,246) I1Th.9435-1219 (PRO533) 5' -GTtAAAiCCATT 31GXXOCI-3' ENA35638-1141 (PiW245) -3 ENA33O89-M12 (PRO221) 5' -3nT~AA~rTorc 1~~~A3# 5' 31 ENA35918-174 (PRO258) 5' -art'ATA3LrcrT 3' 5' T~cnG~CCCG31 EM32286-Ml9 (PRO214) -aMrAA~CtCAN~t~~lZIIC~ft-3' -TTaCc-3' EM~33221-1133 (PRO224) 5' -~a'1ATAC1~CT3' 5' 3' ENA3S557-2.17 (P=~234) 5'xD3xa ~3AA~-3' (SEQ ID NO: 353) (SEQ ID NO:354) (SEQ ID ND:355) (SEQ ID NO:356) (SEQ ID NO:357) (SEQ ID NO:358) (SEQ ID NO:359) (SEQ ID NO:360) (SEQ ID NO.361) (SEQ ID NO:362)

(SEQ

(SEQ

NO: 3 63) NO: 364) p 1 p2

(SEQ

(SEQ

ID NO:365) ID NO: 366) (2.1) p 1 (sEQ ID NO: 367) p2 5' -CAtAT7A 1AANAANU~3AI 3' (SEQ ID No: 368) E3310O-L159 (PRO229) 180 p1 5' -OATICrAATAC ACIAcAa X 03-3' (SE2 ID NO: 369) p2 5' -CTACIAAArTAAICTNAA ACAC TACOC-3' (SEQ ID NO: 370) (13) ENA34431-1177 (PRO263) p1 5' TIAATAACICACTATAOCAA-3' (SEQ ID NO:371) p2 5' -CGArAAATAACICACTAAOGrA X A-3' (SEQ ID NO:372) (14) IM38268-1188 (PRO295) p1 5' -3ATITAATAGACI CACFC3' (SEQ ID 10:373) p2 5' -C A'AAAAACOCICACTAAOGACIAG-3' (SEQ ID NO:374) G. Results In situ analysis mas perforned an a variety of II sequences disclosed herein. The results fran these analyses are as follows.

EMM33094-1131 (PF4217) **Highly distinctive expression pattern, that does not indicate an obvious biological function. In the human embryo it was expressed in outer snooth nuscle layer of the GI tract, respiratory cartilage, branching respiratory epithelium, osteoblasts, tendons, gonad, in the optic nerve head and developing dennmis. In the adult expressin was observed in the epidernal pegs of the chinp tongcue, the basal epithelial/myoepithelial cells of the prostate and urinary bladder. Also expressed in the alveolar lining cells of the adult lung, mesenchymal cells juxtaposed to erectile tissue in the penis and the cerebral cortex (probably glial cells). In the kidney, expression was only seen in disease, in cells surrounding thyroidized renal tubules.

D=n=n fetal tiassues examined (E12-E16 weeks) include: Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus, stonach, small intestine, spleen, thynus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower lixrb.

Adult buann tissues examidned: Kidney (nonral and end-stage), adrenal, mnyocardium, aorta, spleen, lynph node, gall bladder, pancreas, lung, skin, eye (inc. retina), prostate, bladder, liver (norml, cirrhotic, acute failure).

Non-uman prinate tissues exmined: Chinp Tissues: Salivary gland, stomach, thyroid, parathyroid, skin, thynus, ovary, lynph node Rhesus Monkey Tissues: Cerebral cortex, hippocanpus, cerebellum, penis.

180a ENA33223-:M36 (PP0230) Sections show an intense signal associated with arterial and venous vessels in the fetus. In arteries the signal appeared to be confined to snooth-uscle/pericytic cells. The signal is also seen in capillary vessels and in gloueruli. It is not clear whether or not endothelial cells are expressing this nRNA. Expression 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 H"F uncertain histogenesis. In the adult, there was no evidence of expression and the wall of the aorta and most vessels appear to be negative. However, expression was seen over vascular channels in the normal prostate and in S S o •the epithelium lining the gallbladder. Insurers expression was seen in the vessels of the soft-tissue sarcoma and a renal cell carcincma. In sumary, 15 this is a miolecule that shows relatively specific vascular expression in .the fetus as well as in same adult organs. Expression was also eee *4 6 09

S

4 oewo ooo• 4..

4

GS*

55.5 55.5 observed in the fetal lens andi the adult gallbladder.

In a secondary screen, vascular expression was observed, similar to tha observed above. seen in fetal blocks. Expression io n vascular smooth Muscle. rather than endotheliwn. Expression also seen in smooth musc of the developing oesophagus. so as reported previously, this molecule is not vascular specific. Expression was examined i&4 lung and 4 breast carcinomas. Substantial expression was seen in vascular smooth muscle of at least 3/4 hing cncen andl 2/4 breast camers. In addition, in one breast carcinoma, expression was observed in peritumoral acronial cells of uncertain histogenesis (possibly rnyofibroblasts). No eridothelial Cell expression was observed in this study- DNA34435-1 140 (PR0232) Strong expression in prostatic e-pithelium and bladder epitheumn, lower level of expression in bronchial *epithelium. High background low evel expression seen in a number of sites. including among others. bone, blood.

chondhoarcoma. adult heart and fetal liver. It is felt that this level of signal represents background. partly because signal at this level was xee over the blood. All other tissues negative.

Human fetal tissues examined (E12-E6 weeks include: Placenta, umbilical cord, liver, kidney. adrenals, -thyroid.

lungs, heart, great vessls, oesophagus, stomach, small intestine.

spleen, thymus. pancreas, brain, eye, spinal cord, body wall, pelvis, testis and lower limb.

Adul b= Dises xamied:Kidney (normal and enid-stage), adrenal, spleen, lymph node, pancreas, lung, eye (inc.

retina), bladder, liver (normal, cirrhotic, acute failure).

20 Non-human pimatc tissues examined: Tlissues: adrenal Rhesus Monkey Tissues: Cerebral cortex, hippocamnpus In a secondary scen, expression was observed in the epithelium of the prostate, the superficial layers ot '.the uretheliumn of the urinary bladder, the urethelium lining the renal pelvis and the urethiumn of the ureter (I out of 2 experimensa). The urethra of a rhesus monikey was negative; it is unclear whether this represents a true lack at expression by the urethra, or if it is the result of a failure of the probe to cross react with rhesus tissue. The findings in the prostate and bladde arc similar to those previously described using an isotopic detection technique. Expression of the mRNA for this antigen is NOT prostate epithelial specific. 'The antigen may serve as a useful marker for etdil derived tissues. Expresion in the suiperficial, post-mitotic cells, of the urinary tract epithelium also suggest that it is unlikely to represent a specific stem cell marker, as this would be expected to be expressed specifically in basal epithelium.

DNA35639-172 (PR0246) Strongly expressed in fetal vascular endothelium, including tissues of the CNS. Lower level of expression in adult vasculaure including the CNS. Not obviously expressed at higher levels in tumor vascular endothelitini.

Signal also seen over bone matrix and adult spleen, not obviously cell associated, probably related to non-specific background at these sites.

Humn fetal tissues examined 12-F16 weeks) include: Placenta, umbilical cord, liver, kidney. adrenals, thyroid.

kings. heamt great vessels. aesophagus. stomach. small intetine, spleen. thymus. pancreas. brain, eye, spinial cord, body'wall, pelvis, testis and lower limb.

Adakkuma tis= exmine:Kidney (nortnal and end-age), adrenal, spleen, lymph node. pancreas. lung. cyc (nc.

retina), bladder. liver (normal, cirrhotic, acute failure).

Non-human primate tissues examined: ChiinR.ITissues: adrenal Rhesus Monkey Tissues: Cerebral cortex. hippocampus DNA49435-1219 (PROS5331 Moderate expression over cortical nteurones in the feral brain. Expression over the inner aspect of the fetal retina possible expression in tdo developing lenis. Expression over fetal skin. cartilage. small intestine, placental villi and umbilic;al cord. I adult tissues there bs an extremely high level of expression over dhe gallbladder epithelium.

Moderate expression over the adult kidney. gastric and clonic epidWela Low-level expression was observed over many Cell types in many tissues, this may be related to stickiness of the probe, these data should therefore be interpreted with a degree of caution.

Human fetal tissues examined! (El 2-E16 weeks) Inglud: Placenta, umbilical cord, liver, kidney. adrenals, thyroid.

hungs. heart. great vessels. oesophagus, stomach, small inestine. spleen, thymus. panicreas, brain. eye, spinal cord, body wall, pelvis, testis and lower limb.

Adul huan is=LumiW:Kidney (Iwmal andI end-stage), adrenal, spleen, lymph node, pancreas, lung, eye (we.- 20 reemn), bladder, liver (normal, cirrhotic, acute failure).

Non-humran oRimate tismues examined: Rhesus Monkey Tissues: Cerebral cortex. hippocanipus, cerebellum.

DNA35639-1 141 (PR02451 Expression observed in the endothelium linin a subset of fetal and placental vessels. Endothelial expression was confined to these tissue blocks. Expression also observed over intermediate trophoblast cells of placenta. All other tissuies negative.

Fea ise wie E1-I ek)icue Placenta, umbilical cord, liver, kidney, adrenals. thyroid lungs, heart, reat vessels, ocsophagus, stomach, small intestine, spleen, 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 hippocanapus(rni), cere~bclln(rm), penis, eye, bladder, stomach, gastric carcinoma, colon, colonic carcinoman, thyroid (chimp), parathyroid (chimp) ovary (chimp) and chondrosarcorna.

Acetominophena induced liver injury and hepatic cirrhosis DNA330I -132 (PR0221) Specific expression over fetal cerebral white and grey matter, as wefl as over ncurones in the spinal cord.

Probe appears (o crass react with rat. Low level of expression over cerebellar neurones in adult sken brain. All other tissues negative.

Fetal tissues examined (E12-EI6 weeks' include: Placenta, umbilical cord, liver, kidney, adrenals, thyoid, lungs, heart. great vessels. csophagus. stomach. small intestine, spleen thymus, pancreas, brain, eye, spinal cord, body wafl, pelvis and lower limb.

Adult tissues examined: Liver. kidney, adrenal, myocardium, aorta, spleen., lymph node, pancreas. lung, skin, cerebral cortex bippocampus(rm). cerebellum(rm), penis, eye, bladder, stomach, gastric carcixna, colon, colonic carinoa anW diondrosarcoma. Acewoiinophen induced liver injury and bepac cirrhosis flNA3591 &1174 (PRO2581 Strong expression in die. nervous system. In the rhesus monkey brain expression is observed i cortical, hipcmland carebella neurorkcs. Expression over spinal neurotics in the fetal spinal cord, the developing brain and the inner aspects of the fetal retina. Expression over developing dorsal root and autonomic ganglia as well as enteric nerves. Expression observed over ganglion cells in the adult prostate. In the rat, there is strong expression oaver the developing hind brain and spinal cord. Strong expression over interstitial cells in the placental villi. All other tissues were negative.

Fetal tissues examined (E12-EI6 wecks) include: Placenta, umbilical cord, liver, kidney, adrenals. thyroid, lungs, :hear, great vessels, ocsophagus, stomach', small intestine, spleen, thyrnus, pancreas, brain. eye, spinal cord, body wall, pelvis and lower limb.

0 Adult tissues examined: liver, kidney. renal cell carcinoma, adrenal, aorta, spleen, lymph node, pancreas, lung, mnyocardium. skin, cerebral cortex hippocampus(rrn), cerebellum(rm), bladder, prostate, stomach, gastric carcinoma, colon, colonic carcinoma, thyroid (chimp). parathyroid (chimp) ovary (chimp) and chondrosarcoma. Acetorainophen induced liver injury and hepatic cirrhosis.

DNA32296-1 191 (PRO214) Fetal tissue: Low level throughoiut inesenchymei. Moderate expression in placental stromal cells in membranous tissues and in thyroid. Low level expression in cortical neurotics. Adult tissue: all negative.

Fetal tissues examined (E12-Et6 weekcs) include: Placenta, umrbilical 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.

Adul tisue exrnied ~chle: ver, kidney, adrenal, nmyocardium., aorta, spleen, lymph node, pancreas, lung and skin.

(10) DNA3322 1-1133 (PRO224)i Expressioni limnited so vascular endotheliurn in fetal spleen, adult spleen, fetal liver, adult thyroid and adult lymph node (chimp). Additional site of expression is dhe developing spinal ganglia. All other tsissus negative.

Human fetal fitses examined (El2-E16 weeks) includ Place=t, umbilical cord, liver, lddricy. adrecals, thyroid.

hungs, heart, great vessel. csophagus, stomach, small'intestine, spleen, thymnus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult huma tissues examined: Kidney (normal and enid-stage), adrenal, myocardiurn, aorta, spleen, lymph node.

pancreas. ig. sin. eye (inc. retina), bladder, liver (normal, cirrhtidc, acute failure).

Non-humnan primate tissueg examined: iWm2isues: Salivary glandl, stomach, thyroid, parathyroid, sin. thymus. ovary, lymph node.

Rhesus Monkey Tissues: Cerebral cortex, bippocnius, cerebellum, penis.

(11) DNA35557.l1137 (PRO234) Specic exressio over developing motor ourones in ventral aspect of the fetal spinal cord (will develop into ventral horns of spinal cord). All other tissues negative. Possible role i growth, differentiation and/or development of spinal motor neurons.

Fetal tissues examined (El 2-F16 weel include: Placenta, umbilical cord, liver. kidney, adrenals,-thyroid, lungs, heart, great vessels, oesophagus. stomach, small intestine, spleen, thymus. pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult ri~sygs examined: liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex hippocampus(rm). cerebellum(rmn), penis, eye. bladder, stomach, gastric carcinoma, colon, colonic carcinomna and ehomrosarcoma. Acetovmohen induced liver injury and hepatic cirrhosis (12) DNA3100-1 159 (PRO229) Strikin expression in mononuclear phagocytes (macrophages) of fetal and adult spleen, liver, lymph node and aduilt thyamus (in tinglble body macrophages). The highest expression is in the spleen. All other tissues negative.

Localisation and homology are entirely consistent with a role as a scavenger receptor for cells of the retuculoendothelial system. Expression also observed in placental mononuclear cells.- HuMa fet ue examined (1312-1116 weeks) includie: PLacenta, umbilical cod liver, kidney, adrenals, thyroid, hungs, 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), adelnal, inyocardi, aorta, spleen, lymph node, gall bladder, pancreas, lung, sin, eye (inc. retina), prostate, bladder, liver (normal, cirrhotic, acute failure).

Non-human primate tissues examined: Chlft Tisue: Salivary gland, stomach, thyroid, parathyroid, sin, thymus. ovary, lymph node.

Rhestu Mon Tissues: Cerebral cortex, bippocampus, cerebellum, penis.

(13) DNA3443 1-1 177 (PR0263) Wklepread expression in human fetal tissues and placenta over mononuclear cells, probably niacroPhages +.lympocte. The Cclular distribution follows a perivascular pattern in many tissues. Strong expression also seen in cpithelial cells of the fetal adrenal cortex. All adult tissues were negative.

Felal tissues exarnined (E12-EI6 weeks) include: Place=.a umbilical cord. liver. kidney, adrenals. thyroid, lungs.

heart. great vessels. oesophagus. stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and tower limb.

Adult tissues examined: Liver. kidney, adrenal, spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm), hipcmus(rm), cerebellutn(rm), bladder, stomach, colon and colonic carcinoma. Actominophen induced liver injury and hepatic cirrhosis.

A secondary screen evidenced expression ave stromal mononuclear cells probably bistiocyres.

(14) DNA311269-I118 (PRO02951 High expression over ganglion cells in human fetal spinal ganglia and over large uneurones in the anterior horns of the developing spinal cord. In the adult there is expression in the chimp adrenal mrdulla (neurl). neurones of the themu monkey brain (hippocampaas ++]and cerebral cortex) and neurones in ganglia in the niormal adult human prstate (tre only section that contains ganglion cells, je expression in this cell type is presumed NOT to be confined to the prostate). All other tissues negative.

lrtrmn fetal fi-snes examined (E12-EI6 weeks)nrlue Placenta, uimbilical cord. liver. idney, adrenals. thyroid.

lunigs, great vessels. stomach, small intestine, spleen. thymus, pancreas, brain, eye. spinal cord, body wall. pelvis, testis and lower limb.

Adult human tissues examined: Kidne (normal and end-stage), adrenal, spleen, lymph node, pancreas, lung, eye (inc.

retina), bladder, liver (normal, cirrhotic, acute failure).

Non-humnan primate tissues examined: Chi= wJissue: adrenal Rhesus Monlkey Tissues: Cerebral cortex, bippocanipus, cerebellum.

Denosit of Material The following mateials have been deposited with the American Type Cultur Collection, 12301 Parklawn Drive. Rockville, MD, USA (ATCC): 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, 1997 DNA27864--1155 ATCC 20975 October 16, 1997 DNA36350-1158 ATCC 209378 October 16. 1997 DNA3229(1- 164 ATCC 209384 October 16, 1997 DNA35639-1 172 ATCC 20939 October 17, 1997 DNA3309-1202 ATCC 209420 October 28. 1997 DNA49435-1219 ATCC 209480 November 21, 1997 DNA35638-1141 ATCC 20965 September 16, 1997 DNA32298-1132 ATCC 209257 September 16, 1997 DNA33089-1 132 ATCC 20962 September 16, 1997 DNA33786-1 132 ATCC 209253 September 16, 1997 DNA35918-1 174 ATCC 209402 October 17, 1997 186 DNA3 7150-1178 DNA3 8260-1180 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 DNA34407-1169 DNA3 5841-1173 DNA3 3470-1175.

DNA3 4431-1177 DNA3 9510 -71181 DNA3 9423-1182 DNA4 0620-1183 DNA4 0604-1187 DNA3 8268-1188 DNA3 7151-1193 DNA3 5673-1201 DNA40370-1217 DNA42551-1217 DNA39520-1217 DNA4 1225-1217 DNA43318'-1217 DNA40587-1231 DNA41338-1234 ATCC2 09401 ATCC2 09397 ATCC209400 ATCC2093 85 ATCC2093 67 ATCC209432 ATCC209263 ATCC209251 ATCC209255 ATCC209252 ATCC2093 77 ATCC209370 ATCC209523 ATCC2093 72 ATCC209374 ATCC2 093 73 ATCC2093 82 ATCC2093 83 ATCC2 094 03 ATCC209398 ATCC2093 99 ATCC2O93 92.

ATCC2093 87 ATCC2093 88 ATCC2093 94 ATCC2 09421 ATCC2093 93 ATCC209418 ATCC209485 ATCC2 094 83.

ATCC209482 ATCC2 09491 ATCC209481 ATCC209438 ATCC209927 October 17, 1997 October 17, 1997 October 17, 1997 October 16, 1997 October 15, 1997 Novenfiber 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 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 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

C

by the U.S. Commissioner of Patents and Trademarks to be entited thereto according to 35 usc 5 122 and the Commissioner's rules pursuant thereto (including 37 CFR 1.14 with particular reference to8600OG638).

The assignee of tr. present application has agreed that if a culture; of the materials on deposit should die or be lost or destroyed when cltivated under sultable conditions, the materials will be promnptly replaced on notification with another of the same. Xvailability of the deposited material is not to be construed as a li c ense to practice the bwention in contravention of the rights granted under the authority of any g overnment in accordance with its patent' laws.

lit foregoing writtn specification is considered to be sufficient to enable one skilled in die art to practice the invention. The present invention is not to be limited in scope by dhe construct deposited. since the deposited emnbodimen is intended as a single ilustration of certain aspects of the invention and any constructhait ame functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of fte hmmnzon incldin die best mode thereof, nor is it so be construed as limiting the scope of dheclaims to the specific illustrations thdat ft cprestrus Indeed. various roodificatiois of the invention in addition to those shown and described herein will become appazua to those skilled in dhe art from the foregoing description and fall within the scope of the appended claim

Claims (18)

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 38 (SEQ ID NO:104).

2. A nucleic acid molecule according to Claim 1, wherein said nucleotide sequence comprises a nucleotide sequence as shown in Figure 37 (SEQ ID NO:103), 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 37 (SEQ ID NO:103), or the complement thereof.

4. An isolated nucleic acid molecule which comprises the full-length coding sequence of the DNA deposited under accession number ATCC 209400. A vector comprising a nucleic acid according to any one of Claims 1 to 4.

6. A vector according to Claim 5, operably linked to control sequences recognized by a host cell transformed with the vector.

7. A host cell comprising a vector according to Claim or Claim 6.

8. A host is a CHO cell. cell according to Claim 7, wherein said cell

9. A host cell according to Claim 7, wherein said cell is an E. coli. A host is a yeast 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 38 (SEQ ID NO:104).

13. Isolated PRO polypeptide having at least 80% sequence identity to the amino acid sequence encoded by the nucleotide deposited under accession number ATCC 209400. 15 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 25 immunoglobulin. *oo

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 Claims 14 to 16; or 190 d) an antibody according to Claim 17 or Claim 18, together with a pharmaceutically-acceptable carrier.

20. 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 GENENTECH, INC. 15 By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia *o *e