AU772734B2 - 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 reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in 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, 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 S* 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 20 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. P110211 and P110217 Epidemmal growth fb=o (E*GP) is a coniventionial mitogenic factor that stimiulates the Proliferation of various typW Of cells inCluin ePidla cells and fibroblasts. EGF binds to and activates dhe EGF receptor (EGFR). which initiates mntmccllr signaling and subsequent effects. The EGFR is expressed in =1urons of the cerebral cortcx.

cerebellum, Wd hipocamPus in addition to other regions of the centrA nervous system (CNqS). In addition, EGF is also expressed in various regions of the CNS. Thereore, EGF acts not only on mitotic cells, but also on posanitotic neurons. In fact, many studies have indicated dhat EGF has acurotrophic or neuromodulatory cffects 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 septa[ cholinergic and mesencephalic dopaminergic neurons, indirectly through glial ceils. Evidence of the effects of EGF on neurons in the CNS is accumulating. but the mechanisms of action remain essentially unkniown. EGF-induced signaling in miitotic cells is better understood than in potnittiic nerons. Studies ofdconed pheochroznocytonza PC12 cells and cultured cerebral cortical neurons have suggested that the EGF-induced neurotrophic actions are mediated by sustained activation of the EGFR and mitogen-activated protein kinase (MAPK) in response to EGF. The sustained intracellular signalin correlates with the decreased rame 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 postitotic neurons.

EGF is produced by the salivary and Brunner's glands of the gstrointestinal system, kidney, pancreas, thyroid gland, pituitary gland, and the nervous system, and is found in body fluids such as saliva, blood, cerebrospinal fluid (CSP), urine, amniotic fluid, prostatic fluid, pancreatic juice, and breast milk, Plata-Salamn, Peptides 12: 653-663 (1991).

EGF is mediated by its membrane specific receptor, which contins an intrinsic ryrosine kinase. Stoscheck et al., J. Cell Bioclzem. 11: 135-152 (1986). EGF is believed so function by binding to the,extraeeliular portion of its receptor which induces a transmembrane 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 et J. Bio. Chem. 7669-7672 (1979).

It is now generaly known that several other peptides can react with the EGF receptor which share the samte generalized motif X.CXA4,CXIDCXCXsGXICX., where X represent any non-cysteine amin acid, and n is a variable repeat n-nbr. Non isolated peptides having this motif include TGF-a. aniphiregulin, schwannotna-derived growth hictor (SDGI), heparin-bndn EGF-Iike growth factors and certain virally encoded peptides Vaccinia virus, Reisner, Nature W-i 801-803 (1995), Shope fibrowa virus, Chang et al., Mol Cell Biol. 7: 535-540 (1987).

Molhzscum contagiosun Porter and Archard, I. Gen. Virol. 60: 673-682 (198M) and Myxoma virus, Upton er al., J1 Varol. Al: 1271-1275 (1987), Prigent and Lemoine, Prog. Growh Factor Resr. 1: 1-24 (1992).

EGE-like domains are not confined to growth factors but have been observed in a variety of cell-surface and extracellular proteins which have interestinag properties in cell adhesion, protein-protein interaction and development, lLaurence arnd Gusterson, Tumor Biol. 11: 229-261 (1990). These proteins include blood coagulation factors (factors VI. IX, X. Xli, protein C, protein S. protein Z. tissue plasminogen activator, urokinase), extracellular matrix components (laminin, cytotactin, enitactin), coil surface receptors (LDL receptor, thrombomoodulin receptor) and innny-related proteins (Complement Mrt. uronmodulin).

Even more interesting, the general structure pattern of EGF-like Precursors is Preserved through lower organisms as well as in manmalian cells. A number of genes with dcvelopmental significance have been identified in invertebrates with EGF4ike repeats. For example, the notch gene of Drosophila encodes 36 tandcmly arranged amino acid repeats which show homology to EGF, Wharton es aL. Cell 51: 557- '581 (1985). Hydropathy-plots indicate a putative membrane spanning domain. with the EGF-rehated sequences being located on the extraceaular side of the membrane. Other homeotic genes with EGF-likc repeats include Delta, 95F and 5ZD which were identified using probes based on Notch, and the nematode gene Lin-)2 which encodes a putative receptor for a developmental signal transmitted between two specified ceils.

Specifically. EGF has been shown to have potential in the preservation and maintenance of gastrointestinal mucosa and! the repair of auzi and chronic -iucsal lesions, Konturek et aL.. Eur.). (kstroenterol HepatoL. 2 933-37 (1995). including the treatment of necrotizing enterocolidis, Zollinger-Ellison syndrome. gastrointestinal ulceration gastrointestinal ulcerations and! congenital microvilhas atrophy. Guglieuta and Sullivan. Eur. 1.

Qzsvoemerol Hepaol. 2(10). 945-50 (1995). Additionally, EGF has been implicated in hair follicle differentiation; du Cros. Inows. Dennol JU (I Suppi.). 106S-1 13S (1993). Hillier, GIn. EndocrinoL 427-28 (1990); kidney function, Hammera al. Sein. NephroL 13 109-15 (1993), Harris. Am. A. Kiney Dis. 627-30 (1991); tear fluid, van Setten et aL, Int. Ophthalmol 13(6); 359-62 (1991); vitamin K mediated blood coagulation, Stenfo a aL. BloodZ&(7: 1637-51 (1991). EUF is also imnplicated various skin disease characterized by abnormal keratinocyte differentiation, psoriasis, epiteal cancers such as squamous cell carcinomas of the lung, epiderimid carcinoma of the vulva and gliomas. King et aL. Am. J. Med. Sd. 226: 154-158 (1988).

Of great interest is mounting evidence that genetic alterations in growth factors signaling pathways are closely linked to developmental abnormalities and to chronic diseases including cancer. Aaronson, Science 1146-1153 (1991). For example, c-erb-2 (also known as HER-2), a proto-oncogene with close structural similarity to EGF receptor protein, is overexpressed in himan breast cancer. King ar al., Science M2: 974-976 (1985); Gullick, Honmones and their actions, Cooke et al., eds, Amsterdam, Elsevier. pp 349-360 (1986).

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

wherein those polypeptides are herein designated PRO21 1 and PR0217.

2. PRO230 Nephritis is a condition characterized by inflammation of the kidney affecting the structure and normal function of the kidney. This 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 nephits can begin treatment of the condition.

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

Cbm. 270C27):16265-70 (July 1995) (GENBANKU24270). 7bis stud reports that the rabbit TIN-ag is a basement membrane glycoprotein having a predicted amno acid sequence which has a carboxyl-terxninzI region exhibiting homology with human preprocatliepsin 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 epidennal growth factor-like motif that shares homology with laminin A and S chains, alpha 1 chain of type I collagen, von Willebrand's factor and mucin, indicating tructural 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 tubulointerstitial nephritis antigens are of particular interest to the medical and industrial communities. Often, proteins having homology to each other have similar function. It is also of interest when proteins having homology do not have similar functions, indicating that certain structural motifs identify information other than function, such as locality of function. We herein describe the identification and S characterization of a novel polypepide, designated hgcrein as PRO230, which has homology to tubulointcrstitial 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 15 the use of these options. Stem cells often express cell surface antigens which are capable of serving as cell specific markers that can be exploited to identify stem cells, thereby providing a means for identifying and isolating specific stem cell populations.

Having possession of different stem cell populations will allow for a number of important applications. For example, possessing a specific stem cell population will allow for the identification of growth factors and other 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 whim the stem cells ar 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 PRO232 polypeptides..

4. PROI87 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 Iregulators. Theiir biological effects Include proliferation, chemnotaxis and stimulation Of extraceljnjar matrix production. Growth factors can have both stirnulatory and inhibitory effects. For example, transforming growth factor (l'GF-P) is highly pleiotropic anid can stimulate proliferation in 3oMC Cells, especially connective tissue, While being a potent inhibitor of proliferation in others. such as lymphocytes and epithelial cells.

7"he physiological cifect of growth stimulation or inhibition by growth (actors depends upon the state of developmn and differemuation of the target tissuie. The mechanism of local cellular regulation by classical endocrine molecules involves comprehends autocrine (same cell), juxtacrine (neighbor ccli), and paracrinc (adjacent cells) pathways. Peptide growth factors are eazits of a complex biological language, providing the basis for initercellular communication. 7bey permit cells to convey information between each other, mediate interaction between cells and change gene expression. The effect of these multifunctional and pluripotent factors is dependent on the presence or absence of other peptides.

FGF4 is a member of the fibroblast growth factors (FO~s) which are a family of heparin-bitiding. potent miogenLs for both normal diploid fibroblasts and established cell lines, Gospodarowicz et at (1984). Proc. Nail.

Ao~d Sd. USA 11:6963. Ils FGF family comprises acidic FGF (FGF-l), basic FGF (PGF-2), INT-2 (FGF-3). K- F-GF/HST (FGF-4). FGP-S, FGF-6. KGF (FGP.7). AlOP (FOPF-8) among others. All PO~s have two conserved cysteine residues and share 30-50% sequence homology at the amino acid level. These factors are mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, including granulosa cells. adrenal cortical cells, chondrocytes, moyoblasts. cortical and vascular endothelial cells (bovine or human). vascular smooth muiscle cells, lens, retina and prostatic epithelial cells, oligodendrocytes, astrocy=es chrondocytes, myoblasts and osteoblasts.

Pibroblast growth factors can also stimulate a large mnber of cell types in a non-mitogenic manner. These activities incuh promotion of cell migration into wounid area (chemootaxis), initiation of new blood vessel formulation (angiogeticsis). moul~ation of nerve regeneration and survival (neurotrophism), modulation of endocrine functions, *.:and stimulation or suppression of specific cellular protein expression. extrtacellu matrix production and cell survival. Baird Bohlen, Handbook of Exp. Pharrmcol. 95(l): 369-418. Springer, (1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healing, nerve repair, collateral blood vessel formation, and the like. For example, fibroblast growth factors have been suggested to miiiemyocardium damage in heart disease and surgery 4.378,347).

POP-8. also known as androgen-induced growth factor (MAPG), is a 215 amino acid protein which shares 30-40% sequence homology with the other members of the FGF family. FGF-8 has been proposed to be under androgenic regulation arid induction in the mouse mammary carciourna cell line SC3. Tanaka et aL. Proc. Nail.

Aemi. Sd. US A12: 8-3932 (199); Sato et at.. J. Stervid Biochant. Motec. DioL 42: 91-98 (199). As a result, PGF-8 may have a local role in the prostate, which is known to be an androgen-responsive organ. FGF-S can also be oncogeic, as it displays transforming activity when transfected into NIH-3T3 fibroblasts. Kouhara er at., Oncogene 9 455-462 (1994). While FGF-8 has been detected in heart, brain, lung, kiny, testis, prostate and ovary.

expression was also detected in the absence of exogenous androgen. Schmitt ea at., J. Steroid BlocA em. Mo. Blot.

.U 173-78 (1996).

FGF-8 shares the property with several other FGFs of being expressed at a variety of stages of matrine embzyogenesis. which supports the theory that rhe various FGRs have multiple and perhaps coordinated roles in differentiation and embryogeneuis. Moreover. FGF-8 has also been identified as a protooncogenec that cooperates with Wat-I in dhe proces of mammary tumoarigetiesis (Shackleford ea at.. Proc. ai. A cad. Sd. UISA 2Q, 740-744 (1993); Heilcinheimo ea at.. Mech. Dcv. 129-138 (1994)).

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

41(2): 129-38 (1994). Further in slut hybridization assays between day 8 and 16 of gestation indicated initial expression in the surface ectoderm of the first bronchial arche, the frontonasal process, the forebrin and the midbrain-hindbrain junction. At days 10-12, FGF-8 was expressed in the surface ctoderm of the forelimb and hindLimb buds, the nasal its and nasopharynx, the infundibulum and in the telencephalon, diencephalon and n~tncepllon. Expression continues in die developing hindlirnbls through day 13 of gestation. but is undetectable thereafter. The results suggest that FGF-8 has a unique temporal and *spatial pattern in embryogenesis and suggests a role for this growth factor in multiple regions of ectodermaal differentiation in the post-gastrulation embryo.

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

PRO-265f Protein-protein interactions include receptor and antigen comnplexes and signaling mechanisms. As more is known aboiut 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 commnunity.

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

Leucine-rich repeats are short sequience motifs present in a number of proteins with diverse functions and cellu locations. The crystal stnutre of riborsiclease 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. go that the protein acquires an unusual, nonglubular shape. These two features have been indicated as responsible for the protein-binding functions of proteins containing eucine-rich repeats. See. Kobe and Deasenhofer, Trends Riichem- Sdi, 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 stroina formation. lozzo. R. Crit. Rev. figghemn Mol. Biol., 32(2):141-174 (1997). Others studies implicaing leucine rich proteins in wound healing and tissue repair are De IA Salle. ct al., YgnL. ev.

Fr. Hering. (Germany). 37(4):215-2 (1995). reporting mutations in the leucane rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome and Chletnetson, K. TmkHaejos. (Germany).

74(l): 111-116 (July 1995). reporing that platelets have leuicine rich repeats. Another protein of particular interst which has bcen reported io have leucine-rich repeats is the SLIT protein which has been reportod to be useful in araing ncurodgencuarivc diseases such as Alzheimier's disease. nerve damage such as in Parkinson's disease, and for diagnosis of cancer, see. Antavanistsakonas. S. and Rodhberg. J. Md.. W09210518-Al by Yale University. Other saxies reporting on the biological functions of proteins having leucine-rich repeats include: Tayar. et al.. R&ul £QILEngorjzm.. (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropin. receptor involvement); MiUr2, ct al., ft a NMim (Japan), 54(7):1784-1799 (July 1996) (apoptosis ivolvement); Harris. P. et al., J.A .Sc Ncln. 6(4):1125-1133 (Oct. 1995) (kidney disease involvement); and Ruoslahti, E. L. ct al.. W091 10727-A by IA Jolla Cancer Research Foundation (decorin binding to transforming growth factor-P involvement for trcatmnent for cancer, wouad healing and scarring). Also of particular interest is fibromodulin and its use to prevent or reduce dermal scarring. A study of fibromodutin is found in U.S. Patent No. 5,654,270 to Ruoslabui, et al.

Effors are therefor being undertaken by both industry and academia to identify new proteins having leucine.

rich repeats to better understand protein-protein interactions. Of particular interest are those proteins having leucine rich repeats an homoliogy to known proteins having leuine rich repeats suchi as fiwumanuln, the Sz..z protein and platelet glycoprotein V. Many/ efforts are focused on the scrtening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound proteins having leucine rich repeats. We 15 herein describe the identification and characterization of novel polypeptides having homology to fibroniodulin, herein designated as PR0265 polypeptides.

6. PRO2 Human matrilin-2 polypeptide is a member of the von Wdlebrand factor type A-like module superfamily.

von Willebrand factor is a protein which plays an isnporuram role in the inaintcnence 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 irtusy via its ability to interact and form a complex with Factor VIII. Ile 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 iqjuzy. The result is the propensity for brusing, nose bleeds, 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 indtustiy and academia to identify new, native proteins which my be involved in the coagulation process. We herein describe the identification of a novel ful-legth polypeptide which possesses homology to the human matrilin-2 precursor polypeptide.

7. PRO24 The Cell surface protein HCAR is a membrane-bound protein that acts as a receptor for subgroup C of the adenoviruses and subgroup B of the coxsackieviruses. Thus, HCAR may provide a means for mediating viral infection of cells in that the presence 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 membraire-bound proteins and specficially those which serve a cell surface receptor for viruses, efforts are currently being undertaken by both hnustry and academia to identify new, native membrane-bouM receptor proteins. Many of these efforts arm focused on the screening of mnammalian recombinant DNA libraries to i deniyte coding sequzences for novel receptor proteins. We herein describe a novel membrane-bound polypcptd& (designated herein as PR0246) having homology to the cell surface protein HCAR and to various annor antigens including A33 and carcizmembsyonic antigen. wherein this polypeptide may be a novel cell surface virus; receptor or tumor antigen.

8. PRO228 There arc a umrber of knwn seven transmeinhsane proteins and within this family is a group which includes CD97 and EMRI. CD97 is a seven-span transainbrane receptor which has a cellular ligand, CDSS, DAF.

Hamann, et al.. LJ. ERMe. 184(3):1189 (1996). Additionally, CD97 has been reported as being a dedficfretaton marter in Iuna thyroid carcnomas; and as associated with iniflaniiation. Aust, et al. CancrRes.

57(9):1798 (1997); Gray, et al.. J. Inimunol. 157(12):S439 (1996). CD97 has also been reported as being related to the aecretin receptor superfamily, but unlike known members of that family, CD97 and EMRI have extended extracellular regions; that possess several EGF domains at the N-terminus. Hamnann, -et al.. ~xGmkj.ic *32(l):144 (1996); Harmann, et al., J. mmuno1., 155(4):1942 (1995). EMRI is further described in Lin. ct al., Genomics, 41(3)-.301 (1997) and Baud, et al., Czkmi~i. 26(2):334 (1995). While CD917 and EMR1 appear to be related to the secretin receptors. a known member of the secretin family of G protein-coupled receptors includes the alpha-larroxin receptor. latrophilin, which has been described as calcium independent and abundant amiong neuronal tissues. l..lianova. et al.. 1.iogl hem-. 272(34), 21504 (1997); Davletov, et al., J.Ro. hm 271(38):23239 (1996). Both members of the scintin receptor superfamily and non-members which are related to the scretin receptor superfamily, or CRF and calcitonin receptors are of interest. In particular, new members of thes families. identified by their homology to known proteins, are of interest.

Efforts are being undertaken by both industry and academia to identify new membrane-bound receptor proteins, particularly tranamembrane proteins with EGF repeats and large N-terminuses which may belong to the family of sevcn-transmembrane proteins of which CD97 and EMRI are members. We herein describe the identification and charactization of novel polypeptides having homology to CD97 and EMRI. designated herein as PR0228 polypeptides.

P.RS33 Growth factors are molecular signals or mediators that enhance cell growth or proliferation, alone or in conoert, by binding to specific cell surface receptors. however, there are other cellular reactions than only growth upon expression to growth factors. Asa result, growth factors are better characterized as multifunctional and potent cellular regulators. Their biological effects include proliferation, chemotaxis and stimulation of extracliular matrix production. Growth factors can have both stimnulatory andl inhibitory effects. For example, transforming growth factors (I'GF-P) is highly piotropic and can atinulate proliferation in some cells, especially connective tisss whilc being a potent inhibitor of proliferation in others, such as lymphocytes and epithelial cells.

The physiological effect of growth stimulation or inhibition by growth factors depends upon the state Of developmnent and differetiation of the target tisse. The mechanism of local cellular regulation by classical endocrine WO 99/14328 PCT/US98/l 9330 molcules comprehends autocrine (same cell), juxdacinc (neighbor cell), and panacrine (adjacent cell) pathways.

Pcptidc growth factors arc elements of a complex biological lIanguage, providing the basis for intercellular communication. They permit cells to convey information between each other. mediate intcraction between cells and change gene expression. the effect of these multifuznctional anid plunpotent factors is dependent on dhe presence or absence of other peptides.

Fibroblast growth factors (FGFs) are a family of heparia-binding, potent mitogens for both normal diploid fibroblasts and establishied cell lines, (3odpodarowicz, D. et al. (1984), Proc. Natl. Acad. Sci. USA 8 1: 6983. the FOF family comprises acidic FGP (FOP-I1). basic POP (FGF-2). WN-2 (FGF-3), K-FOFIHST (FOP-4). FGP-S, FGF-6, ICOF (FGP-7), AlGF (FGF-8) among others. All FOFs have two conserved cysteine residues and share 30-50%/ sequence homology at the amino acid level. These factors are mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, inducing granulosa cells, adrenal cortical cells, chrondocytes, myoblasts, corneal and vascular cadothelial cells (bovine or human), vascular smooth mscle cells, lens, retina and prostatic epithelia[ cells, oligodendrocytes, astrocytes, chrondocytes, nsyoblasts and osteoblasts.

Fibroblast growth factors can also stimulate a large number of cell types in a non-mitogenic manner. These' activities include promotion of cell migration into a wound are (chemotaxis), initiation of new blood vessel formulation (angiogesss), Modulation of ne-ye regeneration and survival (neurvtrophism). modulation of endocrine functions. and or suppression of specific cellular protein expression, actracellular matnix production and cell survival.' Baird, A. Bohlen, Handbook of Exp. PhrmocoL 21( 369-418 (1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healing, nerve repair, collateral blood ~.vessel formaution, and the like. For example, fibroblast growth factors, have been suggested to minimize myocarditum damage in heart disease and surgety 4,378,437).

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

PRO24~ Some of the most important proteins involved in the above described regulation and modulation of cellular processes arc the emyymes whiich regulate evels of protein phosphorylation in the cell. For example, it is know that the transduction of signals that regulate cell growth and differentiation is regulated at least in part by phosphorylation and dephosphorylation of various cellular proteins. The enzymes that catalyze these processes include the protein kinases, which funcetion 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 many protein kinae enzymrs have been identified, the physiological role played by many of these catalytic proteins has yet to be elucidated. It is well known, however, tha a mnber of the known protein kimaes function to phosphorylate tyrosine residues in proteins, thereby leading to a variety of different effects. Perhaps most importantly, there has been a great deal of interest in the protein tyrosine kinases; since the discovery that many orrogene products and growth factors possess intrinsic protein tyrosine kinase activity. There is, therefore, a desire to identify new members of the protein tyrosine kizrase family.

Given the physlologcal importance of the protein kinases, efforts are being undertaken by both industry and academia to idenify, new. native kinasc proteins. Many of dhese'effarts are focused on the screening of mammalian recombinant DNA Libraries to identify the coding sequences for novel kinase proteins. We herein describe the identification and characterization of novel polypeptides having homology to tyrosine kinase proteins, designated herein as PR0245 polypeptides.

11. PRO220, PR0221and PRO227 Protein-protein interactions include receptor and antigen complexes aixd sialing mechanisms. As more is knoiwn about the structural and functional mechanisms underlying protein-protein interactions. protein-protein interactions can be mome easily manipulated to regulate the particular result of the protein-protcin interaton. 7tus, the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community.

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

0 S Leucine-rich repeats are short sequence motifs present in a numiber of proteins with diverse functions and cellular S loctions. The crystal structure of ribonuelease inhibitor protein has revealed that leucine-uich 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 feanares have been inficated as responsible for the protein-binding functions of proteins containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends fliochem. 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, antumor stroma formation. lozzo, R. Crit. Rev. Hlochem. Mot. Biol., 32(2):141-174 (1997). Others studies implicating leucine rich proteins in wound healing and tissue repair are De LA Salle, et al., !nnLv. v Fzir. Hmato. (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. Thromb-Hajfrgr. (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 SUT protein which has been reported to be useful in treating neAvr-degenerative diseases such as Alzheimner's disease, nerve damage such as in Parkinson's disease, and for diagnosis of cancer. see, Artvanistsakonas, S. and Roth berg. J. W09210519-Al by Yale University. Other sidies reporting on the biological functions of proteins having leucine-rich repeats include: Tayar, et al.. Mni.

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

t±ipgriinshll (Japan), 54(7)-1784-1789 (July 1996) (apoptosis involvement); Harris. P. et al., L Am. Soc.

Nobmul., 6(4):1125-1133 (Oct. 1995) (kidney disease involvement); and Ruoslahti. E. cc al.. W0911O72-A by La Jolla Cancer Research Foundation (decorin binding to transforming growth factor3 involvement for treatment for cancer, wound healing and scarring).

Efforts are therefore being undertaken by both intry and academia to identify new proteins having leucine rich repeats to bette understand protein-protein interactions. Of particular interest ame those proteins having leucine rich repeats and homology to knjown proteins having leucine rich repeats such as the SLIT protein and platelet glycoprotein V.

12. EI)ZsB harrnoglobulins are antibody molecules. the proteins that function both as receptors for antigen on the Bcell membrane and as dhe secreted products of dhe plasma ell. Like all antibody molecules. inlmunoglobulins perform two major functions: they bind specifically to ant antigen and they paticipate in a limited number of biological effector fuinctions. 7berefore, new members of the Ig superfamily are always of interest. Molecules which act as recptor by variots viruses and those which act to regulate immune function are of particular interest. Also of particular interest are those molecules which have homology to known Ig family membcrs which act as virus receptors or regulate imme function. Th~us, molecules having homology to poliovirus receptors, CRTAM and CIDl16 (a ligand for lymphocyte antigen CD6) are of particular interest.

Extracellular and membrane-bound proteins play important roles in the formation. differentiation and maintenance of multiccllular organisms. 71e fan: of many individual cells, proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the finmediar environment- This information is often transmitted by secreted polyeptidecs (for instance, mitogenic factors, survival factors. cytotoxic factors, differentiation factors. neuropeptides, and hormones) which are, in zurn.

received and interpreted by divers 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 cxtraeiular environment, usualy at a membrane-bouni receptor protein.

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

13. PROM~ 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 mome easily manipulated to regulate the particular result of the protein-protein interaction. 7hus, the underlying mechanisms of protein-protein interactions are of interest to the sciertific and medical community.

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

Letucine-rich repeats are short sequence motifs present: in a mnber of proteins with diverse funictions and ceiular locations. The crystal structure of riboruclease: inhibitor protein has revealed that leucixie-rich repeats correspond to beta-alpha structural units. These units ame arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual. noraglobular shape. These two features have been indicated as responsible for the protein-binding functions of proteins containing leucime-rich repeats. See, Kobe and Deisenhofer. Trends iochem. Sdi. 19(10):415-421 (Oct. 199-4).

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 irroma formation. lozzo, Rt. Crit. Rev- liohEm. Mol. ii., 32(2):141-174 (1997). Others stutfics implicating leucine rich proteins in wound healing arnd tissue repair arm De LA Salle. et V2l. Rev ELr matoI. (Germany), 37(4)215-2 (1995), reporting mutations in the leucine rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome and Clemetson, K. JIrmb Qiklaemoii. (Germany).

74(l):Ill.116 (July 1995). reporting that platelets have leucine rch repeats. Another protein of particular interest which has been reported to have leucine-rich repeats is the SIT protein which has been reported to be useful in treating neuro-degeneranvc diseases such as Alzheimer's disease. nerve damage such as in Partinson's disease, and for diagnois of cancer. sme. Arnavanisrama, S. and Rothberg, J. W09210518-Al by Yale University. other studies reporting on the biological fitnctions of proteins having leucine-rich repeats include: Tayar, et al M&ID CeilEndocimi., (Ireland). 125(1-2):65-70 (Dec. 1996) (gon~aepin receptor involvement); Miura. et a1., Niom Rnsg (Japan). 54C7):1784-1789 (July 1996) (apoptosis involvement); Harris, P. et al.. L Am. c N 6(4):1125-1133 (Oct. 1995) (kidney disease involvement); and Ruoslahti, E. L. et al., W09110727-A by La Jolla Cane Research Foundation (decorin binding to transforming growth factor3 involvement for treatment for cancer, wound healing and scarring).

Efforts are therefore being unralen by both imlustry and academia to identify new proteins having leucine rich repeats to better understand protein-protein interactions. reuronal development and adhesin molecules. Of particular interest ame those proteins having leucine rich repeats and homology to known proteins having Ieucine rich repeats much as mhe SMI protein. We herein describe ravel polypqerkles having homology to SLIT, designated herein as PR0266 polypeptides.

14. PROZ2 Thrombomodulin binds to and regulates the activity of thrombin. It is important in the control of blood coagulation. Thrombomodulin functions as a natual anticoagulant by a clrating the activation of protein C by thrombin. Soluble thrombomodulin may have therapeutic use as an azitithrombotic agent with reduced risk for hemorrhage as compared with heparin. Thrombomodulin is a cell surface trans-membrane glycoprotein, present on enidothelial cells and platelets. A smaller, functionally active form of throrobomodulin circulates in the plasma and is also found in urine. (In Haeberli, Human Protein Dama, VCH Oub., 1992). Peptdes3 having homology to thrornbomodulin arc particularly desirable.

We herein describe the identification and characterization of novel polypeptides having homology to thrombornodulin, designated herein as PRO269 polypeptides.

Procoilagen C-proteinase enhancer protein binds to and ethances the activity of bone morphogenic protein "BMP /Procollagen C..proteinase (PCP). It plays a role in extracelluilar matrix deposition. BMPI proteins may be used to induce bone and/or cartilage formation and in wound bealing and tissue repair. Therefore, procollagen Cprotinase wlan protein, BMP I and proteins having homology thereto, are of interest to the scientific and medical communities.

We herein describe the idcrtification and chiaracterization of novel polypeptides having homology to procoilagen C-protsei aseehancer protein preursor and proouagen C-proteinase enhance protein, designated herein as PR0287 polypeptides.

16. PRQ21~ Growth factors are molecular signals or Mediators that enhances cell growth or proliferation. alone or in concert. by binding to specific cell surface receptors. However, there are other cellular reactions than only growth upon espression to growth factars. As a result. growth factors arm better charcterized as multiftunctional and potent cellular regulators. Their biological effects include proliferation, chemotaxis and stimuldation of extracdilular matrix production. Growth factors can have both stimnulatory and inhibitory effects. For example, transforming growth factor P3 (rGF-f3) is highly plejorpc and can stirmulate proliferation in somne cells, especially connective tissue. while being a potent inhibitor of proliferation in others, such as lymphocytes and epithelial cells.

The physiological effect of growth stimulation or inhibition by growth factors depends upon the state of development and differentiation of the target tissue. The mechanism of local cellular regulation by classical endocrine molecules involves comprehends autocrine (sum cell), juxtcrine (neighbor cell), and paracrine (adjacent cells) pathways. Peptide- growth facormn e lements of a corrplex biological language, providing the basis for intercellular comunication. They permit cells to convey information between each other, mediate interaction between cells and change gene expression. Thec effect of these mnultiunctional and phuipotent factors is dependent on the presence or abec of other peptides.

Epidermnal growth factor (BOF) is a conventional mitogaiic; factor that stimulates the proliferation of various types of cells including epithelial cells and fibroblasts. EGF binds to and activates the EGIF receptor (EGPR), which initiates intracellular signaling and subsequent effects. The EGFR is expressed in neurons of the cerebral cortex, cerebellum, and hippocampus; in addition to other regions of the central nervous system (CNS). In addition, EUF is ~also expressedin various regions of theMCS. Thberefore, EGF acts not only on mitotic cells, but also on posmiitotic 20 neurons. In fact, many studies have inicated that EGF has neuronrohic or neuromodulatory effects on various types of neurons in the CNS. For example, EGF acts directly on cultured cerebral cortical and cerebellar neurons, enhancing neurite outgrowth and survival. On the other hand, EGF also acts on other cell types. including septal cholinergic and mesencephalic dopaminergic neurons, indirectly through glial cells. Evidence of the effects of EGF 0on neurons in the 'CNS is accumulating, but the mechanisms of action remain essentially unknown. EUF-induced signaling in mitotic cells is better understood than in postinitotic neurons. Studies of cloned pheochromocytoma PC 12 cells and cultured cerebral cortical neurons have suggested that the EGF-induced neurotrophic actions are mediated by sustained activation of the EGFR and mitogen-activated protein kinase (MAPK) in response to EGF. The sustained intracellular signaling correlates with the decreased rate of EGFR downeecgulation. 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 tnitotic cells and postmnitotic neurons.

EGF is produced by the salivary and Brnner's glands of the gastrointestinal system, idney, pancreas, thyroid gland, pituitary gland, and the nervous system, and is found in body fluids such as saliva, blood.

cerebrospinal fluid (CSF), urine. aniniotic fluid, prostatic fluid, pancreatic juice, and breast nmilk Plata-Salarnan, CR Peptides 12: 653-663 (1991).

EGF is mediated by its aea specific receptor, which contains an intrinsic tyrosine kinase. Stoscheck CM et at.. Cell Biodwem U: 135-152 (1986). EGF is believed to function by binding to the extraceliular portion of its receptor which induces a transmemnbrane signal that activates the intrinsic tyrosine kinase.

.Purification and sequence analysis of the EUF-like domain has revealed the presence of six conserved cysteine: residues which cross-bind to create three peptide loops. Savage CR er at., J. Biol. Chem. 241: 7669-7672 (1979). Itis now generally known that several other peptides can react with the EGF receptor which share the same generalized motif XUCXCXCX,,CXCXGX,CX.. where X represents any non-cysteine amnin acid, and n is a variable repeat rnnmber. Non isolated peptides having this motif include TGF-a. aniphiregulin. schwannorma-derivcd growth ftctor (SDGF), heparin-binding EUF-like growth factors and certain virally encoded pcptides Vaccini virus, Reimrer Ali, Natuire M1.: 801-803 (1985). Shape fibromia virus, Chang et al.. Mol Cell Biol. 7: 535-540 (1987). Moliuscumn contagiosurn, Porter CD Archard LC, I. Gen. Vrol. 0j: 673-682 (1987), and Myxoma virus, Upton C et al.. J. Virtrl. 1271-1275 (1987). Prigent SA Lemoine Prog. Growh Factor Res. 4: 1-24 EGF-Iike domains are zn confined to growth factars but have been observed in a variety of cell-surface and extracellular proteins which have interesting properties in celi adhesion, protein-protean interaction and development.

Laurence DIR Gusterson BA. Tumor Biol. 11: 229-261 (1990). These proteins include blood coagulation (actors (factors VI. DCX. X X3. protein C, protein S. protein 7, tissue plasminogen activator, uroltirase). extracellular matrix components (laminin, cytotactin. entactin). cell surface receptors (LDL receptor. dfirombornodulin receptor) and immunity-related proteins (complement CIr, urornodulin).

Even more interesting, the general structure pattern of EGF-Iike precursors is preserved through lower organisms as well as in mamnmalian celis. A number of genes with developmental significance have been identified in invertebrates with EUF-like repeats. For example, the notch gene of Drosophila encodes 36 tandemly arranged aino acid repeals which show homology to EGF. Wharton W et al.. Cell 42- 557-581 (1985). Hydropathy plots indicate a putative membranc spanning domain, with the EGF-related sequences being located on the extracelar side of the membrane. Other homeotic genes with EGF-Iike repeats include Delta. 95F and 5ZD which were identified using probes based on Notch. and the nematode geme Lin-2 which encodes a putative receptor for a developmenal signal transmitted between two specified cells.

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

1 933-37 (1995), including the treatment of necrotizing enterocolitis, Zollinger-Ellison syndrome, gasrontestmal ulceration gastrointestinal ulcerations aW congenital microvilius atrophy, A. Guglietta PB Sulivan, Eur. L. Gasiroenterol Hepatol, 2(10). 945-50 (1995). Additionally, EGF has been implicated in hair follicle differentiation; C.L dum Cros, J. Invest. Dermazol. .IQ (I Suppi.), 106S-1 13S (1993), SG Hillier, Cin. FEndocrinol.

33(4). 427-28 (1990); idney function, L.L. Hanmm at at.. Semnin. Nephrol. 1. 109-15 (1993), RC Harris, Am.

J. Aidney Dir. 627-30 (1991); tear fluid, GB van Setteri et al., Int. OplahakrzolJ.5(6); 359-62 (1991): vitamin K mediated blood coagulation, 1. Stenflo at al., Blood 2f1(7): 1637-51 (1991). EGF is also implicated various sin disease characterized by abnormal kcratinocyte differentiation. psoriasis, epithelial rancers such as squamous cell carcinomas of the lung, epidermoid carcinomna of the vulva and gliomas. King, LE a al.. Am. J. Med. Sci. M: 154-158 (1988).

Of great interest is mounting evidence that genetic alterations in growth factors signaling pathways are closely linked to developmental abnormalities and to chronic diseases icuding cancer. Aaronson SA, Science 754: 1146-1153 (1991). For example. c-crb-2 (also known as HER-2). a proto-oieogene with close structural sinilarity to EGF reeptor Protein, is ov'erexpressed in huan brmt cr.r K 8 g al Science 222: 974-976 (1985); Gullick.

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

17. PRO317 MiE TG"~ supegcne family, Or simply TG&-j superfamily, a group of secreted proteins, includes a large number of related growth and differentiation factors expressed in virtually all phyla. Superfamily members bind to specific cell surface receptors that activate signal transduction mechanisms to elicit their multifunctional cytokine effects. Kolodziejczyk and Hall. Biachem- Cell Blot.l. 7A: 299-314 (1996); Attisano and Wrana, CvoicCml Factore.. Z: 327-339 (1996); and Hill, CelhzlulrigmaljnE. 1: 533-5"4 (1996).

Members of this family inriele five distinct forms of TGF-P (Spore and Roberts. in Pcnrdec Growth Factors and Thiry Recntr, Sporn and Roberts, eds. (Spriqger-Verlag: Berlin, 1990) pp. 419-472). as well -as the differentiation factors vgl (Weeks and Melton. QcL 51: 861-867 (1987)) and DPP-C polypeptide (Padgett ei al..

N1ature, M2: 81-84 (1987)), the hormones activin and inhibin (Mason et al., Niature 3n: 659-663 (1985); Mason et al., GrwhFcos 1: 77-8(1987)), the Mullerian-inhibiin substance (MI) (Cate eta!., fCll 45: 685-698 (1986)). die bone morphogenetic proteins (BMPs) (Wozney ei al., Scec 2VZ: 1528-1534 (1988); PCT WO 88100205 published January 14. 1988; U.S. 4.877.864 issued October 31, 1989), the developmentally regulated proteins Vgr-1I (Lyons et Proc. Na!. Acad. Sdi. USA. U: 4554-4558 (1989)) and Vgr-2 (Jones et al.. Molcc.

EndocrinnL I: 1961-1968 (1992)), fte mouse growth differentiation factor (ODF). such as GDF-3 and GDF-9 ,:(Kingsley, Q~e De. 133-146 (1994); McPherron and Lce, L Biol-Chem.. 22: 3444-3449 (1993)), the mouse lefty/Stral (Meno ar al., Nature. MJJ: 151-155 (1996); Bouillet al. BioI. 17: 420-433 (1995)). glial cell .*.line-derived euotrophic factor (GDNr-) (Lin ar al., Scir=. 2fQ: 1130-1132 (1993), neurturin (Kotzbauer ef al., kNaMlz, 2M.- 467470 (1996)), and aidonietrial bleeding-associated factor (EBAF) (Kothapalli ei al., L Clin. nvest-, 2342-235D0(1997)). The subse BMP-2A and BMP-2B is approximately 75 homologous in sequence to DPP-C and may represent the mammalian equivalent of that protein.

The proteins of the TGF-P superfamily are disulfide-lonked homo- or heterodirners encoded by larger precursor polypeptide chains containing a hydrophobic signal sequence, a long aix! relatively poorly conserved Nterminal pro region of several hunrdred amino acids, a cleavage site (usually polybasic). and a shorter and more highly conserved C-terminal region. T7his C-termizial region corresponds to the processed matuire protein and contains aproxi- tly 100 anow acids with a characteristic cysteine motif, the conservation. of seven of the nine cysteine residues of TO"- amoeng all kown family members. Although the position of the cleavage site betweecn the mature aix! pro regions varies among the family members, the C-terminus of all of the proteins is in the identical position, ending in t sequence Cys-X-Cys-X, but differing in every case from. the TOP consensus C-terminus of Cys-Lys- Cys-Ser. Spore and Roberts, 1990, supra.

There art at least five forms of TGP currenly identified, TGF-P I, TGF-P2. TGP3, TGrP4. and TOP- P5. The activated form of TOFPIl is a homodimer formed by dimerization of the carboxy4erminal 112 amino acids of a 390 amino acid precursor. Recombinant TGFPfl has been -cloned (Derynck et al.. N~atuzre, M.-701-705 (1985)) and expressed in Chinese hamster ovary cells (Gentry at al.. MoL. Cel.Rio 2: 3418-3427 (1987)). Additionally, recombinant humn TGF-P2 (deMartin a EMBO J. fi: 3673 (1987)). as well as human and porcine TGF-P3 (De'ynmker aL. EMNBO L. 2: 3737-3743 (1988); ten Dij er aL. Proc. Nati Acad. Sci. USA, 4715 (1988)) have been cloned. TGF-t32 has a precursor form of 414 amino acids and is also processed to a homodimer from the carboxy-termina 112 amino acids that share approximately 70% homology with the active form of TGF-13 (Marquardt er al.. LBiolani CbeM 12127 (1987)). See also EP 200,341; 169,016; 268.561; and 267,463; U.S.

Pat. No. 4.774.322; Cheifetx d aL. £gll. 4: 409-415 (1987); Iakowlew et al.. Molecular Endocriii 2: 747-755 .(1988); Detynck er al.. I Rio!.Cem. ZJR: 4377-4379 (1986). Sharples et al., DEA. 6: 239-244 (1987; Derynck fal.. Nul AcidLs, Res-, 3188-3189 (19p7; Derynck ar al., NucI. Acids. Res., 1.5: 3187 (1987). Seyedin er at., L~fi.hL=. 5693-5695 (1986); Madisen ar al.. DfrIA 2: 1-8 (1988); and Hanks et Prc at.Aa Si 79-82 (1988).

TGF-P4 and TGF-PS were cloned from a chicken chondrocyte cDNA library (Jakowlew et Moe EndocinoL., 2: 1186-1195 (1988)) and from a frog oocyte cDNA library, respectively.

The pro region of TGF-P associates non-covaiemly with die manure TGF-P dimer (Wakefield et Bil Ckg. 263: 7646-7654 (1988); Wakefield ar al., Growth Factors, 1: 203-218 (1989)). and the pro regions ame found to be necessary for proper folding and secretion of the active mature dimers of both TGF-P and activin (Gray and Mason, sc 242: 1328-1330 (1990)). The association between the mature and pro regions of TGF-P masks the biological activity of the mature dimer, resulting in formation of an inactive latent form. Latency is not a constant of die TG"- 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. TGFP has been shown to have numrerous regulatory actions on a wide variety of both normal and neoplastic cells. TGF-P is niultilimcdtonal, as it can either stimulate or inhibit cell proliferation.

differentiation, and other critical processes in cell fuanction (S porn and Roberts, saqra).

One memuber of the TG"- superfamily, EBAF. is expressed in endontiwn only in the late secretory phase and during abnormal enidornetrial bleeding. Kothapalli ar al., J.CiLJD Ist, 22: 2342-2350 (1997). Human adontiuw is unique in that it is the only tisse in the body that bleeds at regular intervals. In addition, abnormal endometriaJ bleeding is one of the most common manifiestations of gynecological diseases, and is a prime indication for hysterectomy. In situ hybridization showed that the rnRNA of EBAF was expressed in the stroma without any significant mRNA expression in the endonietrial glands or endothelial cells.

The predicted protein sequence: of E1BAF showed a strong homology to the protein encoded by mouse lfty/sirc of the TOF-fi supertumily. A motif search revealed that the predicted EBAF protein contains most of the cysteine residues which are conserved among the TGr-p-related proteins and which are necessar for the formation of the cysteive knot structure. The EBAP sequence contains an additional cysteine residue. 12 amino acids upstream from the first conserved cysteine residue. The only other family members known to contain an additional cysteine residue are TGF-~s, inhibins, and GDF-3. EBAF. similar to LEFT"Y, GDF-3lNgr2, and GDF-9. lacks -the cysteine residue that is known to form the intermolecular disulfide bond. Therefore, EBAF appears to-be an additional member of the TGF-P superfamily with an unpaired cysteine residue that may not exist as a dimer. However, hydrophobic contacts between the two monomer subunits may promote dimer formation. Fluorescnce in situ hybridization showed that the eba/ gene is located on human chromosome 1 at band q42. 1.

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

18. PR0301 The widespread occurrnce of cancer has prompted the devotion of considerable resources and discovering new treatments of treatment. One particular method involves the creation of tumor or cancer specific monoclonal antibodies (mAbs) which are specific to tumor antigens. Such mAbs, which can distinguish between normal and cancerous cells are useful in the diagnosis, prognosis and treatment of the disease. Particular antigens are known 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 epithelimn. Since colon cancer is a widespread disease, early diagnosis and treatment is an imporant medical goal. Diagnosis and treatment of colon cancer can be implemented using monoclonal antibodies (mAbs) specific therefore having fluorescent, nuclear magnetic or radioactive tags.

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

15 mAbs can also be used to diagnose during the diagnosis and treatment of colon cancers. For example, when the scrurn 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 indicate 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 cpithelial origin have adenovirus receptors. In fact, adenovirus-dcrived vectors have been proposed as a means of inserting antisense nucleic acids into tumors 5,518,885). Thus, the association of Sviral receptors with neoplastic tumors is not unexpected.

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

19. PRO224 Cholesterol uptake can have serious implications on one's health. Cholesterol uptake provides cells with most of the cholesterol they require for membrane synthesis. If this uptake is blocked, cholesterol accumulates in the blood and can contribute to the formation of atherosclerotic plaques in blood vessel walls. Most cholesterol is transported in the blood bound to protein in the form of complexes known as low-density lipoproteins (LDLs). LDLs are endocytosed into cells via LDL receptor proteins. 'Terefore. 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

C

zuczbrane-bound proteins act as receptors such as die LDL receptor. These receptors can function to endocytosc substrates or they can futnction as a receptor for a channel. Othei membrane-bound proteins function as signals or antigens.

Membrane-boun proteins and receptor molecules have various industrial applications. including as pharmceuz~eal and diagnostic age=t. *TIe membrtanc-bound proteins can also be employed for screening of potential peptide or small molecule regulators of the relevant receptor/ligand interaction. In the case of the LDL receptor. it is desirable to find molecules which enhance axiacyosis so as to lower blood cholesterol levels and plaque formation.

It is also desirable to identify molecule which inhibit cixiocytsis so that these molecules can be avoided or regulated by individuals having high blood cholesterol. Polypetddes which amt homologous to lipoprotein receptors but which do not lUrction as lipoprtxezn receptors ame also of interest in the determination of' the futnction of the fragments which show homology.

The following studies report on previously known low density lipoprotein receptors and related proteins including apolipoproteins: Sawartara, ct al.. blipon Chemniphar Co, Japan patent application 309098787: Novak, S..

*etal., J. iol.Chem-. 271:OM0)1732-6 (1996); Blaas, J.Iirl., 69(11)7244-7 (Nov. 1995); Scott, L. nheit.

Metab. Dfis. 9ISupp. 1 (1986); Yamamoto. et al., fal 39:27-38 (1984); Rebece, ct al., Ntnbiol.

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

Cll t1l...Bioli.. 40(4):461-81 (June 1994). These publications and others published prior to the filing of this application provide further background to peptdes already known in the art.

Efforts are being undertaken by both industry and academia to identify new, native membrane-bound II:: ~receptor proteins, particulauly those having homology to lipoprotein receptors. We herein describe the identification and characterization of novel polypeptdecs having homology to lipoprotein receptors, designated herein as PR0224 polypeptides.

PROMZ

Complement is a group Of Proteins found in the blood that are important: in huoral immunity and inflammation. Complement proteins are sequentialy activated by antigen-antibody complexes or by proteolytic: eazymes. When activated, conmlae proteins HUl bacteria and Other microorganisms, affect vascular permeability, release histamin and attract white blood cells. Complement also enhances phagocytosis when bound to target cell.

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

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

Proteins known to be important in regulating complemnent activation in humans include Factor H and Complement receptor type I (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 1-mediated cleavage of compleenit protein COb. 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 C3b. C4b. and iC3b to accelerate dissociation of C3 convertases, acts as a cofactor for Factor I-mediated cleavage of C3b and C4b. and binds immune complexes and promotes their dissolution and phagocytosis.

Proteins which have homology to cotpl- proteis am of partcular interest to the medical and industriial communities. Often. proteins having homology to each other have sirniiar fuinction. It is also at interest when proteins having homology do not have similar functions, indicating that certain structural motifs identify information othcr than function. such as locality of function.

Efforts arc being undertaken by both industry and academia to identif new, native secreted and membrane-bound proteins. particularly those having homology to known proteins involved in .thc complement pathway. Proteins involved in the complement pathway were reviewed in Birminghamt DJ (1995), Criicl evew jJH~qj2S 15(2):133-154 and] in Abbas AK, ct al. (1994) Cellular and Molecular Immunology, 2nd Ed. WAB 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. PRO23a4 The successful function of many systems within rmticellular organisms is dependent on cell-cell interactions. Such interactions are affected by the alignment of particular ligands with particular receptors in a manner which allows for ligand-receptor binding and thus a 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. Brandley Ce al., J. Leuk. iol. 4: 97 (1986) and N. Sharon a aL. Sdence 24": 227 (1989). Oligosaccharides are well positioned to act as recognition novel lectins due to their cell surface location and] structural diversity. Many oligosaccharide structures can be created through the differential activities of a smaller number of glycosyltransferases. The diverse structure of oligosaccharides can he generated by transcritfion of relatively few gene products, which suggests that the oligosaccharides are a plausible mechanism by which is directed a wide range of cell-cell interactions. Examples of differential expression of cell surface carbohydra=e and putative carbohydrate binding proteins (lectins) on interacting cells have been described QI. Dodd T.M. Jessel, J. Neurosci. 1: 3278 (1985); LJ. Regan a1 al., Proc. Nail. Acri. Sci. UISA U: 2248 (1986); M.

Constantin -Paton et al.. Nature 3Z4: 459 (1986); and M. Tiemneyer et al.. J. Biol. Chzem. W: 1671 (1989). One interesting member of the lectin family are selecdmn.

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

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

71e selectins are cell adhesion molecules that are unified both structurally and functionally. Strcturally.

selectins are characterized by the inclusion of a domain with homology to a calchim-dependent lectin (C..lectin). an epidermal growth factor (egt)-likc domain and several complement binding-Like domains, Bevilacqua. M.p. eal., Sdea 2M3: 1160-1165 (1989); Johnston ar al, CeU 1033-1044 (1989); Lasky af al, Cell 1045-1055 (1989); Siegalman, M. et al.. Science 2&3: 1165-1172 (1989); Stoolman. LM., Cell 5f: 907-910 (1989). Functionally.

selcctins sharm the common property of their ability to mediate cell binding through interactions between their lectin domains and! cell surface carbohydrate ligands (Brandlcy. B. ef al.. Cell 62, 861-863 (1990); Springer. T. and Laskcy, Nature 3a2. 19-1917 (1991); Bevilacqua, M.P. and Nelson, J. Clin. Invest. 21 379-387 (1993) and Tedder at al., J. Eri,. Med. InQ: 123-133 (1989).

There are thre members identified so far in the selecin family of cell adhesion molecules: L-selectin (also called periphecral lymph node homing receptor (pnHR). LEC-CAM-1. LAM-I. gp9Obef. gpi00lL. gp MEL, 14 antigen, Lew-S antigen TIQ-1 antigen, DREG antigen), E-selectin (LEC-CAM-2, LECAM-2, ELAM-[I) and Pselectin (LEC-CAM-3. LECAM-3. GMP-140. PADGEM).

The identification of the C-lectin domain has led to an intense effort to define carbohydrate binding ligands for proteins containing such domains. E-selectin is believed to recognize the carbohydrate sequence NeuNActi2- 3GaI1-4(FucaI-3)GIcNAc (sialyl-Lewis x, or tLe') and related oligosaccharides. Berg ar al.. ARiol. ac~m. 2M: 14869-14M7 (1991); Lowe i al.. CeilO: 475-484 (1990); Phillips el Science 2MQ: 1130-1132 (1990); 7tiemqyer al.. Proc. Nall. Acad. Sd. UISA JI: 1138-1142 (1991).

L-selectin. which comprises a lectin domain, performs its adhesive function by recognizing carbohydrate- 20 containing Ligands on endothelial cells. L-selectin is expressed on the surface of leukocytes. such as lymphocytes, neutrophils, monocytes and eosinophils, and is involved with the trafficking of lymhocytes to peripheral lympboid tissues (Gallatin ar aL. Nature 3U: 30-34 (1983)) and with acute neutrophil-medicated inflammatory responses (Watson, S.L, Nature 342.:164-167 (1991)). Thc amino acid sequence of L-seleetmn and the encoding nucleic acid sequence are, for example, disclosed in U.S. patent No. 5.098,833 issued 24 March 1992.

L-selectin (LECAM-1) is particularly interesting because of its ability to block neutrophil influx (Watson et al.. Nature 3A2: 164-167 (1991). It is expressed in chronic lymphocytic leukemnia cells which bind to HEV ~(Spertini a aL..Natur 32:691-694(1991). It is also believed that HEY structures at sites of chronic inflammation are associated with the symptoms 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 ILA or ThF. 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 futher been reported that and-ELAM-1 antibody blocks the influx of neutrophils in a primate asthma model and thus is beneficial for preventing airway obstruction resulting from the inflammatory response. Guuidel at al., J.

Clin. Invest. A8: 1407 (1991).

The adhesion of circulating neutrophils to stimulated vascular endothelium is a primary event of the inflammatory response. P-selectin has been reported to recognize the Lewis x structure (Galfll-4(Fucal-3) GIcNAc).

Larsen eal.. Cell 467-474(1990). Others report that an additional termina linked sialic acid is required for high affinity binding, Moore al., J. Cell. Not. 31: 491-499 (1991). P-selectin has been shown to be significant in acute lung injuzy. Anti-P-scectin aribody has been shown to have strong protective effects in a rodent lung injury model.

M.S. Mulligan et al. J. Clin. Inveit. 20: 1600 (1991).

We herein describe the identification and chanrcerization of novel polypeptides having homology to Icctin proteins, herein designated as PR0234 polypeptides.

22. PR231.

Some of the most important proteins involved in the above described regulation and modulation 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 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 finction to.

remove phosphate residues from vaious 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.

Prote= phosphatases represent a growing fumily of enzymes that are found in many diverse forms, including both membrane-bound and soluble forms. VWil many protein phosphatases have been described, the funoctions of only a very few ame beginning to be understood (ronics, Semin. Cell Biol. 4:373A453 (1993) and Dixon, Recent Prog.

Ilonn Res. 51.405-414 (1996)). However. in general, it appears that many of the protein phosphatases function to modulate the positive or negative signals induced by various protein kinases. Thecrefore, it is likely that protein phosphatases play critical roles in nume rous and diverse cellular processes.

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

Examples of screening methods and techniques arc described in the literature (see, for example, Klein et al., EM Nati. Acad. Sci., 21:7108-7113 (1996); U.S. Patent No. 5,536,637)].

We herein describe the identification and characterization of novel polypeptides having homology to acid phosphatascs, designated herein as PR0231 polypeptides.

23. PRO229 Scavenger receptors are known to protect IgG molecules from catabolic degradation. Riechann and Hollinger, Nature Biotechnology, 15:617 (1997). In particular, studies of the 012 and CH3 domains have shown that specific sequences of these domains are important in determining the half-lives of antibodies: Ellerson, ct al., JLlIninoil.. 116:510 (1976); Yasmeen, et al.. Llmmllnol. 116:518 (1976; Pollock, et al., BurLJ. mm1.. 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-fif of polypeptides and their involvement in immune function, molecules having homology to scavenger receptors are of importance to the scientific and medical community.

Efforts are being undertaken by both indlustry and academia to identify new, native secreted and membranebound rceptor 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 tar novel secreted and memnbrane-bound receptor proteins. Examples of screening methods and techniques ame described in the literature (sec. for example. Klein et al.. Poc. Nail- Acad. Sci., 21:7109-7113 (1996); U.S. Patent No. 5,536.637)1.

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

24. flR23 Oxygen free radicals and antioxidants appear to play an importan role in the central nervous system after cerebral iscbezriia and reperfusion. Moreover, cardiac injury, related to ischaemia and reperfusion has been reported to bc caused by the action of free radicals. Additionally, saud;e& have reported that the redox stare of the cell is a pivotal determinant of the fAme of the cells. Furthermore. reactive oxygen spedies have been reported to be cytotoxic, causing inflammatory disease, including tissue necrosis. organ failure. atherosclerosis, infertility.- birth detects, pranr aging, nmaaions and malignancy. 1hus, the control of oxidation and reduction is important for a number of measons 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 matter include Kelsey. et al., Br J ac.76(7):&52.4 (19917); Friedrich and Weiss, J~g. o. iot., 197(4):529-40 (1997) and Pieulle, et al.. 1. Bactiol.. 179(lS):5684-92 (1997).

Efforts are being undertaken by both irdustry and academia to identify new, native secreted and membrane-.

bound receptor proteins. particularly secreted proteins which have homology to reductase. Many efforts are focused on the screening of mnunalian 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. NatI. Acad. Sci., 2:7109-7113 (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 PR0238 polypeptides.

25. PRO233 Studies have reported thac the redox state of the cell is an important determinant of the fate of the Cell.

Furthermore, reactive oxygen species have been reported to be cytotoxic. causing inflammatory disease. including tisse necrosis, organ failure, atherosclerosis, infertility. birth defects, premature aging, mnutations; 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 ischernia and reperfusion. Moreover, cardiac injury, related to ischaemia and reperfusion has been reported to be caused by the action of free radicals. In this regard, reductases, and particularly, oxidoreductases, arc of interest. in addition, the transcription factors, NF-kappa B and 1 w ae 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.. B.L. Cance 76(7):852-4 (1997); Friedrich and Weiss, -L.

Tfeor-Bio 1S7(4):529-40 (1997) andi Pierille, et al., L.Datiol., 179(18):5684-92 (1997). Given the physiological imnportazx of rodox reactions in vivo. efforts arm currently being under taken to identify new, native proteins which amt involved in redox reactions. We dcscribe herein the identification of novel polypeptides which have homology to reductase, designated herein as PR0233 polypeptides.

26, PR0223 The carboxypeptidase family of exopeptidascs constitutcs a diverse group of enzymes that hydrolyze carboxyl-terminal amide bonds in polypeptides, wherein a large number of mammalian tisses produce these enzymes. Many of the carboxypeptidase enzymes that have been identified to date exhibit rather strong cleavage specificities for certain amino acids in polypeptides. For example. carboxypeptidase enzymes have been identified which prefer lysine. arginine, serine or amino acids with either aromatic or branched aliphatic side chains as substrates at the carboxyl termims of the polypecptide.

With regard to the scrinc carboxypeptidases, such amino acid specific enzymes have been identified from a var iety of diflerent -fm~ian and non-mazwnalian organisms. The mammalian scrine carboxypeptidase enzymes play important roles in many different biological processes including, for example, protein digestion, activation.

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

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

27. PRO23S Plexin was first identfied in Xeriopus tadple nervous system as a membrane gtycoprotein which was shown to mediate cell adhesion via a homophilic binding mechanism in the presence of calcium ions. Strong evolutionary conservation between Xenopus. mouse and human homologs of plexin has been observed. [Kaneyama et al..

Biochem. And Biophys. Res. Comm. 226: 524-529 (1996)). Given the physiological importance of cell adhesion mechanisms In iWvo, efforts are currently being under taken to identify new, native proteins which arc involved in cell adhesion. We describe herein the identification of a novel polypeptide which has homology to plexin, designated herein as PR0235.

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

P-galactosidase has been employed for a variety of different applications. both in Wro and In vo 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 ji-galactosidasc polypeptide.

Given the Wtong interest in obtaining novel polypeptides having homology to P-galactosidase, efforts arc currently being undertaken by both industry and academnia to identify new, native P -galactosidase bornolog proteins.

Many of these efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel P-gahactosjdase-lke proteins. Examples of screening methods and techniques are described in the literature [see, for example. Klein et al.. Proc. Nat!. Aad. Sci,. 23:7109-7113 (1996); U.S. Patent No.

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

29. PRO239 Dens in is a glycoprotei which has been isolated from the brain which has all the hallmarks of an adhesion molecule. it is highly concentrated at synaptic sites in the brain and is expressed prominently in dendritsc processes in developing neurons. Densin has been characterized as a member of the 0-linked sialoglycoproteins. Densin has relevance to medtically imporant processes such as regeneration. Given the physiological importance of synaptic processes and cell adhesion mnechanismas in vivo, efforts are currently being under taken to identify new, native proteins which are involved in synaptic machinery and cell adhesion. We describe herein the Identification of novel polypeptides which have homology to densmn, designated herein as PR0239 polypetdes.

PR02h2 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 possess sequence homology to cell surface proteins such as ebnerin. Many of these efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel receptor proteins.

We herein describe the identification at novel polypeptides having sigmificant homology to the von Ebner's gland- *.:associated protein ebnerin, designated herein as PRO2S7 polypeptides.

31. PR26 .too.:Fucosidases are enzymes that remove fuicose residues from fuicose containing proteoglycans. In some pathological conditions. such as cancer, rheumatoid arthritis. and diabetes, there is an abnormal fucosylahian at 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 Nas. 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.. J.YimIo..

71(9):6796 (1997), Aktogu. et al.. Moanidi. Arch- Cest Dig. (Italy), 52(2):1 18 (1997) and Focarelli, et al., Bio2hn.

Bioohvs Res. Commun. 234(1)M4 (1997).

Effort are being undertaien by both industry anid academia to identify new, native secreted and membranebound receptor proteins. Of particular interest are proteins having homology to the alpha-I-fucosidase precursor.

Many efforts are focused on the scning 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 e( al.. Proc. Nat!, Acad. Sci 23:7108-7113 (1996); U.S. Patent No. 5.536,637)].

We herein describe the Idntfication and charcterization of novel polypcpddc-s having homology to (ucosidases. designated herein as PRO260 polypeptides.

32. PRO263 CD44 is a cell surface adhesion molecule involved in cell-cel and cell-matrix interactions. Hyaluronic acid.

a component of the extracellular matrix is a major ligand. Other ligands: include collagen, fibronectin, laminin, chuondroitin sulfate, mucosal addressin. serglycmn and osteoponin. CD44 is also important in regulating cell traffic, lymph node homning. transmission of growth signals, and presentation of chemokines and growth factors to traveling cells. CD44 surface proteins arc associated with mietastatic tumors and CD44 has been used as a marker for HIV bnfction. Certain splice variants are associated with metastasis and Poor prognosis of cancer patients. 7Terefore, molecules having homology with CD44 arm of particular interest, as their homology indicates that they may have Ametions related to those functions of CD44. CD44 is furher described in U.S. Patent Nos. 5,506,119. 5.504.194 and 5.108,904; Gerberick, et al., Toxicol. al, Phamcol., 146(1):1 (1997); Wittig. et al., Junol.Ietters (Netherlands). 57(1-3):217 (1997); and Oliveira and Odell, Oal Onc. (England), 33(4).-260 (1997).

Efforts are being undlertaken by both industry and academia to identify new, native secreted and membranebound receptor proteins, particularly transinembrane proteins with homology to CD44 antigen. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel :secreted and memtbrane-bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Nafl. Acad. Sci., 9:710&7113 (1996); U.S. Patent No.

5,536.637)].

We herein describe the Identification and characterization of novel polypeptides having homology to CD44 antigen, designated herein as PR0263 polypeptides.

33. PR270Z Thioredoxdns effectreduction-oxidation(redox) state. Many diseases are potentially related to redox state and reactive oxygen species may play a role in many important biological processes. Ihe transcription factors, NF-kappa B and AP- 1. are regulated by redox state and are known to affect the expression of a large variety of genes though 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 stress such as stroke and inflammation in addition to having a role in apoptosis. Therefore, thioredoxins, and proteins having homology thereto.

are of interest to the scientific and medical communities.

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

34. PROl21 The proteoglycan link protein is a protein which is intimately associate with vatious etaeilrmti proteins and more 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 glycwazinoglycan hyaluronan tbrtugh the Amino terminal G1 globular domain of the core protein. This binding is Stabilized by the protcoglycan link protein which is a protein that is also associated with other tissucs containing hyaluronan binding proteoglycans such as versican.

Link protein has been identified as a potential target for autoirninune antibodies in individuals who suffcr from juvenile rheumatoid Arthritis (see Guerassimov ct al. J. Rhwnmatology 24(5):959-964 (1997)). As such, therc is strong interest in identifying novel proteins having homology to link protein. We herein describe the identification and characerization of novel polypeptides having such homology. designated herein as PR0271 polypeptides.

S35.

PROM

Reticulocalbin is an endoplasmic reticular protein which may be involved in protein transport and luminal protein processing. Reticulocalbin resides in the lumen of the endopLadsmic rerticulum, is known to bind calcium, 6:0.15 and may be involved in a tumninal retention mechanism of the endoplasmic reticulum. It contains six domains of the EF-hand motif associated with high affinity calcium binding. We describe herein the identification and characterization of a novel polypeptdec which has homology to the reticulocalbin protein, designated herein as PROM7.

36. PRZ24 Collagen, a naturally occurring protein, finds wide application in indusuy. Chemically hydrolyzed natural collagen can be denatred and renatred by heating and cooling to produce gelatin, which is used in photographic and medical. among other applications. Collagen has importan properties such as the ability to form interchain having a conformation designated as a triple. helix. We herein describe the identification and characterization of a novel polypeptide which has homology to portions of the collagen molecule, designated herein as PRO294.

37. QZ The integmns comprise a supergene famiily of cell-surface glycoprotein receptors that promote cellular adhe-sion. Each cell has inumerous receptors that define its cell adhesive capabilities. Integrins are involved in a wide variety of bntraction between cells ind other cells or matrix components. T-he integrins are of particular importance in regulating movement and function of immune system cells The platelet IMh/IIA integrin complex is of particular impqortance in regulating platelet aggregation. A member of the integrin family, integrin P-6, is expressed on epithelial cells and modulates epithelial inflammation. Another integrin. leucocyte-associated antigen-I (LFAk-l) is important in the adhesion of lymphocytes during an immune response. 'The integrins are expressed as heterodimers of noncovalently associated alpha and beta subunits. Given the physiological importance of cell adhesion mechanisms in Wv, efforts are currently being under taken to identify new, native proteins which are involved in cell adhesion. We describe hecrein the identification and characteriation of a novel polypeptide which has homology to integrin.

designated herein as PRO295.

38. PR029 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, 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 are short sequence motifs present in a number of proteins with diverse functions and cellular locations. The crystal strhcture of ribormiclease inhibitor protein has revealed that leucine-rich repeats correspond sees 0:to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface 0 exposed to solvent, so that the protein acquires an unuasual. nonslubular shape. These two features have been indicated as responsible for the protein-binding functions of proteins containing leucine-rich repeats. See, Kobe and BeeS 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 tmor stroma formation. lozzo. R. Crit. Rev. Biocdieni Mo!- Biol 32(2):141-174 (1997). Others studies implicating leucine rich proteins in wound healing and tissue repair are Dc La Salle, et al.. Vouv. Rev.

JFr Hrnt. (Germariy), 37(4):215- 222 (1995), reporting nmutations in the leucine rich motif in a complex associated '20 with the bleeding disorder Bcrnard-Soulicr syndrome and Chlemetson, K. Thromb.Haernost. (Germany), Good 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 B B treating neuro-degenertive diseases such as Alzheimer's disease, nrve damage such as in Parkinson's disease, and for diagnosis of cancer, see, Artavanisisaima, S. adRothberg. 3. W09210518-A yYl nvriy te 25 studies reporting on the biological functions of proteins having leucine-ich repeats include: Tayar, et al.. MgI.

£nild~gjngj., (Ireland). 125(1-2):65-70 (Dec. 1996) (gonadotrojin receptor involvement); Miura, et al., kWigplnnibt (Japan), 54(7):1784-1789 (July 1996) (apoptosis involvement), Harris, P. et alLn.. J iSoc.

NokbEW.. 6(4):1 125-1133 (Oct. 1995) (kidney disease involvement); and Ruoslahti, E. et al.. W091 10727-A by La Jolla Cancer Research Fouindation (decorin binding to transforming growth factorfi involvement for treatment for cancer, wound healing and scarring).

Efforts are therefore being undertake 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 andI homology to known ricurons] Icucin rich repeat proteins. Many efforts are focused on the screening of marmmalian recombiant DNA libraries to identify the coding sequences for novel secreted and membrane-bound proteins having leucine rich repeats. Examples of screening methods and technique s are described in the literature (see, for example, Klein et al.. Pmo. Nal. Acad 91:7108-7113 (1996); U.S. Patent No. 5,536,637)].

Wc describe herein thc identification and characterization of a novel polypeptide which has; homology to leucie rich repeat proteins. designated herein as PR0293.

39. PRQO2 Procein-protein interactions include receptor and antigen complcxes and signaling mechanisms. As more is known about the structural and functional mechanisms underlying prin-procein interactioris. protein-protein interactons can be more easily manipulated to regulatc the particular result of the protein-protein interaction. Thus, the undeulying mechanisms of protein-protein interactions arc of' interest to the scientific and medical community.

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

Lcucine-rich repeats are short sequence motifs present in a mnmiber of proteins with diverse functions and cellular locations. The crystail structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha strucural units. These units are arranged so that they form a parallel beta-sheet with one surfaceexposed to solvent, so that the protein acquires an untuual, oonglubular shape. These two features have been idicated as responsible for the protein-binding functions of proteins containing leucine-rich repeats. See, Kobe and Deisenhoter, Trends Biochem Sci., 19(10):415-421 (Oct. 1994).

A study has been reported on leticine-rich proteoglycans which serve as tissue organizers, orienting and ordering collagen fibrils during ontogeny and are involved in pathological processes such as wound healing, tisse repair, and tumor stroma formation. lozzo. ft. Crit. Rev- Riocheni. Mot. Biol., 32(2):141-174 (1997). Others studies implicating leuicine rich proteins in wound healing and tissue repair are De La Salle, et al., Vouv- Rev, Fr He matw1. (Gerinany), 37(4):215-222 (1995). reporting muitations In the leuicine rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome and Chlcrmtson, K. L. Thb H~jaemm. (Germany), 74(1):111-1 16 (July 1995), reporting that platelets have leucine rich repeats. Another protein of particular interest which has been reported to have leuicine-rich repeats is the SUll 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 fordiagnosis oftcancer, see. Aravanistsakonas, S. and Rothberg, I. W09210518-Al by Yale University. Other studies reporting on the biological functions of proteins having leucine-rich repeats include: Tayar, et al., M.91- 0 Cell EndocrinoI., (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor involvement); Mura, et al1., NipnRnh (Japan), 54(7):1794-1789 (July 1996) (apoptorsis involvement); Harris. P. et al.. 1LAm-'Soc- Nezhrol., 6(4):1125-1 133 (Oct. 1995) (kidney disease involvemn~t);. and Ruoslahti, E. et al., W091 10727-A by La Jolla Cancer Research Foundation (decorin binding to tanasforming growth factoul3 involvement for treatment for cancer, wound healing and scarring).

Densin is a glycoprotein which has been isolated fi-r the brain which has all the hallmarks of an adhesion mnolecuzle. Itis highly concentrated at synaptic sites in the brain and is expressed prominently in dendritic processes in developing neurons. Densin has becn characterized as a member of the 0-inked sialoglycoproteins. Densin has relevance to medically important processes such as regeneration. Given the physiological importance of synaptic processes and cell adhesion mechanism In vivo. eforts are currently being under taken to identify new, native proteins which are involved in synaptic machinery and cell adhesion. Densin is further described in Kennedy, M.B.

Trnd u osc. (England), 20(6):264 (1997) and Apperson, et al., J. NurosL*, 16(21):6839 (1996).

Efforts mre therefore being undrtaken by both industry aril academia to identify new proteins having leucanec richrepeatsto betterundrttnd protein-protein intcetions. Of Particular interest arc those proteins having Ieucine rich repeats and homology to known proteins having leucine rich repeats such as KLAAO231 and dcnsin. Many efforts arc focused on the screning of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound proteins having leucine rich repeats. Examrples of screening methods and techniques am described in thieliterature [see, for example. Klein et al.. Proc. Nat] Acad. Sci- 91:7108-7113 (1996); U.S. Patent No. 5,536,637)].

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

40. PR0302. PR0303. PR0304. PR307 and PR0343 Proteases ame enzymatic proteins which are involved in a large number of very importan biological processes in mammlian and non-manmmalian organisms. Numerous different proteiae enzymes from a variety of different mammalian and non-mamimalian organisms have been both identified and characterized. The mammnalian.

**..protease 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 enzymes.

In light of the important physiological roles played by proteae enzymes, efforts are currently being undertaken by both industry and academia to identify new, native protease homologs. Many of these efforts are focused on the screening of mamnmalian recombinant DNA libraries to Identify the coding sequences for novel 20 secreted and membrae-bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Na]. Acad. Sci. 9:7108-71 13 (1996); U.S. Patent No.

5,536,637)]. We herein describe the identification of novel polypeptides having homology to various prote=s *.:enzymes, designated herein as PR0302. PR0303, PR0304, PR0307 and PR0343 polypeptides.

41. JR2 The GLIP protein family has been characterized as comprising zinc-finger proteins which play important roles in embryogenesis. These proteins may function as transcriptional regulatory proteins and are known to be amplified in a subset of human tumors. Glioma pathogenesis protein is structurally related to a group of plant pathogenesis-rclated proteins. It is highly expressed in glioblatomi. See US Pat. Nos. 5,582,981 (issued Dec. 1996) and 5.322.801 (issued June 21, 1996), Ellington, A.D. et al., N~ature, I:818 (1990). Grindley. J.C. et al..

Den. Biiil. 18L2)337 (1997). Marine, et at., MhOcxj2M f=2:2 11 (1997), The CRISP or cysteine rich secretory protein family ame a group of proteinis which am also stunrally related to a group of plant pathogenesis proteins. (Schwidetzky, BiocmJ-, n21:325 (1997), Pfiterer. hMoL.CjfljLBo. I1=f:6160 (1996), Krtscmr Rxj 236) 827 (1996)]. We describe herein the identification of a novel polypeptide which has homology to GLIP and CRISP, designated herein as PR0328 polypeptides.

42. PR033S. PR0331 And PR0326 Protein-protein interactions include receptor anal antigen complexes and signaling mechanisms. As more is known about the structural and functional mechanisms underlying protein-protein interactions. protein-protein interacions can be more easily manipulated to regulate dic particular result of te protein-protein interaction. Thus.

the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical comuiy AllI proteins containing leucine-rich repeats are thought to be involved in protein-protein interactions.

Lzucine-rich repeats are short scquence. motifs present in a number of proteins with diverse functions and cellular locations. The crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arran~ged so that they form a parallel beta-heet with one surface exposed to solvent, so that die protein acquires an unusual, nonglubular shape. These two features have been indicated as responsible for the protein-binding functions of proteins containing Ileucizie-rich repeats. See, Kobe and Deisenhiofer. Trenids Biochem ScL. 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 stroina formation. lozzo, R. Crit. Rev. Riochein. Mol. Biol.. 32(2):141-174 (1997). Others studies implicating Icucne rihproteins in wdhalnnd ts e pln air areeLae, C. etal., Vot. Rev Fr. Hcatol. (Germany), 37(4):215-m (1995). reporting mutations in the leuane rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome, Chlemetson, K. Thromb.liacmosi. (Germany). 74(1): 111 116 (July 1995), reporting that platelets have leucine rich repeats and Ruoslahti. E. et al.. W091 10727-A by La Jolla Car Research Foundation reporting that decorin binding to transforming growth factorp has involvement in 20 a treatment for cancer, wound healing and scarring. Related by function to this group of proteins is the insulin like ~growth factor (IUF). in doa it is useful in wound-healig and associated therapies concerned with re-growth of tissue.

such as connective tissue, skin andi bone; in promoting body growth in humans andi aqn;ias; and in stimulating other growth-related processes. The acid labile subunit of IGF (AIS) is also of interest in that it increases the half-life of lOP and is part of the lOP complex in vivo.

Another protein 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 Alzlheimer's disease, nerve damage such as in Parkinson's disease, and for diagnosis of cancer, see, Artavanistsakonas. S. and Rothbcrg, J. W09210518-Al by Yale University. Of particular interest is UG-l, a membrane glycoprotein that is expressed specifically in glial cells in the mouse brain, and has leucine rich repeats and iminunoglobulin-like domins. Suzuki, et al., 1. Biol.

Chem. 271(37)72522 (1996). Other studies reporting on the biological functions of proteins having Ieucinc rich repeats include: Tayar. ct al.. Mol. Cell ndocrnol., (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor involvement); Miura, et al., Nbgj~f (Japan), 54(C7):1784-1789 (July 1996) (apoptosis involvemen); Harris, P. ct al, 1. Am. Soc. NeRol., 6(4):1 125-1133 (Oct. 1995) (kidney disease involvemnent).

Efforts are therefore being undertaken by both industry andi academia to identify new proteins having leucixie rich repeats to better undersAnd protein-protein interactions. Of particular interest are those proteins having leucine rich repeats and homology to known proteins having leucine rich repeats such as LIG-1, ALS and decorin. Many efforts ame focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound proteins having leucine rich repeats. Examples of screening methods and tehiusaedsrbe nteltrtr (see, for example. Klict Proc. Natl. Acad. Sci 21:7108-7113 (1996).

U.S. Patent No. 5,536.637)).

We describe herein the identification and characterization of novel polypeptidcs which have homology to proteins of the leucine rich repeat superfamnily, designated hereinas PR0335, PR0331 and PR0326 polypeptides.

43. ER0332 Secreted proteins comprising a repeat characterized by an arrangement of conserved leucine residues (leucine-rich repeat mnotif) have diverse biological roles. Certain proteoglycans, such as biglycan, fibromodulin and decorin. are, for example, characterized by tie presence of a leucine-rich repeat of about 24 amino acids (Ruoslaliti, Ann, Rev. Cell. iol A 229-255 (1988): Oldberg er al.. _EMO J. 1,2601-2604 (1989)). Ia general. proteoglycans arc believed to play a role in regulating extraceiular matrix, cartilage or bone function. The protooglycan de-orn bindis to collgen type I and Hl and affects the rate of fibril formation. Fihrornodulin also binds collagen and delays fibril formation. Both fibromodulin and decorin inhibit the activity of transforming growth factor beta (TUE-f (U.S.

Patent No. 5.583,103 issued December 10. 1996). TGF-P is known to play a key role in the induction of extracellular matrix and has been implicated in the development of fibrotic diseases, such as cancer and glorneruonephritis. Accortdingly, proneogycans 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 treatroent 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-anigioplasty 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 reconstructive :surgery Patent No. 5,654.270, issued August 5. 1997).

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

*44. PRO334 Microfibril bundles and protcins 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 continuous elastic network of sktin. and are present in dermis as niicroflbril bundles devoid of measurable elastin extending from the dermal-epithelial Junction and as components of ct thickc elastic fibres present in t deep reiazlar dermis. Moreover, Marfan syndrome has been linked to mutations which interfere with multimerization of fibrillin monomers or other connective tissue 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 extracellujar matrix protein freqluently found in close association with microfibrils containing either fibronectin or fibrillin. Thus, fibrillin, fibulin. and molecules related thereto are of interest, particularly for tbe use of preventing skin from being damaged from aging, injuries or the sun, or for restoring skin damaged from same. Moreover, these molecules are gaeerally of interest in dre Study Of connective tissue and attachment moleaules and related r-echanais FibriiI, fibulin and relaied rnokes are (ilier described in Adams. et LMDLQiaI..

272(2):226-36 (1997); Kielty and Shuttlcworth. Microsc. Res- Tech.. 38(4):413-27 (1997); and Ch~bi _CL..

Su& 12(2Supp.):131-S (1997).

Currntly. efforts art being undertaken by both industry and academia to identify new, native sccrete d and mnbran-bu receptor proteins, paricularly screted pm=on which have homology to fibulin an~fbrilrLn Many efforts amt focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and memrbrane-boud receptor proteins. Examples of screening methods and tchniques are described in the literature (see, for example. Klein et al.. Proc. Nati- Acad, Sci 22:7108-7113 (1996); U.S. Patent No.

5,536,637)].

We herein describe the identification and characterization of novel polypeptides having homology to fibulin and fibrillin, designated herein as PRO334 polypeptides.

PR0346 The widespread occurrnc of cancer has prompted fth devotion of considerable resources and discovering .15 new treatments of treatment. One particular method involves the creation of tumor or cancer specific monoclonal antibodies (niAbs) which are specific to tumor antigens. Such snAbs. which can distinguish between normal and cancerous cells are useful in the diagnosis, prognosis and treatment of the disease. Particular antigens are known to be assciated with neoplastic diseases. such as colorectal and breast cancer. Since eolon cancer is a widespread disease, early diagnosis and tr-eatmnent 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 Ptien deciptonryn antigen (CEA) is a glycoprotemn found in human colon cancer and the digestive organs of a 2-6 month human embryos. CEA is a known human tumor marker and is widely used in the diagnosis of neoplasuic diseases, such as colon cancer. For example, when the serum levels of CEA are elevated in a patient, a drop of CEA levels after surgery would indicat the tumor resectron was successful. On the other band, a subsequent rise in serum CEA levels after surgery would indicate tat metastases of the original tumor may have formed or that new primary tumors may have appeared. CEA may also be a target for mAlb, antisense nucleotides P6 R268i Protein disulfide isomease is an enzymatic proten which is involved in the promotion of correct refolding of proteins through the establishment of correct disulfide bond formation. Protein disulfide: isomerase was initially identiuid based upon its ability to catalyze the renaturahion of reduced denatured RNAse (Goldberger et al.. Biol.

OeM. 23 9 :1 4 0 6 -1 4 10 (1964) and Epstein et al. Col Spdqn Harbor S wr. Quant. Biol. 28:439-449 (1963)). Protein disulfide isomerase has been shown to be a resident enzynte of the endoplasmic reticulum which is retained in the endoplaintic reticulumn 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 tIh coding sequences for novel protein disulfide isomerase homologs. We herein describe a novel polypeptide having homology to protein disulfide isomerase, designated herein as PR0268.

47. ERO330 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 procollagen. Hydroxyladon 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 threcodimensioal triple-helical conformation. If hydroxylation does not occur, synthesized collagen polypeptides remain non-helical, are poorly secreted by cells and cannot assemble into stable functional S.collagen fibrils. Vuorio et al., Proc. Natl. Acad. Sci. USA 89:7467-7470 (1992). Prolyl 4-hydroxylase is comprised 15 of at least two different polypeptide subunits, alpha and beta.

S fforts are being undetakn by both industry and academia to identify new, native secreted and membranebound receptor proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein ct al., Proc. Natl. Acad. Sci., 20 23:7108-7113 (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-hydroxylasc. designated herein as S. PR0330.

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

49. PR0244 Leins are a class of proteins comprising a region that binds carbohydrates specifically and non-covalently.

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

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

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

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

This group includes L-selectin (peripheral lymph node homing receptor (pnHR), LEC-CAM-1, LAM-1. gp90MEL, gpl00ME 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).

25 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 PRO244 polypeptides.

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

SUMMARY OF THE INVENTION 1. PRO211 and PR0217 Applicants have identified cDNA clones that encode novel polypeptides having homology to EGF, designated in the present application as "PRO211" and "PRO217" 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 EGFake homologue polypeptides. which in one embodiment. includes an amino acid sequenct comprising residues: 1 to 353 of Fig. 2 (SEQ ID NO:2) or i to 379 of Fig. 4 (SEQ ID NO: 4).

2. PROZ~ Applicants have identified a eDNA clone that encodes a novel polypeptide. wherein the polypeptide is designted in the present application as PR0230'.

In one embodiment, the invention provides an isolated nucleic; acid molecule comprising DNA encoding a PR0230 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0230 polypeptidc having amino acid residues 1 through 4.67 of Figure 6 (SEQ ID NO: 12). or is complementary to such encoding nuclcic acid sequence. and rcinains stably bound to it under at least moderate, andl optionally, under high stringency conditions.

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

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

3. PRO232 Applicants have identified a eDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0232'.

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

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

4. 1PRO187 Applicants have identified a cDNA clone that encodes a novel polypeptide. designated in the present application as "PR0187'.

In one embodiment, die invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO187 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PROW18 polypeptide of Figure 11 (SEQ 1ID NO:.23), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the invention provides a rucleic acid comprising the coding sequnc 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 DNA27864-1 155, deposited with the ATCC under accession number ATCC 209375, alternatively tie coding sequence of clone DNA27864-1lSS.

deposited under accession number ATCC 209375.

In yet another embodiment, the invention provides isolated PRO187 polypcptidc. In particular, the invention provides isolated native sequence PRO187 polypcptidc. which in one embodiment, includes an amino acid sequence comprising residues 1 to 205 of Figure 11 (SEQ ID NO:23). Altematively. 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 polypeptide, 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 polypeptide having amino acid residues I to 660 of Figure 13 (SEQ ID NO:28), or is complementary to such encoding nucleic acid sequcnce, 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 N028). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0265 polypeptide.

6. EBQ219 o 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 S *PR0219 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0219 polypcptide 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. PRO246 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as 'PR0246".

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

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

S. RO228 Applicants have identified a cDNA clone doat encodes a novel polypeptide having homology to CD97, EMR1 and latrophilin. wherein the polypeptide is designated in the present application as *PR0228".

In one embodiment, dhe invention provides an isolated nucleic adid molecule comprising DNA encoding a PR0228 polypeptidc. In one aspect, the isolate nucleic acid comprises DNA encoding the PRO229 polypeptide having amino acid residues i to 690 of Figure 19 (SEQ ID NO:49). or is coruplemientrty 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 PR0228 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues I to 690 of Figure 19 (SEQ ID NO:49). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PRO228 polypeptide.

In another embodiment, the invention provides an expressed sequence tag (EST) comprising the nucleotide :sequence of SEQ ID NO:50. designated herein as DNA2i95I.

9. PRO533~ Applicants have identified a eDNA clone (DNA49435-12 19) that encodes a novel polypeptide, designated in the present application as PROM3.

In one embodiment, the invention provides -an isolated nucleic acid molecule having at least about sequence identity to a DNA molecule encoding a PR0533 polypeptide comprising the sequence of aminto acids 23 to 216 of Figure 22 (SEQ ID N0~59). or the complement of the DNA molecule of The sequence identity preferably is about 85%, moue 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 amin acid residues 23 to 216 of Figure 22 (SEQ ID NO:59). Preferably, the highest degree of sequence identity ocmzrs 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 polypeptide having amino acid residues 1 to 216 of Figure 22 (SEQ ID NO:59), or is com~plemntary to such encoding nucleic acid sequence, and remtains; stably bound to it under at least moderate, and optioMaly, under high stringency conditions. In another aspect, tie invention provides a nuecic acid of the full length protein of clore DNA4943S41219. deposited with the ATCC under aiccession mnber ATCC 209480.

Ini yet another embodiment the ivenion provides isolated PRO533 polypeptide. In particular, the invention provides isolated native sequence PR0533 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 23 So 216 of Figure 22 (SEQ ID NO.59). Native PR0533 polypeptides with or without the native signal sequenc (amino acids 1 to 22 in Figure 22 (SEQ ID NO:S9)). and with or without the iitiating methionine are specifically included. Alternatively, the invention provides a PR0533 polypepid&encoded by t nucleic acid deposited under accession number ATCC 209480.

10. PR245 Applicants have identified a cDNA clone that encodes a novel polypepide, wherein the polypeptide is designated in the present application as "PRO245".

In one embodiment. the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0245 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0245 polypeptide havng aino acid residues Ilto 312of Fig. 24(SEQID NO:64), or is complemenay to suchbencodingnuicleic 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 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. PR0220. PR0221 and PR0217 Applicants have identified eDNA clones thnt each encode novel polypeptides, aLl having leuicine rich repeats.

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

:In one embodiment, the invention provides isolated nucleic; acid molecules comprising DNA respectively encoding PR0220, PR0221 and PROW,7 respectively. In one aspect, provided herein is an isolated nucleic acid comprises DNA encoding the PR0220 polypeptide having amino acid residues I through 708 of Figure 26 (SEQ ID NO:69), 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. Also provided herein is an isolated nucleic acid comprises DNA encoding the PR0221 polypeptide having amnino acid residues 1 through 259 of Figure 28 (SEQ ID NO:71), or is complemientary 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 acid comprises DNA encoding the PROW2 polypeptide having amino acid residues I through 620 of Figure 30 (SEQ ID NO:.73), or is complemerazy to such encoding nucleic add sequence, and remains stably bound to it under at least moderate, and optionally. under high stringency conditions.

In anotr embodiment, the: invention provides isolated Pk0220, PR022 and PR0227 polypeptides. In particular, provided herein is the isolated native sequence for thc PRO220 polypeptd&. which in one embodiment, includes an amino acid sequence comprising residues I to 708 of Figure: 26 (SEQ ID NO:69). Additionally provided lbein 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 polypeptde, which in one embodiment, includes an amino acid sequence comprising residues I to 620 of' Figure 30 (SEQ ID NO:73).

12. PR02SS Applicant have identified a cDNA cdone dont encodes a novel polypeptide having homology to CRTAM and poliovims receptor precursors. wherein the polypeptide is designated in the present application as "PR0258.

In onc embodiment. the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO259 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0258 polypeptide having amino acid residues I to 398 of Figure 32 (SEQ ID NO:84). or is comnplementry to suciecoding nucleic acid sequence, and remains "tlly bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO258 polypeptide. In, particular. the invention provides isolated native: sequence PR0258 polypeptidc, 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 extracellular domain of a PR0258 polypeptide.

13. PRO Applicants have identified a cDNA 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 nucleic acid molecule comprising DNA encoding a PR0266 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0266 polypeptide having amino acid residues 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 moderate, and optionally, under high stringency conditions.

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

14. PR.O269 Applicants have identified a eDNA 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 residues I to 490 of Fig. 36 (SEQ ID NO:96), or is complenenry 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 copiigresidues I to 49D of Figure 36 (SEQ ID NO:96). An additional embodiment of the present invention is directed to an isolated extraceliular domain of a PR0269 polypeptide.

is. PRO2ZB7 Applicants have identified a cDNA clone that encodes a novel polypeptide. wherein the polypeptide: is designated in the present application as *PR0287".

In one embodiment. the invention provides an isolated aucleic acid molecule comprising DNA encoding a PRO297 polypep tide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0287 polypeptide having amino acidresidues I to 415 of Fig. 38 (SEQ ID) NO: 104). or is complemetr tsuh oding 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 PR0287 polypeptide. In particular, the invention provides isolated native sequence PR0287 polypeptide. which in one embodiment includes an amino acid sequence comprising residues I to 415 of Figure 38 (SEQ ID NO: 104).

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

In one embodiment, the invention provides an isolated cucleic acid nxilecule comprising DNA encoding a PR0214 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding die PR0214 polypeptide of sees*:Fig. 40 (SEQ ID NO: 109). or is complementary to suci encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. [n another aspect, the invention provides a nuceleic acid comprfising the coding sequence of Fig. 39 (SEQ ID NO: 108) or its complement In another aspect, dhe invention provides a nucleic acid of the ful length protein of clone DNA32286..1191. deposited with ATCC under accession number ATCC 209385.

In yet another embodiincr the invention provies isolated PR0214 polypeptide. In particular, the invention provides isolated native sequence PR0214 polypeptide, which in one embodiment. includes an amino acid sequence comprising the residues of Figure 40 (SEQ MD 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 polypeptide, designated in the present application as "PR0317".

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

In another embodiment, the invention provides isolated PROW1 polypeptde. In particular, the invention provides isolated native-sequence PR0317 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 366 of Figure 42 (SEQ ID NO: 114).

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 anwi-PRO3 17 antibody with a sample suspected of containing PRO3 17; and b) detecting binding of the antibody to the sample; wherein dhe sample is selected from the group consisting or a body fluid, a tissue samplc. a cell extract, and a cell culture medium.

In a still feullier 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 PR0317 toRNA with a detectable nucleft acid probe that hybridizes under moderate to stringent conditions to PR0317 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 siru hybridization, or the sample is a cell extract and detection is by Northern analysis.- Further, dhe invention provides a methodl for treating a PRO3 17-associated disorder comprising administering to a mammal an effective amount of the MR0317 polypeptide or a composition thereof containing a carrier, or with an effective amount of a PR0317 agonist or PR0317 antagonist, such as an anribody which binds specifically to PR0317.

18. PR030 Applicants have identified a eDNA clone (DNA40628-1216) that encodes a novel polypeptide, designated in the present application as *PR0301".

In one embodiment, the invention provides an isolated nucleic acid molecule having at least about sequence identity to a DNA molecule encoding a PRO301 polypeptide comprising the sequce of amino acids 28to 258-of Fig. 44 (SEQ ID NO: 119). or ie complement of the DNA molecule of The sequence identity preferably is about S5 more preferably about 90%, motpreferbly about 95 In one aspect, the isolated nuceic 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 extracellular domains (amino acids 00*025 28 to 258 of Fig. 44, SEQ ID NO:1 19). In a further embodiment, the isolated nucleic acid molecule comprises DNA 0 *00.:encoding a PRO301 polypeptide having amino acid residues 28 to 299 of Fig. 44 (SEQ ID NO:119). or is complcmentiy to such eccoding nucleic acid sequence, and remains stably bound to It under at least moderate, and 0000optionally, une high Stringency conditions. In anoche aspect, the invention provides a nucleic acid of the full lengt protein of clone DNA4062& 1216, deposited with the ATCC under accession number ATCC 209432, alternatively the coding seuenice of clone DNA40628-1216, deposited under accession number ATCC 209432.

In Yet another embodiment, the inion 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 cxtracIuiiar domain residues 28 to 258 of Figure 44 (SEQ ID NO: 119). Native PRO301 polypeptides wihhofrwittthe native signal sequenc (amino acids 1 to 27 in Figure 44 (SEQ ID NO: 119). and with or without the initiating meth onine are specifically included. Additionally, the sequences of the invention may also comprise the transrnbrane domain (residues 236 wo about 258 in Figure 44; SEQ ID NO:! 119) and/or the intracellular domain (about residue 259 to 299 in Figure 44; SEQ ID .NO:119). Alternatively, the invention provides a PRO301 polypeptide encoded by the nucleic acid deposited under accession number ATCC 209432.

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

In one embodiment, the invention provides an isolated nuclic acid molecule comprising DNA encoding a PRO224 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO224 polypeptide 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 PRO224 polypeptide. In particular, the invention provides isolated native sequence PRO224 polypcptide, which in one embodiment, includes an amino acid sequence comprising residues I to 282 of Figure 46 (SEQ ID NO:127).

20. PR0222 15 Applicants have identified a cDNA clone that encodes a novel polypcptide, wherein the polypeptide is designated in the present application as "PR022'.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO222 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0222 polypeptide 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 PRO222 polypeptide. In particular, the invention provides isolated native sequence PRO222 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 490 of Figure 48 (SEQ ID NO: 132).

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

In one embodiment, the invention provides an isolated nucleic acid encoding a novel lectin comprising DNA encoding a PRO234 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 sequene, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the invention provides an isolated nucleic acid molecule comprising the nucleotide sequence of Fig. 49 (SEQ ID NO:136).

In another embodiment, the invention provides isolated novel PR0234 polypeptides. In particular, the invention provides isolated native sequence PRO234 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 382 of Figure 50 (SEQ ID NO:137).

In yet ano8dr embodiment. the invention provides oligomicleotide probes useful for isolating genonic and cDNA nuclcotidc sequences.

n. PRO231 Applicants have idcndticid a cDNA clone that encodes a novel polypeptide having homology to a putative acid phosphatase. wherein dhe polypeptide is designated in the present application as "P 21 In one embodiment. the invention provides an isolated nucleic acid molecule comprising DNA ecoding a PR0231 polypeptide. In one aspect, dhe isolated nucleic acid comprises DNA encoding the PRO231 polypeptide having anoacid residues 1 to 428 of Fig. 52 (SEQ ID NO-.142). or is complementary to such ecxoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

Ia anothe embodiment. die invention provides isolated PR0231 polypeptide. In particular, die invention provides isolated native sequence PR0231I polypeptide. which in one embodiment. includes an amino acid sequence comprising residues I to 428 of Figure 52 (SEQ ID NO:142).

23. PR12 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to scavenger receptors wherein the polypeptide is designated in the present application as "PR0229'.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0229 polypeptide. In one aspect. the isolated nucleic acid comprises DNA encoding the MR0229 polypeptide having amin acid residues Ito 347 of Figure 54 (SEQ ID NO:148). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0229 polypeptide. In particular, the invention provides isolated native sequence PR0229 polypeptide. which inoune embodiment, includes an amino acid sequence comprising residues I to 347 of Figure 54 (SEQ ID NO: 148).

24. PRO238 Applicants have identified a eDNA clone tat encodes a novel polypepude having homology to reductase, wherein die polypeptide is designated in the present application as "Pft0238".

In one embodiment. the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0238 polypeptide. In one aspect, t isolated nucleic acid comprises DNA encoding the PR0239 polypeptide having am-ino acid residues I to 310 of Figure 56 (SEQ ID NO: 153). or is complementary to such encoding nucleic acid sequenice, 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, die invention provides isolated native sequence PR0238 polypeptide. which in one embodiment, inchtdes an amino acid sequence comprising residues I to 3 10 of Figure 56 (SEQ ID NO: 153).

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

In one embodiment, the invention provides an isolated mucleic acid molecule comprising DNA encoding a PR0233 po.lypeptide. In one aspect. the isolated nucleic acid comprises DNA encoding the PR0233 polypptide having amino acid residues 1 to 300of Figure 58(SEQ ID NO:1S9), or iscomnplementary to suchcncoding nucleic acid sequence. and remains stably bound to it under at least mnoderate, and optionally. under high stringency conditions.

In another embodiment. the invention provides isolated PR0233 polypeptide. In particular, the invention provides isolated native sequence PR0233 polypeptide, which in one embodiment. includes an amino acid sequence conmprising residues 1 to 300of Figure58 (SEQ ID NO:159).

26. PRO223 Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to serine carboxypeptidase polypeptides, wherein the polypeptd& is designated in the present application as "PR0223'.

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

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

27. PR73 Applicants have Identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as *PR0235'.

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

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

28. PRO236 and pR0262 AppliCant have identified eD)NA clones that cncde novel polypeptides having homology to p-galacosjdasc.

wherein those polypeptides are designated in the present application as UPR0236. and *PR0262'.

In oe embodiment, the invention provides an isolated nucic acdd molecule cotnprnsing DNA encoding a PR0236 polypeptide. In one aspect. the isolated nucleic acid comprises DNA encoding the PR0236 polypeptide having aminoacid residues I bo 636 of Figure 64 (SEQD NO:l5) orisom letry tosuch noig Mleic acid sequence, and remains stably bound to it undler at least moderate, and optionally, under high stringency conditions.

In another embodiment. the invention provides an isolated micleic acid molecule comprising DNA encoding a PR0262 polypeptide. In one aspect the isolated nucleic acid comprise DNA encoding the PR0262 polypeptide having amino acid residues I to 654 of Figure 66 (SEQ MD NO: 177). or is complemenuasy to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate. and optionally, under high stringency conditions.

In another emibodiment, the invcntion provides isolated PR0236 polypeptide. In particular, the invention provides isolated native sequence PR0236 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 £o 636 of Figure 64 (SEQ ID NO: 175).

In another embodiment, the invention provides isolated PR0262 polypeptide. In particular, the invention 0 provides isolated native sequence PR0262 polypeptide. which in one embodiment, includes an amino acid sequence comprising residues I to 654 of Figure 66 (SEQ ID NO: 177).

20 29. PRO739 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 PR0239 polypeptide. In one aspect. the isolated nucleic acid comprises DNA encoding the PR0239 polypetide 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 leat moderate, and optionally. under high stringency conditions.

In another embodiment, the invention provides isolated PR0239 polypeptide. In particular, the invention provides isolated native sequence PR0239 polypeptide, which in one embodimfent, includes an amino acid sequence comprising residues I to 501 of Figure 68 (SEQ ID NO: 185).

PRO?27 Applicants have identified a eDNA clone that encodes a novel polypeptide, wherein the polypcptide is designated in dhe present application as *PR0257".

In oe enbodinment, the invention provides an isolated nuecic acid molecule comprising DNA encoding a PRO257 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0257 polypeptide having amino add residues I to 607 of Figure 70 (SEQ ID NO: 190). or is complementary to such encoding nucleic acid sequence. andi remains stably bound to it under at least moderate, and optionally. under high stringency conditions.

in another embodiment. the invention provides isolated PRO2S7 polypeptidc. In particular, the invention provides isolated native sequence PR0257 polypeptide, which in one embodiment, includes an amnino acid sequence comtprisingyreidues Ito 607of Figure70 (SEQ ID NO: 190). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0257 polypeptide.

31. PR0260 Applicants have identified a eDNA clone that encodes a amve polypeptide, wherein the polypeptide is designated in the present application as *PR0260'.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0260 polypeptide. In one aspect, the isolated nucleic acid comprises DNA enoding the PR0260 polypeptide having amino acid residues I to 467 of Figure 72 (SEQ ID NO: 195). or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under.high stringency condiions 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. PRO26 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 molec comprising DNA encoding a :PR0263 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0263 polypeptide having amino acid residues I 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 provides isolated native sequence PR0263 polypepuide, which in one embodiment, includes an amino acid sequence comprising residues I to 322 of Figure 74 (SEQ ID N0:201). An additional embodiment of the present invention is directed to an isolated extraceilular domain of a PR0263 polypeptide.

33. PRO270I 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 polypcptide. In one aspect, tie isolated nucleic acid comprises DNA whivch includes the sequence encoding the PR0270 polypeptide having amino acid residues I to 296 of Fig. 76 (SEQ ID 140:207), 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 polypeptdc, which in one embodiment, includes an amino acid sequence comprising residues 1 to 296 of Figure 76 (SEQ ID NO:207).

34. PRO271 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".

SIn 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 acid residues 1 to 360 of Figure 78 (SEQ ID NO213), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

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

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

In one embodiment, the invention provides an isolated nuclcic 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 1 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 polypcptide, 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 PRO294 polypeptide. In one aspect, the isolated nucleic add comprises DNA encoding the PR0294 polypeptide having amino acid residues 1 to 550 of Figure 82 (SEQ ID NO:227), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

48 In another embodiment, the invention provides isolated PRO294 polypeptide. In particular, the invention provides isolated native sequence PR0294 polypeptide, which in one embodiment, includes an amino acid sequence ccnprising residues 1 to 550 of Figure 82 (SED ID NO:227).

37. PRO295 Applicants have identified a cENA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0295.

In one embodiment, the invention provides an isolated nucleic acid molecule ccnprising LNA encoding the PRO295 polypeptide. In one aspect, the isolated nucleic acid cnaprises ENA encoding the PR0285 polypeptide having amino acid residues 1 to 350 of Figure 84 (SE) ID ND:236), or is carplementary to such encoding nucleic acid sequence, and 15 renains 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 PRO295 polypeptide, which in one embodiment, includes an amino acid sequence carprising residues 1 to 350 of Figure 84 (SED ID NO:236) *38. PR293 Applicants have identified a cIEA clone that encodes a novel human neuronal leucine rich repeat polypeptide, wherein the polypeptide is designated in the present application as "PR0293".

In one embodiment, the invention provides an isolated nucleic acid nolecule carprising ENA encoding a PRO293 polypeptide. In one aspect, the isolated nucleic acid carprises ENA encoding the PR0293 polypeptide having amino acid residues 1 to 713 of Figure 86 (SEQ ID N0:244), or is ccnplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO293 polypeptide. In particular, the invention provides isolated native sequence PR0293 polypeptide, which in one embodiment, includes an amino acid sequence ccnprising residues 1 to 713 of Figure 86 (SEQ ID ND:244) An additional aebodiment of the present invention is directed to an isolated extracellular docain of a PR0293 polypeptide.

49 39. PRO247 Applicants have identified a clA clone that encodes a novel polypeptide having leucine rich repeats wherein the polypeptide is designated in the present application-as '*iP247".

In one aebodient, the invention provides an isolated nucleic acid nmolecule ccuprising IEA encoding a PfRO)247 polypeptide. In. one aspect, the isolated nucleic acid couprises EA encoding the PR0247 polypeptide having amino acid residues 1 to 546 of Figure 88 (SED ID NO:249), or is ccplementary to such encoding nucleic acid sequence, and remain stably bound to it under at least moderate, and optionally, under high stringency .conditions.

In another enbodinent, the invention provides isolated PR0247 **gpolypeptide. In particular, the invention provides isolated native ~sequence PR0247 polypeptide, which in one edbodinent, includes an amino acid sequence carprising residues 1 to 546 of Figure 88 (SBQ ID NO:249).

g nAn additional enbodiment of the present invention is directed to an isolated extracellular domain of a PRO247 polypeptide.

PR302, PRO303, PER304, PRO307 and PRO343 Applicants have identified cMlA clones that encode novel polypeptides having haology to various proteases, wherein those polypeptide are designated in the present application as "PR0302", "PR0303", "PRO304", "PRO307" and "PR0343- polypeptides.

*g ~In one enbodiment, the invention provides an isolated nucleic 25 acid nmolecule ccrprising IM encoding a PERO302 polypeptide. In one aspect, the isolated nucleic acid carprises IIM encoding the PRO302 polypeptide having amino acid.residues 1 to 452 of Figure 90 (SEP ID ND:254), or is carplementary to such encoding nucleic acid sequence, and renains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodinent, the invention provides an isolated nucleic acid nolecule carprising EA encoding a PRO0303 polypeptide. In one aspect, the isolated nucleic acid ccnprises EN. encoding the PR0303 polypeptide having amino acid residues 1 to 314 of Figure 92 (SED ID NO:256), 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 yet another aebodiment, the invention provides an isolated nucleic acid nolecule ccnprising EM encoding a PR0304 polypeptide. In one 50 aspect, the isolated nucleic acid molecule ccurprises EM encoding a PR~O304 polypeptide having amino acid residues 1 to 556 of Figure 94 (SE2 ID) NDK:258) or is complemenitary to such enicoding nucleic acid sequence, and remains stably bound to it under at least zmderate, and optionally, under high stringency conditions.

In another emkxodinent, the invention provides an isolated nucleic acid mrolecule ccmprising EM encoding a PRO307 polypeptide. In one aspect, the isolated nucleic acid comprises fl'A encoding the PR0307 polypeptide having amino acid residues 1 to 383 of Figure 96 (SE)Q ID N2):2 60), or is ccztplementary to such encoding nucleic acid sequence, and remiains stably bound to it under at least moderate, and cptionally, under_high stringency conditions.

In another ertbodimint, the invention provides an isolated nucleic acid molecule caruprisingj EM~ encoding a PR0343 polypeptide. In one aspect, the isolated nucleic acid ccrnprises EMA encoding the PR0343 00::0*polypeptide having amino acid residues 1 to 317 of Figure 98 (SE)Q ID NO:262), or is ccrrplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

00.. 20 In another embodimrent, the invention provides isolated PR0302 .ooopolypeptide. In particular, the invention provides isolated native Poo. sequence PRO302 polypeptide, which in one ertnodinmnt, include an amino acid sequence comprising residues 1 to 452 of Figure 90 (SEp ID NO:254). in aothr enxdimtthe invention provides isolated PO0 .00. 25 polypeptide. In particular, the invention provides isolated native sequence PIR0303 polypeptide, which in one ewbodinent, includes an amino 0 0 acid sequence ccmprising residues 1 to 314 of Figure 92 (SE)2 IM IK:256).

In another entodiirent, the invention provides isolated PRO3 04 polypeptide. In particular, the invention provides isolated native sequence PRO304 polypeptide, which in one emb~odiment, includes an amino acid sequence comprising residues 1 to 556 of Figure 94 (SEQ ID NOf:258).

In another embodimnit, the invention provides isolated PI)3 07 polypeptide. In particular, the invention provides isolated native sequence PRO307 polypeptide, wich in one embodiment, includes an amino acid sequence ccxprising residues I. to 383 of Figure 96 ID NO:260).

In another embodiment, the invention provides isolated PR0343 polypeptide. In particular, the invention provides isolated native sequence PR0J343 polypeptide, which in one enbodiment, includes an amino acid sequence ccxrprising residues 1 to 317 of Figure 98 (SE)2 ID N):262).

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

In one embodiment, the invention provides an isolated nucleic acid molecule cacprising EM encoding a PRO328 polypeptide. In one aspect, the isolated nucleic acid ccnprises EM 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 embodiment, 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 caoprising residues 1 to 463 of Figure 100 (SEQ ID N3:284).

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

42. PRO335, PRo331 and PR0326 Applicants have identified three cENAM clones that respectively encode three novel polypeptides, each having leucine rich repeats and homology to LIG-I and ALS. These polypeptides are designated in the present application as PRO335, PRO331 and PRO326, respectively.

*0 000 25 In one enbodiment, the invention provides three isolated nucleic acid molecules comprising IM respectively encoding PRO335, PRO331 and PRO326, respectively. In one aspect, herein is provided an isolated nucleic acid comprising INA encoding the PR0335 polypeptide having amino acid residues 1 through 1059 of Figure 102 (SEQ ID ND:289), or is cmaplementary 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 cacprises IMA encoding the PRO331 polypeptide having amino acid residues 1 through 640 of Figure 104 (SEQ ID NO:291), 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. Additionally provided herein is an isolated nucleic acid ccrprises EAI encoding the PR0326 polypeptide having amino acid residues 1 through 1119 of Figure 106 (SEQ ID NO: 293), or is complementary to such encoding nucleic acid sequence, and remains 52 stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO335, PR0331 and PR0326 polypeptides or extracellular domains thereof. In particular, the invention provides isolated native sequence for the PR0335 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 through 1059 of Figure 102 (SEQ ID ND:289). Also provided herein is the isolated native sequence for the PR0331 polypeptide, which in one eabodiment, includes an amino acid sequence croprising 1 through 640 of Figure 104 (SEQ ID NO: 291). Also provided herein is the isolated native sequence for the PRO326 polypeptide, which in one embodiment, includes an amino acid sequence coaprising residues 1 through 1119 of Figure 106 (SEQ ID NO: 293).

15 43. PR0332 Applicants have identified a cEINA clone (EIA40982-1235) that encodes a novel polypeptide, designated in the present application as "PRO332".

In one embodiment, the invention provides an isolated nucleic 20 acid molecule carprising ENA having at least about 80% sequence identity to a IiA molecule encoding a PRO358 polypeptide ccrprising the sequence of amino acids 49 to 642 of Fig. 108 (SEQ ID NO:309), or the ccnplement of the DA molecule of The sequence identity preferably is about nore preferably about 90%, most preferably about 95%. In one aspect, the 25 isolated nucleic acid has at least about 80%, preferably at least about more preferably at least about 90%, and most preferably at least about 95% sequence identity with a polypeptide having amino acid residues 1 to 642 of Fig. 108 (SBE ID N0:309). Preferably, the highest degree of sequence identity occurs within the leucine-rich repeat domains (amino acids 116 to 624 of Fig. 108, SEQ ID ND:309). In a further embodiment, the isolated nucleic acid olecule comprises INA encoding a PRO332 polypeptide having amino acid residues 49 to 642 of Fig. 108 (SEQ ID ND:309), or is ccmplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least noderate, and optionally, under high stringency 3 5 conditions.

In another erbodiment, 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 ccuprising residues 49 to 624 of Figure 108 (SEQ ID ND:309).

53 Native PRO332 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 hcuology to fibulin and fibrillin, wherein the polypeptide is designated in the present application as "PRO334".

In one embodiment, the invention provides an isolated nucleic acid molecule caoprising EMA encoding a PRO334 polypeptide. In one aspect, the isolated nucleic acid carprises EA encoding the PRO334 polypeptide having amino acid residues 1 to 509 of Figure 110 (SEQ ID. N:314), or is ccplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another enbdinment, the invention provides isolated PRO0334 polypeptide. In particular, the invention provides isolated native sequence PRO334 polypeptide, which in one embodiment, includes an amino acid sequence corprising residues 1 to 509 of Figure 110 (SEQ ID NO:314).

2 0 45. PRO346 Applicants have identified a cElA clone (ENA44167-1243) that encodes a novel polypeptide, designated in the present application as "PRO346".

In one embodiment, the invention provides an isolated nucleic acid molecule 25 having at least about 80% sequence identity to a EVA molecule encoding a PRO346 polypeptide ccaprising the sequence of amino acids 19 to 339 of Fig. 112 (SEQ ID NO:319), or the coplement of the DIA molecule of The sequence identity preferably is about 85%, more preferably about most preferably about 95%. In one aspect, the isolated nucleic acid has at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity with a polypeptide having amino acid residues 19 to 339 of Fig. 112 (SEQ ID NO:319). Preferably, the highest degree of sequence identity occurs within the extracellular damains (amino acids 19 to 339 of Fig. 112, SEQ ID N3:319). In alternative embodiments, the polypeptide by which the homology is measured carprises the residues 1-339, 19-360 or 19-450 of Fig. 112, SEQ ID ND: 319). In a further embodimnent, the isolated nucleic acid molecule canprises DOA 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 ccuplementary 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 11A44167-1243, deposited with the ATCC under accession number A'CC 209434, alternatively the coding sequence of clone EIA44167-1243, deposited under accession number ATOC 209434.

In yet another embodiment, the invention provides isolated PR0346 polypeptide. In particular, the invention provides isolated native sequence PRO346 polypeptide, which in one embodiment, includes an amino acid sequence ccmprising 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 (SEQ ID NO: 319), with or without the initiating nethionine, with or without the transmabrane donain (residues 15 340 to 360) and with or without the intracellular domain (residues 361 to 450) are specifically included. Alternatively, the invention provides a PR0346 polypeptide encoded by the nucleic acid deposited under accession number AITC 209434.

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

In one embodiment, the invention provides an isolated nucleic acid molecule ccmprising MNA encoding a PRO268 polypeptide. In one aspect, the isolated nucleic acid ccmprises ENA encoding the PR0268 polypeptide having amino acid residues 1 to 280 of Figure 114 (SEQ ID N:324), or is camplementary to such encoding nucleic acid sequence, and renains 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 PRO268 polypeptide, which in one ennbodiment, includes an amino acid sequence conprising residues 1 to 280 of Figure 114 (SEQ ID ND: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 clNA clone that encodes a novel 54a polypeptide having harology to the alpha subunit of prolyl 4-hydroxylase, wherein the polypeptide is designated in the present application as "PRO330".

In one embodiment, the invention provides an isolated nucleic acid molecule carprising ENA encoding the PR0330 polypeptide. In one aspect, the isolated nucleic acid molecule ccnprising IA encodin a PR0330 polypeptide having amino acid residues 1 to 533 of Figure 116 (SEQ ID N0:331), or is ccnplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under S 10 high stringency conditions.

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

In one embodiment, the invention provides isolated nucleic acid nolecules caprising INA encoding the PR0339 and/or a PRO310 polypeptide.

.In one aspect, the isolated nucleic acid ccnprises INA encoding the PR0339 polypeptide having amino acid residues 1 to 772 of Figure 118 (SEQ ID 10:338), or is carplementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the isolated nucleic acid ccnprises IMA encoding the PRO310 polypeptide having amino acid residues 1 to 318 of Figure 120 (SEQ ID N0:340), 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 eabodiment, the invention provides isolated PR0339 as well as isolated PRO310 polypeptides. In particular, the invention provides isolated native sequence PR0339 polypeptide, which in one embodiment, includes an amino acid sequence ccprising residues 1 to 772 of Figure 118 (SEQ ID NO: 338). The invention further provides isolated native sequence PRO310 polypeptide, which in one embodiment, includes an amino acid sequence cacprising residues 1 to 318 of Figure 120 (SEQ ID END:340).

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

In one embodiment, the invention provides an isolated nucleic acid molecule ccnprising ENA encoding PR0244 polypeptide. In one aspect, the isolated nucleic acid comprises Ca encoding PR0244 polypeptide having amino acid residues 1 to 219 of Fig. 122 (SEQ ID NO:376), or is complementary 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 PRO244 polypeptide. In particular, the invention provides isolated native sequence PRO244 polypeptide, which in one embodiment, includes an amino acid sequence cacprising residues 1 to 219 of Figure 122 (SEQ ID N0:376).

Additional anEodiments In other emnbodiments of the present invention, the invention provides vectors coaprising INA encoding any of the above or below described polypeptides. A host cell conprising any such vector is also provided. By way of example, the host cells nay be CHO cells, E. coli, or yeast. A process for producing any of the above or below described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide fran the cell culture.

25 In other erbodiments, the invention provides chimeric molecules ccuprising any of the above or below described polypeptides fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises any of the above or below described polypeptides fused to an epitope tag sequence or a Fc region of an imnunoglobulin.

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

Optionally, the antibody is a monoclonal antibody.

In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genanic and cENA nucleotide sequences, wherein those probes may be derived fran 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.

S* 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.

20 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".

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

Figure 8 shows a nwcboddc acquenc (SEQ MD NO: 17) of a native sequence PR0232 cDNA. wherein SEQ ID NO: 17 is a clone designated herein as 'UNQ206* and/or 'DNA34435-1140'.

Figure 9 shows te amino acid sequence (SEQ ID NO: IS) derived from the coding sequence of SEQ ID NO: 17 shown in Figure 8.

Figure 10 shows a zmclode sequence (SEQ ID NO:22) ofta nativ sequence PROIS87 cDNA. wherein SEQ ID NO:22 is a clone designated herein as "UNQ161' and/or "DNA27864-115S'.

Figure I11 shows the amino acid sequence (SEQ MD NO:23) derived from the coding sequence of S EQ ID NO:22 shown in Figure Figure 12 shows a axeoid sequnce (SEQ ID N027) of a native sequence PR0265 cDNA, wherein SEQ MD NO:27 is a clone designated herein as "UNQ232' arid/or "DNA36350-1158'.

Figure 13 shows the amino acid sequence CSEQ ID NO:28) derived from the coding sequence of SEQ ID N0277 shown in Figure 12.

Figures 14A-B show a uxleotide sequence (SEQ ID NO:33) of a native sequence PR0219 eDNA. wherein *SEQ ID NO.33 is a clone designated herein as ULNQ193* and/or 'DNA3229-1 164.

Figure 15 shows the amino acid sequence (SEQ MD NO:34) derived from the coding sequence of SEQ ID NO:33 shown In Figures 14A-B.

Figure 16 shows a ni~detde sequene (SEQ ID NO38) of a native sequence PR0246 cDNA, wherein SEQ ID N0:38 is a clone designated herein as 'UNQ220' and/or 'DNA35639-1172'.

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

20 Figure 18 shows a nucleotide sequence (SEQ ID NO:48) of a native sequence PR0228 cDNA, wherein SEQ ID NO0:8 is a clone designated herein as 'UNQ202" anid/or IDNA33092-1202".

Figure 19 shows the amino acid sequence (SEQ ID 140:49) derived from the coding sequence of SEQ ID NO:48 shown in Figure 18.

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

Figure 21 shows a nucleoide sequence (SEQ MD NO.58 of a native sequence PR0533 cDNA, wherein S EQ ID N0:58 is a clone designated herein as 'UNQ344* and/or 4DNA49435-1219'.

Figure 22 shows the amino acid sequence (SEQ ID NO0:59) derived from the coding sequence of SEQ ID NO:58 shown in Figure 21.

Figure 23 show a ncloodde sequence (SEQIM NO:63) of a native sequence PRO245 cDNA, wherein SEQ ID N0:63 is a clone designated herein as "UNQ219' and/or *DNA35638-1141".

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

Figure 25 shows a mideotide sequenc (SEQ ID NO:68) of a native seqence PR0220 cDNA, wherein SEQ 1D NO-68 is a clone designated herein as 'UNQ194' and/or "DNA32298-1132".

Figure 26 shows the amino acid sequence (SEQ ID NO:69) derived from the,coding sequence of SEQ ID 140:68 shown in Figure Figure 27 shows a nloodde sequaesii (SEQ U) NO:70) of a native sequence PR0221 eDNA. wherein S EQ ID NO:70 is a clone designated herein as 'UNQ195" and/or 'DlA33089-1 132'.

Figure 28 shows the amino acid sequence (SEQ MD NO:71) derived from the coding sequence of SEQ MD N0:7O shown in Figure 27.

Figure 29 shows a aloodde sequence (SEQ ID NO:72) of a native sequence PR0227 cDNA. wherein SEQ IM NO:72 is a clone designated herein as "UNQ201 and/orDNA337S6-l 132'. t Figue 30 shows the amino acid sequence (SEQ ID NO:73) derived from the coding sequence of SEQ ID NO:72 shown in Figure 29.

Figure 31 shows a nucloodde sequence (SE ID NO:83) of a nanve sequence PR0258 eDNA, wherein SEQ ID NO:83 is a clone designated herein as 'UNQ225" and/or'DNA359IS.1174'.

Figur 32 shows the amino acid sequence (SEQ MD NO:84) derived from the coding sequence of SEQ MD NO-.83 shown in Figure 31.

Figure 33 shows a nucloodde sequence (SEQ 11) NO:90) of a native sequence PR0266 cDNA. wherein SEQ *ID NO:90 is a clone designated herein as OUNQ233' arid/or ODNA37150-1178".

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 nuclodde sequence (SEQ ID NO:95) of a native sequence PR0269 cDNA, wherein SEQ MD 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 NO:95 shown in Figure Figure 37 shows a nucleotide sequence (SEQ ID NO:103) of a native sequece= PR0287 cDNA. wherein SEQ ID NO.103 is a clone designated herein as 'UNQ250" and/or 'DNA39969-118S".

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

Figure 39 shows a nucleodide sequence (SEQ ID NO:108) of a native sequence PR0214 cDNA, wherein SEQ ID NO:108 is a clone designated herein as *UNQ188* and/or *DNA32286.1191".

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

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

Figure 42 shows the amino acid sequence (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 MD 1401IS) of a native sequence PRO301 eDNA, wherein SEQ ID NO:118 is a clone designated herein as KJNQ264' and/or *DNA40628-1216'.

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

Figure 45 shows a nueceotide sequence (SEQ ID NO0126) of a native sequence PR0224 cDNA. wherein SEQ ID NO:126 is a clone designated herein as 'U1Q198' and/or "DNA33221-1133'.

Figur 46 shows thc aimino acid sequence (SEQ MD NO:127) derived from the coding sequence of SEQ ID NO: 126 shown in Figure Figure 47 shows a nucleotide sequence (SEQ ID NO: 13 1) of a native sequence PR0222 cDNA, wherein SEQ ID NO: 131 is a clone designated herein as *UNQ196' and/or "DNA33107-1135" Figure 48 shows the amino acid sequence (SEQ ID NO: 132) derived trom the coding sequee of SEQ MD NO:131 shownin Figure 47.

Figure 49 shows a nucleotde sequence (SEQ MD NO: 136) of a native sequence PR0234 cDNA, wherein SEQ ID NO: 136 is a clone designated herein as 'UNQ208* and/or "DNA35557.1137".

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

Figure 51 shows a nucleotide sequence (SEQ M) NO. 14 1) of a native sequence PiZ0231 eDNA, wherein SEQ ID NO: 141 is a clone designated herein as 'UNQ205' and/or "DNA34434-1 139'.

Figure 52 shows the anin acid sequence (SEQ ID NO: 142) derived from die coding sequence of SEQ ID bob NO: 141 shown in Figure 5 1.

Figure S3 shows a nucleotide sequence (SEQ ID NO: 147) of a native sequence PR0229 cDNA. wherein SEQ ID NO: 147 is a clone designated herein as 'UNQ203" and/or "DNA33100d1159".

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

Figure 55 shows a nueleotide sequence (SEQ ID NO: 152) of a native sequence PR0238 eDNA, wherein 0 SEQ ID NO: 152 is a clone designated herein as "UNQ212" and/or "DNA35600-1162".

Figure 56 shows the amino acid sequence (SEQ ID NO: 153) derived from dhe coding sequence of SEQ ID NO: 152 shown in Figure Figure 57 shows a nucleotide sequenice (SEQ ID NO: 158) of a native sequence PRO233 eDNA, wherein SEQ ID NO:.158 is a clone designated herein as 'UNQ207" and/or uDNA34436-1238".

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

Figure 59 shows a nucleoodde sequence (SEQ ID NO: 163) of a native sequence PR0223 eDNA. wherein SEQ ID NO-:163 is a clone designated herein as *UNQlgrT and/or *DNA33206-l 165".

Figur 6D shows the ainino acid sequence (SEQ ID NO: 164) derived from the coding sequence of SEQ MD NO: 163 shown in Figure 59.

Figure 61 shows a nucleouide sequence (SEQ ID NO: 169) of a native sequence PR0235 cDNA. wherein SEQ ID NO:169 is a cloaw designated herein as UNMQ209' and/or ODNA35558-1167'.

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

Figure 63 shows a nucleotide sequence (SEQ ID NO: 174) of a native sequence PR0236 eDNA. wherein SEQ ID NO: 174 is a clone designated herein as OUNQ210" and/or IDNA35599-l 168'.

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

Figure 65 shows a nucleodde sequec (SEQ ID NO:176) Of a native sequence PR0262 eDNA. wherein SEQ ID NO: 176 is a clone designated herein as *UNQ229 and/o r *DNA36M921 168% Figure 66 shows the amino acid sequence (SEQ ID NO: 177) derived from the coding sequence of SEQ ID NO: 176 shown in Figure Figure 67 shows a nuiccotide 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 "DNA3440Y7.1169'. t' Figurc 68 shows the amino acid sequence (SEQ 13D NO:IM5 derived from the coding sequence of SEQ ID NO: 184 shown in Figure 67.

Figure 69 shows a nucleotide sequence (SEQ MD NO: 189) of a nativc sequence PR0257 cDNA i wherein SEQ ID NO: 199 is a clone designated herein as "UNQ224' and/or *DNA3841-1173'.

Figure 70shows the amino acid sequence (SEQ ID NO:190) derived from the coding seqence ofSEQ I NO: 189 shown i Figure 69.

Figure 71 shows a nucleotide sequence (SEQ ID NO: 194) of a native sequence PR0260 cDNA, whercin SEQ ID NO:l194 is a clone designated 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: 194 shown in Figure 7 1.

Figure 73 shows a nucleodde sequence (SEQ ID N0200) of a native sequence PR0263 cDNA, wherein SEQ ID NO:200 is a dlone designated herein as OUNQ230' and/or *DNA34431-1 17?".

Figure 74 shows the amino acid sequence (SEQ ID NO:201) derived from the coding sequence of S EQ ID NO:200 shown in Figure 73.

20 Figure 75 shows a nueceotide sequence (SEQ WD NO:206) of a native sequence PR0270 cDNA. wherein SEQ ID NO:206 is a clone designated herein as '1JNQ237' anid/or *DNA39510-1181.

Fgr 76 O:06soni shows the amino acid sequence (SEQ ID NO:207) derived from the coding sequence: of SEQ ID shon inFigure Figure 77 shows a nucleotide sequence (SEQ MD NO:212) of a native sequence PR0271 eDNA. wherein SEQ ID NO:212 is a clone designated herein as *UNQ238* and/or "DNA394231182" Figure 78 shows the amino acid sequence (SEQ ID NO:2 13) derived from the coding sequence of SEQ ID NO0:212 shown in Figure 77.

Figure 79 shows a nueleotide sequence (SEQ ID NO:.220) of a native sequence PR0272 eDNA, wherein SEQ ID P40:220 is a clone designated herein as 'UNQ239" and/or "DNA40620-1183".

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

Figure 81 shows a nucleotide sequence (SEQ ID NO :226) of a native sequence PR0294 cDNA, wherein SEQ ID NO:226 is a clone designated herein as "UNQ257* anid/or "DNA4O604-11Sr".

Figure 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 OUNQ2SS" and/or "DNA38268-l 188'.

59 Figure 84 shows the amino acid sequence (SEQ ID ND:236) derived frcn the coding sequence SEQ ID ND:235 shown in Figure 83.

Figures 85A-B show a nucleotide sequence (SED ID NO:243) of a native sequence PRO293 cUA, wherein SEQ ID N3:244 is a clone designated herein as 'UN256 and/or "EIA37151-1193".

Figure 86 shows the amino acid sequence (SEQ ID N3:244) derived frnom the coding sequence of SEQ ID ND:244 shown in Figures Figures 89A-B show a nucleotide sequence (SEQ ID NO:248) of a native sequence PRO247 cIA, wherein SEQ ID N:248 is a clone designated herein as "UN221" and/or "IEM35673-1201".

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

Figure 89 shows a nucleotide sequence (SEQ ID ND:253) of a native sequence PR0302 cIIA, wherein SEQ ID NI:253 is a clone designated herein as "IU265" and/or "IA40370-1217".

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

Figure 91 shows a nucleotide sequence (SEQ ID NO:255) of a native sequence PR0303 cA, wherein SEQ ID NO:255 is a clone designated 20 herein as "Ulg266" and/or "NA42551-1217.

Figure 92 shows the amino acid sequence (SED ID NO:256) derived from the coding sequence of SED3 ID M0:255 shown in Figure 91.

Figure 93 shows a nucleotide sequence (SEQ ID NO:257) of a native sequence PR0304 cENA, wherein SEQ ID NO:257 is a clone designated herein as "UNQ267" and/or "mEA39520-1217".

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

Figure 95 shows a nucleotide sequence (SEQ ID N3:259) of a native sequence PRO307 cEA, wherein SE3 ID ND:259 is a clone designated herein as "UNQ270' and/or "EIA41225-1217'.

Figure 96 shows the amino acid sequence (S3EQ ID NO3:260) derived fram the coding sequence of SED3 ID NO:259 shown in Figure Figure 97 shows a nucleotide sequence (S3EQ ID N:261) of a native sequence PRO343 crM, wherein SEQ ID ND3:261 is a clone designated herein as "UMq302' and/or "MIA43318-1217".

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

Figure 99 shows a nucleotide sequence (S3 ID NO:283) of a native sequence PRO328 cIENA, wherein SEQ2 ID ND:283 is a clone designated 60 herein as "UN0289" and/or "EN40587-1231'.

Figure 100 shows the amino acid sequence (SEQ ID D:284) derived from the coding sequence of S2 ID N:283 shown in Figure 99.

Figures 101A-B show a nucleotide sequence (SE* ID ND:288) of a native sequence PR0335 cA, wherein SEQ ID NO:288 is a clone designated herein as "UNQ287' and/or "'M41388-1234".

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

Figure 103 shows a nucleotide sequence (SEQ ID 1D:290) of a native sequence PR0331 cEMA, wherein SEQ ID ND:290 is a clone designated herein as 'UN292 and/or 1'M40981-1234.

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

Figure 10A-B show a nucleotide sequence (SEQ ID N: 293) of a native sequence PR0326 ciNA, wherein SEQ ID 1ND:293 is a clone designated herein as "UNQ287" and/or "I'A37140-1234.

Figure 106 shows the amino acid sequence (SEQ ID No3:294) derived fraom the coding sequence of SEQ ID NO:293 shown in Figures 105A-B.

Figures 107A-B show a nucleotide sequence (SEQ ID 1N:309) of a 20 native sequence PR0332 clEM, wherein SEQ ID NO3:309 is a clone designated herein as "UNQ293" or "[IA40982-1235.

Figure 108 shows the amino acid sequence (SEQ ID NO3: 310) derived from the coding sequence of SEQ ID NO: 309 shown in Figure 107.

Figure 109 shows a nucleotide sequence (SEQ ID 1:314) of a native sequence PR0334 cIA, wherein SEQ ID N:314 is a clone designated herein as "UNQ295" or "IMA41379-1236".

Figure 110 shows the amino acid sequence (SE ID 1ND:315) derived fran the coding sequence of SEQ ID N:314 shown in Figure 109.

Figure 111 shows a nucleotide sequence (SEQ ID ND:319) of a, native sequence PRO346 cI, wherein SEQ ID D :318 is a clone designated herein as "UNQ305" or "E'M44167-1243.

Figure 112 shows the amino acid sequence (SEQ ID ND:319) derived from the coding sequence of SEQ ID 1N:318 shown in Figure 111.

Figure 113 shows a nucleotide sequence (SEQ ID 1:323) of a native sequence PRE268 cEIM, wherein SED ID N1:323 is a clone designated herein as "UN235 or IIA39427-1179".

Figure 114 shows the amino acid-sequence (SEQ ID N0:324) derived fran the coding sequence of SEQ ID 103:323 shown in Figure 113.

Figure 115 shows a nucleotide sequence (SEQ ID 13:330) of a 61 native sequence PR0330 cIMA, wherein SEQ ID NO:330 is a clone designated herein as "UNQ290" or "1N40603-1232.

Figure 116 shows the amino acid sequence (SEQ ID O: 331) derived fran the coding sequence of SED ID N3:330 shown in Figure 115.

Figure 117 shows a nucleotide sequence (SEQ ID 13:337) of a native sequence PRO339 cI1, wherein SEQ ID 130:337 is a clone designated herein as "UNQ229 or "IIM43466-1225".

Figure 118 shows the amino acid sequence (SEQ ID NO:338) derived froman the coding sequence of SE2 ID NO: 337 shown in Figure 117.

Figure 119 shows a nucleotide sequence (SE ID 10:339) of a native sequence PRO310 crM. wherein SPM ID M1:339 is a clone designated herein as UN,1273" or "IE43046-1225".

Figure 120 shows the amino acid sequence (SEQ ID 13:340) derived fron the coding sequence of SEQ ID NO: 339 shown in Figure 119.

Figure 121 shows a nucleotide sequence (SEQ ID N13:375) of a native sequence PR0244 cUXA, wherein SEY ID 10:375 is a clone designated herein as "UNQ2181 or "1N35668-1171".

Figure 122 shows the amino acid sequence (SEQ ID 10:376) derived fran the coding sequence of SEQ ID NO:375 shomn in Figure 121.

E 3 SEcRIPTIO OF T13E PREFERRED EMBODIMEN I. Definitions The terms %PRO polypeptide" and 'PRO as used herein and when immediately followed by a nurerical designation refer to various polypeptides, wherein the carplete designation PRO/nurber) refers to specific polypeptide sequences as described herein. The tensr "PRO/nurber polypeptide" and PRO/rnuxber" as used herein encarpass native sequence polypeptides and polypeptide variants (which are further defined herein). The PRO polypeptides described herein may be isolated fra a variety of sources, such as fron human tissue types or frcm another source, or prepared by reccnbinant or synthetic mrethods.

A "native sequence PRO polypeptide- ccrprises a polypeptide having the sarre amino acid sequence as the corresponding PRO polypeptide derived froman nature. Such native sequence PRO polypeptides can be isolated fran nature or can be produced by recambinant or synthetic mreans. The term "native sequence PRO polypeptide" specifically enccarpasses naturallyoccurring truncated or secreted forms of the specific PROC) polypeptide and extracellular domain sequence), naturally-occurring variant forms of alternatively spliced forms) and naturally-occurring 61a allelic variants of the polypeptide. In various enbodinents of the invention, the native sequence PRO211 is a mature or full-length native sequence PRO211 polypeptide canprising amino acids 1 to 353 of Figure 2 (SEQ ID NO:2), the native sequence PRO217 is a nature or full-length native sequence PR0217 polypeptide caoprising amino acids 1 to 379 of Figure 4 (SEQ ID ND:4), the native sequence PRO230 is a mature or full-length native sequence PRO230 polypeptide carprising amino acids 1 to 467 of Figure 6 (SEQ ID ND:12), the native sequence PR0232 polypeptide is a mature or fulllength native sequence PRO232 polypeptide ccaprising amino acids 1 to 114 of Figure 9 (SEQ ID ND:18), the native sequence PRO187 is a mature or fulllength native sequence PR0187 ccrprising amino acids 1 to 205 of Figure 11 (SEQ ID N0:23), the native sequence PRO265 polypeptide is a mature or fulllength native sequence PRO265 polypeptide is an extracellular damain of the full-length PRO265 protein, wherein the putative transmeabrane drmain of 15 the full-length PRO265 protein is encoded by nucleotides beginning at nucleotide 1969 of SEQ ID N0:31, the native sequence PR0219 polypeptide is mature or full-length native sequence PRO219 polypeptide crprising amino acids 1 to 915 of Figure 15 (SEQ ID NO:34), the native sequence PRO246 is a mature or full-length native sequence PRO246 polypeptide ccnprising amino 20 acids 1 to 390 of Figure 17 (SEQ ID NO:39) or the native sequence PRO246 polypeptide is an extracellular damain of the full-length PR0246 protein, wherein the putative transnmabrane domain of the full-length PRO246 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 native sequence PR0228 polypeptide carprising amino acids 1 to 690 of Figure 19 (SEQ ID NO: 49) or the native sequence PRO228 polypeptide is an extracellular domain of the full-length PR0228 protein, the native sequence PR0533 is a mature or full-length native sequence PRO533 carprising 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 PRO245 polypeptide is a mature or full-length native sequence PRO245 polypeptide comprasinon acids Ito 312 of Figure 24 (SEQ MD NO:64), thc native sequence of each PR0220. PR0221 and PR0227 polypeptides is a mature or full-lengt native sequence PR0220. PR0221 and PR0227 polypeptide cmprismg amincids I through 708of Figure 26 (SEQ ID NO:69), I through 259of Figure 28(SEQ ID NO:7 1), and 1 through 62D of Figure 30 (SEQ ID NO.73). the native sequence PRO258 po4lyppde hs a mature or ful-length native sequepce PR0258 polypeptde comprising amino acids I to 398 of Figure 32 (SEQ ID NO:84) or dhe native sequece PRO258 polypepdde Is an emtaceliular domain of the full-length PR0258 protein, wherein die putative transineinranc domain of the fuli-length PR0258 protein is enicoded by nucleoddes beginning at nucleotide 1134 of SEQ ID NO:83. the native sequence PR0266 polypeptide is a -aure or filcgth native sequence PR0266 polypeptide comprising amino acids I to 696 of Figure 34 (SEQ ID NO:91) or the native sequec PR0266 polypeptide is an extraccihular domain of the ful-~cth PR0266 protein, wherein the putative transmembrane domain of the L1-lcogth PRO266 protein i encoded bymaucleotides beginning at about nuicleotide 2009 of SEQ MD NO: 104.

the native eece PR0269 polypptide is a mture or full-lengtht native sequence PR0269 polypepde comprising aminm acids I to 490 of Figure 36 (SEQ MD NO:96) or doe native sequence PR0269 polypeptide is an eztracellular domain of die full-length PR0269 protein, wherein the putative transmbrae domain of the full-length PR0269 protein is encoded by nicleotides; beginning at nucleotide 1502 as shown in Figure 35. the native seqluece~ PRO28 polypeptide is a mature or ful-length native seque PR0287 polypeptide comprising amino acids 1 to 415 of Figure 38 (SEQ ID NO: 104), dhe native sequence PR0214 is a mature or full-length native sequence PR0214 comprising amino acids 1 to 420 of Fig. 40 (SEQ ID NO: 109). the native-sequence PR0317 is a full-length native-pre-sequenice: PR0317 comprising amin acids 1 to 366 of Fig. 42 (SEQ ID 140:114) or a mature native-sequence PRO317 20 comprising amino acids 19 to 366 of Fig. 42 (SEQ ID NO.:114), the native sequence: PRO301 is a mature or fulll0 ength native sequence PRO301 comprising amino acids 1 to 299 of Fig. 4 (SEQ ID) NO: 119). with or without the N-terminal signal sequence, with or without die initiating ruethionine at position 1. with or without the potential transmembrane domain at position 236 to about 258. and with or without the intracellular domain at about position *.:259 to 299. the native sequence PR0224 polypeptide is a mature or full-length native sequence PR0224 polypeptide comprising amino acids 1 to 282 of Figure 46 (SEQ ID NO:127). doe native sequence PRO=2 polypeptide is a mature or full-length native sequence PR0222 polypeptide comprising amino acids I to 490 of Figure 48 (SEQ ID N0 .132). the native sequence PR0234 is a mature or fluli-letigth native: sequence novel lectin comprising amino acids I to 382 of Fig. 50 (SEQ ID NO: 137), the native sequence PR023 1 polypeptide is a mature or fuall-length native sequecec PR0231 polyptdec comprising amino acids 1 to 428 of Figure 52 (SEQ ID NO: 142). the native sequence PRO229 polypeptde is a matue or fil-Imgth native sequence: PR0229 polypeptdec comprising amin acids I to 347 of Figure 54 (SEQ ID NO:148). the native sequence PR0239 polypeptide is a mature or full-length native sequence PR0238 polypeptide comprising amino acids I to 310 of Figure 56 (SEQ IM NO: 153), the native sequence PR0233 polypeptie is ammmr rful-length nativesequ=PR0233 polypeptide comprisin~g amin acids 1 to 300of Figure 58 (SEQ ID NO: 159). the native sequence PR0223 polypeptde is a mature or full-length native sequence PR0223 polypeptide comprising amio acids I to 476 of Figure 60 (SEQ ID NO:164), the native sequence PR0235 polypeptde is a mature or Mlencgth native sequeocc PR0235 polypeptide comprising amino acids I to 552 of Figure 62 (SEQ U) NO: 170), the native sequence PR0236 polypeptide 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 carprising amino acids 1 to 654 of Figure 66 (SEQ ID N:177), the native sequence PR0239 polypeptide is a mature or full-length native sequence PR0239 polypeptide ccaprising amino acids 1 to 501 of Figure 68 (SED ID NO:185), the native sequence PRO257 polypeptide is a mature or full-length native sequence PR0257 polypeptide carmprising amino acids 1 to 607 of Figure 70 (SEQ ID N3:190) or the native sequence PRO257 polypeptide is an extracellular dcmain of the full-length PRO257 protein, wherein the putative transmrbrane daain 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 nmature or full-length native sequence PRO260 polypeptide cacprising amino acids 1 to 467 of Figure 72 (SEQ ID NO:195), the native sequence PRO263 polypeptide is a mature or full-length native sequence PRO263 polypeptide caiprising amino acids 1 to 0 o. 15 322 of Figure 74 (SEQ ID N: 201) or the native sequence PRO263 polypeptide is an extracellular dnarain of the full-length PRO263 protein, wherein the putative transmaxtrane darain of the full-length PR0263 protein is encoded by nucleotides beginning at nucleotide 868 of SEQ ID ND:200, the native sequence PRO270 polypeptide is a mature or full-length native sequence 20 PRO0270 polypeptide ccrprising amino acids 1 to 296 of Figure 76 (SEQ ID ND:207), the native sequence PR0271 polypeptide caiprising amino acids 1 to 296 of Figure 76 (SEQ ID ND:207), the native sequence PRO271 polypeptide is a nmature or full-length native sequence PRO271 ccrprising amino acids 1 to 360 of Figure 78 (SEQ2 ID N):213), the native sequence PR0272 polypeptide is a mature or full-length native sequence PR0272 polypeptide carprising amino acids 1 to 328 of Figure 80 (SEQ ID NO:221), the native sequence PRO294 0polypeptide is a mature or full-length native sequence PiRO294 polypeptide carprising 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 ccarprising amino acids 1 to 350 of Figure 84 (SEQ3 ID NO:236), the native sequence PRO293 polypeptide is a nature or full-length native sequence PR0293 polypeptide ccarprising amino acids 1 to 713 of Figure 86 (SEQ ID ND:244) or the native sequence PRO293 polypeptide is an extracellular domain of the full-length PR0293 protein, wherein the putative transmabrane danain of the full-length PRO293 protein is encoded by nucleotides beginning at nucleotide 2771 of SE3 ID NO:243, the native sequence PRO247 polypeptide is a mature or full-length native sequence PRO)247 polypeptide carprising amino acids 1 to 546 of Figure 88 (SEQ ID NI:249), the native sequence PRO302 polypeptide is a nmature or full-length 64 native sequence PRO302 polypeptide caprising amino acids 1 to 452 of Figure 90 (SED ID NO:254), the native sequence PR0303 polypeptide is a nature or full-length native sequence PR0303 polypeptide carprising amino acids 1 to 314 of Figure 92 (SEQ ID NO:256), the native sequence PRO304 polypeptide is a nature or full-length native sequence PRO304 polypeptide carcprising amino acids 1 to 556 of Figure 94 (SEQ ID ED:258), the native sequence PR0307 polypeptide is a mature or full-length native sequence PRO307 polypeptide caprising amino acids 1 to 383 of Figure 96 (SEQ ID NO:260), the native sequence PRO343 polypeptide is a nature or full-length native sequence PR0343 polypeptide carcnprising amino acids 1 to 317 of Figure 98 (SEQ ID ND:262), the native sequence PRO328 polypeptide is a nature or full-length native sequence PR0328 polypeptide ccnprising amino acids 1 to 463 of Figure 100 (SEQ ID N3:284) or the native sequence PR0306 polypeptide is an extracellular darrin of the full-length PRO306 protein, wherein the putative extracellular darain of the full-length PR0306 protein, the native sequence PRO335 polypeptide is a nature or full-length native sequence PR0335 polypeptide carprising amino acids 1 through 1059 of Figure 102 (SEQ ID N:289), the native sequence PR0331 polypeptide is a nature or full-length native sequence PR0331 polypeptide carprising amino 20 acids 1 through 640 of Figure 104 (SED ID 1N:291), the native sequence PRI326 polypeptide is a mature or full-length native sequence PR0326 *too polypeptide canprising amino acids 1 through 1119 of Figure 106 (SE2 ID too* NO:293), wherein additional erbodinents include wherein the transmanbrane *0 -regions are deleted or the peptides are truncated, so as to not include the transmrane regions for each of PR0335, PRO331, and PR0326, the native sequence PRO332 is a nature or full-length native sequence PRO332 caprising amino acids 49 to 642 of Figure 180 (SD ID ND:309), with or without the N-terminal signal sequence, and with or without the initiating nethionine at position 1, the native sequence PRO334 polypeptide is a nature or full-length native sequence PR0334 polypeptide carprising amino acids 1 to 509 of Figure 110 (S2EQ ID D:314), the native sequence PRO346 is a mature or full-length native sequence PRO0346 carprising amino acids 19 to 339 of Figure 112 (SEQ ID ND:319), with or without the N-terminal signal sequence, with or without the initiating nethionine, with or without the transmrbrane dciain at positions 340 to 360 and with or without the intracellular doain at positions 361 to 450, the native sequence PRO268 polypeptide is a mature or full-length native sequence PRO268 polypeptide camprising amino acids 1 to 280 of Figure 114 (SED ID N3:324) or the native sequence PRO268 polypeptide is an extracellular damin of the full-length 64a PRO268 protein, wherein the putative tran~artrane dnmain of the fulllength PRO268 protein is encoded by nucleotides beginning at nucleotide 559 as shown in Figure 113, the native sequence PRO330 polypeptide is a mature or full-length native sequence PRO330 polypeptide ccaprising amino acids 1 to 533 of Figure 116 (SEQ ID N0:331), the native sequence PRO339 polypeptide is a mature or full-length native sequence PRO319 polypeptide conprising amino acids 1 -to 772 of Figure 118 (SEQ ID NO:338), the native sequence PRO310 polypeptide is a mature or full-length native sequence PRO310 polypeptide ccnprising amino acids 1 to 318 of Figure 120 (SEQ ID N0:340) and the native sequence PRO244 is a mature or full-length native sequence PRO244 caprising amino acids 1 to 219 of Figure 122 (SEQ ID SND:376), wherein the nature, full-length native sequence PRD244 protein carprises a cytoplasmic dtnain (about amino acid positions 1 to 20), a ransmerbrane domain (about amino acid positions 21 to 46), and an 15 extracellular drmain (about amino acid positions 47 to 219). Within the extracellular domain, the C-lectin daomain is between about amino acid positions 55 and about amino acid position 206. Native sequence PR0244 as shown in Figure 122 naps to chraosome 12, bands p12-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 o 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.

*W e 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 nore preferably at least about 95% amino acid sequence identity, with the amino acid sequence of the full-length native l amino acid sequence as disclosed herein.

30 "PR0317 variants" or 'PR0317 sequence variants" as defined herein mean biologically active PRO317s as defined below having less than 100% sequence identity with the PRO317 isolated fran recambinant cell culture or fromn mamalian 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 more preferably at least about 80%, still nore preferably at least about 85%, even more 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 PR0317 fragments and glycosylation variants thereof. PR0317 fragments have a consecutive sequence of at least 10, 15, 20, 25, 30, or 40 amino acid residues, preferably about 10-150 residues, that is identical to the sequence of the PR0317 shown in Figure 42.

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

A "chimeric PR03170 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 PR0317. The second protein will typically by a cytokine, growth factor, or hormone such as a neurotrophic or angiogenic factor such as GDNF or VEGF, or another member of the TGF-superfamily such as EBAF-1. Another exemplary preferred *0 PR0317 chimera is a "domain chimera" that consists of the Nterminal residues-substituted with one or more, but not all, of the residues of the human EBAF-I. In this embodiment, the PR0317 chimera would have individual or blocks of residues from the human EBAF-l sequence added or substituted into the PR0317 sequence. For example, one or more of those segments of EBAF-1 that are not homologous could be substituted into the corresponding segments of PR0317. It is contemplated that this "PR0317-EBAF-1 domain chimera" will have an agonist biological activity.

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

"Percent nucleic acid sequence identity" with respect to PRO-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in the PRO sequence of interest which are identical with the nucleotides in a candidate nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of ~determining percent nucleic acid sequence identity can be 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.

20 "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 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 internal amino acid sequce by use of a spinning cup sequenator. or to homogeneity by SDS-PAGE under nonreducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypcptide includes polypeptide in siu within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypcpide will be prepared by at least one purification step.

An "isolated" PRO polypeptide nucleic acid is a nuclcic acid molecule that is identified and separated from at least one contaminat ucleic acid molecule with which it is ordinarily associated in the natural source of the PRO polypeptide nucleic acid. An isolated PRO polypeptide nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated PRO polypcptide nuclic acid molecules therefore are distinguished from the specific PRO polypcptide nucleic acid molecule as it exists in natural ccls. However, an isolated PRO polypeptide nucleic add molecule includes PRO polypeptide nucleic acid molecules contained in cells that ordinarily express the PRO polypeptidc 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 restriction cndanuclease digest of DNA or a DNA-comaining composition is confirmed by hybridization to a known, labeled oligonucleotide or DNA fragment. Southern analysis typically involves electrophoretic separation of DNA 15 digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nirocelhilose, nylon or another suitable membrane support for analysis with a radiolabeled, biotinylated, or enzymelabeled probe as described in sections 937-9.52 ofSambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press. 1989).

'Northern analysis' or "Northern blotting" is a method used to identify RNA sequences that hybridize to a known probe such as an oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment. The probe is labeled with a radioisotope such as 2P, or by biodinylation, 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 et al., supm.

25 The term control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic add 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 oligonuclcotide adaptors or linkers are used in accordance with conventional practice.

The term 'antibody' is used in the broadest sense and specifically covers single ant-PRO polypeptide monoclonal antibodies (including agoist, antagoist, and neutralizing 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 arc identical except for possible natrally-occuring mutations that may be present in minor amounts.

"Active* or "activity" for the puposes herein refers o form(s) of PRO polypeptide which fetain the biologic and/or immunologic activities of the specific native or narally-occurring PRO polypcptide. The activity of a PR0332 polypeptide 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 underexreessed. Such disorders include diseases of the female genital tract or of the endometrium of a mammal, including hyperplasia. endometris, endometriosis, wherein the patient is at risk for infertility due to endometrial factor, endomctrioma, and endomtrial cancer, especially those diseases involving abnormal bleeding such as a gynecological disease. They also include diseases involving angiogensis, wherein the anglogenesis results in a pathological condition, such as cancer involving solid tumon (the therapy for the disorder would result in decreased vascularization and a decline in growth and metastasis of a variety of tumors). Alternatively, the angiogenesis may 15 be beneficial, such as for ischemia, especially coronary ischemia. Hence, these disorders include those found in patients whose earts 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 Sinvolving the kidney or originating from the kidney tissue, such as polycystic kidney disease and chronic and acute renal failure.

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

"i :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 glycinc, glutamine, asparagine, arginine or lysine; monosacchardes disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannltol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEi", polyethylene glycol (PEG). and PLURONICS T m The term "agonist" is used to refer to peptide and non-peptide analogs of the native PRO polypcptidcs (where native PRO polypeptide refers to pro-PRO polypeptide, pre-PRO polypeptide, prepro-PRO polypeptide, or manture PRO polypeptide) of the present invention and to antibodies specifically binding such native PRO polypeptides. provided that they retain at least one biological activity of a native PRO polypeptide. Preferably, the agonists of the present invention retain the qulitative binding recognition properties and receptor activation propertes of the native PRO polypeptide.

The term *antagonist* is used to reficr wo a molecule inhibiting a biological activity of a native PRO polypeptide of the present invention wherein native PRO polypeptide refers to pro-PRO polypeptide. pre-PRO polypeptide. prepro-PRO polypeptide. or mature PRO polypeptide. Preferably, the antagonists herein inhibit the binding of a native PRO polyped of the present invention. Preferred antagonists essentially completely block die binding of a native PR0317 polypeptde to a PR031,7 polypeptide receptor to which i otherwise binds. Such receptors may include the Type I and Type and possibly Type MI receptors identified for the TOF- superfamily.

KolodzieJczyk and Hall, srepra. 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 polypeptide). Such molecules can be screened for their ability to competitively inhibit PRO polypeptide receptor activation by mnonitoring binding of native PRO polypeptdec in tie 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 polynucleocide blocks transcription or translation of the PRO polypptde gene, thereby inhibiting its expression and biological activity.

"Stringent conditions" means employing low ionic strength and high temnperature for washing, for example, 0.015 sodium cbloride/0.0OlS M sodium eitrateff.1 sodium dodecyl sulfate at SO*C. or employing during hybridization a denatuing agent, such as farnumide, for example, 50% (vollvol) formamnide with 0.1 bovine serum albwninlO.lI% FieollIO.1 polyvinylpyrrolidone/50 old sodium phosphate buffer at pH 6.5 with 750 mM :sodium chloride, 75 miM sodium citrate at 42'C. Another example is usc of 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate). 50 mM sodium phosphate (pH 0.1 sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (SO pg/l). 0. 1% SDS, and 10% dextran sulfate at 42'C. with washes at 42*C in 0.2 x. SSC and 0.1 SDS. Yet another example is hybridization using a buffer of 10% dextra sulfate. 2 S x SSC (sodium diloridedsodium cuam) and 50% formamide at 55*C. followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 550C.

'Moderately stringent conditions* ame described in Sambrook et al., szqra, and include the use of a washing solution and hybridization conditions temperature, ionic strength, and %SDS) less stringent than described above. An example of moderately stringent conditions is a condition such as overnight incubation at 37*C in a solution compnisii. 20% foriramide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL 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 accommodate; factors such as probe length and the like.

II. Cormpositions mnid Methiods of the byewntion- 1. FalI-length PR0211 and PR0217 Polypcntides The present invention provides newly identified and isolated nueceotide sequences encoding polypetides referred to in t present application as PRO211I and PRO2 17. In particular, Applicants have identi fied and isolated cDNA encoding PRO211 and PRO217 polypeptides. as disclosed in further deti in th Eamls below. Using BLAST (FastA format) seqluence alignment computer programs. Applicants found that cDNA sequences encoding full-length native sequence PRO211 and PR0217 have homologies to known proteins having EGF-like domains.

Specifically, the cDNA sequence DNA32292-1 131 (Figure 1, SEQ ID NO:I) has 36 identify and a Blast score of 209 with PAC6 -RAT and 31 identify and a Blast score of 206 with Fibulin-l. *isoform c precursor. The cDNA sequence DNA33094-1131 (Figure 3. SEQ ID NO:3) has 36% identity and a Blast score of 336 with eastern newt tenascin, and 37% identity and a Blast score of 331 with human tenaseip-X precursor. Accordingly, it is presently believed that PRO21 1 and PR0217 polypeptides disclosed in the present application are newly identified members of the EGF-like family and possesses properties typical of the EGF-ike protein family.

2. Full-length PR0230 Povoentides The present invention provides newly identified and isolated nucleotide sequences encoding polypetides referred to in the present application as PROM3. In particular, Applicants have identified and isolated cDNA encoding a PR0230 polypqxtide. as disclosed in further detail in the Examples below. Using known programs such as BLAST andi FastA sequence alignment computer programs. Applicants found that a cDNA sequemc encoding fulllength native sequence PR0230 has 48% amino acid identity with the rabbit tubulointerstitial nepliritis antigen *20 precursor. Accordingly. it is presently believed that PR0230 polypeptide disclosed in the present application is a newly identified member of the tuuointerstitial nephiritis antigen family and possesses the ability to be recognized by human autoantibodies in certain forms of tubulointerstitial nephritis.

3. Full-lenpth PR232 Poloentides The present invention provides newly identified and isolated nudleotide sequences encoding polypeptides referred to in the present application as PR0232. In particular. Applicants have identified and'isolated eDNA encoding a PR0232 polypeptde,. as disclosed in further detail in te Examples below. Using BLAST and FassA sequence alignment computer programs, Applicants found that a portion of the full-length native sequence PR0232 (shown in Figure 9 and SEQ ID NO:I18) 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-length PRO187 Pftveptides The present invention provides newly identified and isolated nucleotide sequences encpding polypeptides referred to in the present application as PR0187. In particular. Applicants have identified and isolated cDNA encoding a PRO187 polypeptide. as disclosed in furthe detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants ftini that a fulllengh native sequence PRO 187 (shown in Figure has 74% amino acid sequence identity and BLAST score of 310 with various andzngcnminduced growth factors and FGF-8. Accordingly. it is presently believed that PRO 197 polypeptide disclosed in thc present application is a newly identified member of the FGF-8 protein family and may possess identify activity or property typical of the FGF-8-Iikc protein family.

5. Full-lend'h -PR0265 Polynnide Mie present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in dhe present application as PR0265. 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 conmtr programs, Applicants found that various portions of the PR0265 polypeptide have significant homology with die fibroniedulin protein and fibromiodulin precuror protein. Applicants have also found din the DNA encoding dhe PR0265 polypeptide has significant homology with platelet glycoprotein V. a member of the leuciu rich related protein family involved in shin and wound repair. Accordingly. it Is presently believed that PR0265 polypeptide disclosed in the present application is a newly identified member of the leucine rich repeat family and possesse protein protein binding capabilities, as well as be involved in sktin and wound repair as typical of this family.

6. Ful.Iennth PR0219 Pobvpntldes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0219. 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 programs. Applicants found that various portions of the PR0219 polypeptide have significant homology with the mousec and human matrilin-2 precursor polypeptides. Accordingly. it is presently *,**,*believed that PR0219 polypeptide disclosed in the present application is related to the nuatrilin-2 precursor polypeptide.

7. Full-lenrth P!R0246 Polvuptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROM4. In particular. Applicants have idntified and isolated cDNA encoding a PR0246 polypeptide, as disclosed in firthe detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that a portion of the PR0246 polypeptide has significant homology with the lazm cell surface Protein HCAR. Accordinly it is presently believed that PR0246 polypeptide disclosed in t present application may be a newly identified uembrane-boundl virus receptor or tumor cell-specific antigen.

8. Full-lenrh ZR0228 P oftde The present invention provides newly identified and isolated nucleotide sequence encoding POlYPeptides referred to in the present application as PR022s. 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 programs, Applicants found that various partions of the PR0228 polypeptide have significant homology with the EMRl protein. Applicants have also found that the DNA encoding the PR0228 polypeptide has significant honmology with lacrophilin. muerophage-resericted cell surface glycoprotein. B0457.1 and leucocyte andgen CD97 precursor. Accordingly. it is presently believed that PR0228 polypeptide disclosed in the present applicatin is a newly identified member of the seven transmembrane superfamnily and possesses characteristics and funrctional propertics typical of this henily. In particular, It is believed that PR0228 is a new member of the subgroup within this family to which CD97 and EMR1 belong.

9. Fuflleng-th PRO533 Polypeptides ihe present invention provides newly identified and isolated nmeloide sequences encoding polypeptides referred to in the present application as PROM3. In particular. Applicants have identilied and isolated cDNA axenoding a PRO533 polyetide, as disclosed in further detail in the Examles below. Using BLAST-2 and FastA sequence aignmaz computer progrs. Applicants found that a full-length native sequence PR0533 (shown in Figure 22 ZandSEQ ID NO.59) has a Blast score of 509 and 53 amino acid sequence identty with fibroblast growth factor 15 (FGP). Accordingly, It is presently believed that PR0533 disclosed in the present application is a newly identified member of the fibroblast growth factor aily and may possess activity typical of such polypeptides.

10. Fufl-Iensgh FROM4 PolynetideS ***The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROM4. In particular. Applicants have identified and isolated cDNA encoding a PR0245 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequemc al;gaz computer programs. Applicants found that a portion of the amino acid sequence of the PR0245 polypeptide has 60% amino acid identity with the human c-myb protein. Accordingly. it is presently believed that the PRO245 polypeptide disclosed in the preset application may be a newly identified member of the transmnembrane protein tyrosine kinase family.

11. Full-length PR020 PR22 nd PROM2 Polvnentides The present invention provides newly Identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0220. PR0221 and PR0227. In particular. Applicants have identified and isolated cDNAs encoding a PR0220, PR0221 and PR0227 polypeptide. respectively, as disclosed in further detall in the Examples below. Using BLAST andl FastA sequence alignment computer programs, PR0220 has amn acid identity with the amin acid sequence of a laticine rich protein wherein the identity is 87 PR0220 additionally has a-in acid identity with the neuronal Icucioe rich protein wherein the identity is 55 The neuronal leucine rich protein is futher described in Taguchi, ez al., Mg1.JjMia.R=. 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 PR0227 has amino acid identity with dhe armino acid sequence of platelet glycoprotein V precursor. Thc same results were obtained for humnan glycoproein 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 PRO220, 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-proteiii binding capabilities typical of die leucine rich repeat protein supeffamily. h t aalso believed that they have capabilities simnila to those of SLIT. the leucine rich repeat protein and human glycoprotein V.

12. Fufl4erngth PRO258 Pobvptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in dhe present application as PR0258.. In particular, Applicants have identified and isolated cDNA encoding a PRO2S8 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence aliginent computer programs, Applicants found that various portions of the PR0258 polypeptide have signlfi-am hoemology with the CRTAM and poliovirus receptors. Accordingly, it is presently believed that PR0258 polypeptide disclosed in the piesenst application is a newly identified member of the Ig superfamily and possesses virus receptor capabilities or regulates immuine function as typical of this family.

13. Full4erwth PR0266 Polvnentides The present invention provides newly identified and isolated nucleoide 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 various portions of die PR0266 polypeptide have significant homlogy with the SLIT protein from Drosophilia. Accordingly, it is presently believed that PR0266 polypeptide disclosed in the present application is a newly identified member of the leucine rich repeat family and possesses ligand-ligand binding activity and ncuronal developmet typical of this family. SLIT has been shown to be useful in the study and treatment of Alzheimner's disease, supra, and thus, PR0266 may have involvement in the study and cure: of this disease.

14. Full-length PRO269 Poynentides The present invention provides newly identified and isolated nucleotide sequences encoding polypcptidcs referred to in the present application as PR0269. In particular, Applicants have identified and isolated cDNA encoding a PR0269 polypeptide, as disclosed in fturthr detail in the Examples below. Using BLAST. FastA and sequence alignment computer programs. Applicants found that the amino acid sequence encoded by nucleotides 314 to 1783 of the full-length native seaqnc PRO269 (shown in 35 and SEQ ID NO:95) has significant homnology to human urinary thrombomodulin and various thromboniodulin analogues respectively, to which it was aligned.

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

is. Full~leneth PR0297 PolvtIetides The present invention provides newly identified and isolated nrucleotide sequences encoding polypeptides referred to in the present application as PROW9. In particular. Applicants have identified and isolated cDNA encoding a PRO28 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 PROW9 polypeptide have significant homology with the type I procoliagen C-proteinase enhancer protein precursor and type I procollagen Cproteinase enhancer protein. Accordingly, it is presently believed tham PRO28 polypeptide disclosed in the present application is a newly identified member of the C-proteinase enhancer protein family.

16. Pullength ER0214 Poblvetides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0214. In particular, Applicants have identified and isolated cDNA encoding a PR0214 polypeptide, as disclosed in further detail in dhe Examples below. Using BLAST and FastA.

sequence alignment computer programs. Applicants found that a full-length native sequence PR0214 polypeptide (shown in Figure 40 and SEQ IM NO: 109) has 49% amino acid sequence identity with HT protein. a known member of the EGF-family. The comparison resulted in a BLAST score of 920. with 150 matching nucleouides. Accordingly, it is presently believed that the PR0214 polypeptide disclosed in the present application is a newly identified member of the family comprising EGF domains and may possess activities or properties typical of the EGF-domain containing x: 20 f al.17. Pall-lengjth PR0317 Polvoetildes The present invention provides newly identified and isolated micleodde sequences encoding polypeptides referred to in the present application as PROM1. In particular. eDNA encoding a PR0317 polypeptide has been identified and isolated. as disclosed in fuirther detail in the Examples below. Using BLASTI' and FastA' sequence alignment comuputer programs. it was found that a full-length native-sequence PR0317 (shown in Figure 42 and SEQ ID NO: 114) has 92% amino acid sequence identity with EBAF-1. Further. it is closely aligned with many other members of the TGF- superfamily.

Accordingly, it is presently believed that PRO3 17 disclosed in the present application is a newly identified member of the TGF- superfamily and may possess properties thag are therapeutically useful in conditions of uterine bleeding. etc. Hence, PR0317 may be useful in diagnosing or treating abnormal bleeding involved in gynecological diseases, for example. to avoid or lessen the need for a hysterectomy. PR0317 may also be useful as an agent that affects angiogenesis in general, so PR0317 may be useful in anti-tumor indications, or conversely, in treating coronary ischemic conditions.

Library sources reveal that ESTs used to obtain the consensus DNA for generating PR0317 primers and probes were found in normal tissues (uterus, prostate, colon. and pancreas), in several tomors (colon, brain (twice), 35 pancreas, and mullerian cell), and in a heart with iscbemia. PR0317 has shown up in several tissues as well, but it does look to have a greater concentration in uterus. Hence, PR0317 may hive a broader use by the body than EBAP-l. It is contemplated that, at least for some indications. PR0317 nay have opposite effects from EBAF-1.- 1s. Full-length PRO301 Polvuentjde The present knton provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0301. In particular, Applicants have identified and isolated cDNA encoding a.PR0301 polypeptide, as disclosed in furthier detail in the Examples below. Using BLAST and PastA sequence alignxmnt comiputer pograms, Applicants hind diat a full-length native sequence PRO301 (shown in Figure: 44 andSEQ ID NOA9) hasa Blast score of246corresponingto 30% amiino acid sequence ldeatity with human A33 antigen precursor. Accordingly, it is-presently believed thai PRO301 disclosed in the present application is a newly ktnuiffed mber of the A33 antigen protein family and may be expressed in human neoplastic diseases such as colorctal cancer.

19. Fufllerwth PRO224 PolXoentides; The present invention provides newly identified and isolated mucleotide sequences encoding polypeptides referd to in the present application as PR0224. In particular, Applicants have identified and isolated cDNA encoding a PRC)224 polypeptide. as disclosed in further detail in the Examples below. Using known programs such as BLAST and FastA sequence alignment computer programs, Applicants found that full-length native PR0224 (Figure 46. SEQ ID NO:127) has amino acid identity with apolipoprotein E receptor 2906 from homo sapiens. The aligazments of different portions of these two polypeptides show amino acid identities of 37%, 36%, 30%. 44%, 44% and 28% respectively. Full-length native PR0224 (Figure 46. SEQ ID NO:127) also has amino acid identity with very low-density lipoprotein receptor precursor from gall. Thec alignmnts of different portions of these two polypeptides show amino acid identities of 38%, 37%, 42%, 33%. and 37% respectively. Additionally, full-length native PR0224 (Figure 46, SEQ ID NO: 127) has amino acid identity with the chicken oocyte receptor P95 from Callus gallus. The alignments of different portions of these two polypeptides show amino acid identities of 38% 42%, 33%, and 37% respectively. Moreover, fuI-length native PR0224 (Figure 46, SEQ In) NO:127) has amino acid identity with very low density lipoprotein receptor short form precursor from humans. The alignmenits of different portions of these two polyppcs show amn acid identities of 32%, 38%, 34%, 45%. and 31%, 25 respectively. Accordingly, it is presently believed that PR0224 polypeptide disclosed in the present application is a newly identified nubrof the low density lipoprotein receptor family and possesses the structural, characteristics required to have the funrctional ability to recognize and endocytose low density lipoproteins typical of the low density lipoprotein receptor family. (Mc alignments described above used the following scoring parameters: T=7. S S2=36, Matrix BLOSUM62.) Full-leng'th PRO222 PovuetIdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0222. In particular, Applicants have identified and isolated cDNA encoding a PR0222 polypeptide. as disclosed in fiuiher detail i the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that a sequence encoding full-length native sequence PR0222 (shown in Figure 48 and SEQ ID NO: 132) has 25-26 amino acid identity with mouse complement factor h precuror. has 27-29% amino acid identity with complement receptor, has 25-47% amino acid identity with mouse complement COb receptor type 2 long form precursor. has 40% amnino acid identity with human hypoteical protein kiaaO247. Accordingly, it is presently believed that PR0222 polypeptide disclosed in dhe present application is a newly identified memnber of the cmqplerrza rceptor family and possesses activity typical of the conpleanent receptor family.

21. FulIlength PRO234 Polypeotdet The present invention provides newly identified and isolated nucleoade sequences encoding polypepuides referred to in the present application as PR0234. In particular, Applicants have identified arnd isolated cDNA encoding a PR0234 polypepRic. as disclosed in further detail in the Examples below. Using BLAST (FastA-forniat) sequence alignmn computer programs. Applicants foux that a cDNA sequnce encoding full-length native sequence PR0234 has 31 identity and Blast score of 134 with E-Weectin precursor. Accordingly, it is presently believed that the PR0234 polypeptdecs disclosed in the present application are newly identified members of the lectin/selectin family and possess activity typical of the lectin/selectin family.

22. Full-leng~th PRO231 Polvuentides The present invention provides newly identified and isolated nucleodde 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 sequence alignment computer program, Applicants found that the full-length native sequence PRO231 polypeptide (d*w inRuc5 n E O 4)hs3 and 31 %amnino acid identity with human and rat prostatic acid phosphatase precursor proteins, respectively. Accordingly, it is presently believed that the PR023 1 polypeptide disclosed in the present application may be a newly identified member of the acid phosphatase protein family.

23. Full-Iength PR10229 Polypfide The present invention provides newly identified and isolated nucleoide sequences encoding polypeptides referred to in the present application as PR0229. In particular, Applicants have identified and isolated cDNA C 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 significant homology with antigen wcl.l, M130 antigen. T cell surface glycoproteint CD6 and CD6. It also is related to Sp-alpha. 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 imnmune function and thus possesses immune function and /or segment which resist degradation, typical of this family.

24. Full-ength PROZadPolynetides The present invention provides newly identified and isolated nrucleotide sequences encoding polypeptides referred to in the present application as PROM3. In particular, Applicants have identified and isolated cDNA encoding a PR0239 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignmnent computer program. Applicas= found that various portions of the pR0238 polyepidc have significant homology with reduCtases. including oxidoreductase and fatty acCCOA reductase. Accordingly, it is presently believed that PR0238 polypeptide disclosed in the present application is a newly identi fied member of the reductase family and possesses reducing activity typical of the reductase family.

2S. Full-lenah PRO233 Pollnentdes The present invention provides newly identified and isolated. nucleoide sequences encoding polypeptides referred to in the pret application as I'R0Z33. In particular, Applicants have identified and isolated eDNA encoding a PR0233 polypeptide. as disclosed in furthcr detail in the Examples below. Using BLAST and FastA sequence-alignment computer programs, Applicants found that various portions of the PR0233 polypeptdec have significant homology with the reductase protein. Applicants have also found that the DNA encoding the PR0233 polypeptide has significant homology with proteins from Caenodwabdii elegans. Accordingly, it is presently believed doat PR0233 polypeptide disclosed in dhe present application is a newly identified member of the reductase family and possesses die ability to effect the redox state of the cell typical of the reductase: family.

26. Full-Ien~tl PR223 Povnentktes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0223. In particular, Applicants have identified and isolated cDNA encoding -a PR0223 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequaa alignment computer program. Applicants fbind that the PR0223 polypeptide has significant homology with various senine carboxypeptidase polypeptides. Accordingly, it is presently believed that PR0223 polypeptide disclosed in the present application is a newly identified serine carboxypeptidase.

S Full-length PRO235 Pol3Mcentides The present invention provides newly identified and isolated nucotide sequences encoding polypeptides; referred to In the present application as PR0235. In particular, Applicants have identified and isolated cDNA encoding a PR0235 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 PR0235 polypeptide have significant homology with the various plexin proteins. Accordingly, it is presently believed that PR0235 polypeptide 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. Fulllength PR236 and ZE0262 Poloetp-ides 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 identified and Msoated cDNA encoding PR0236 and PR0262 polypeptides, as disclosed in further detail in tie Examples below. Using BLAST and FastA sequence alignment computer program, Applicants found that various portions of the PR0236 and PR0262 polypeptidecs have significant homology wish various k-alactosidase and Il-galactosidase precursor polypepddrs. Accordingly, It is presently believed tiat the PR0236 and PR0262 polypptcs disclosed in dhe present application ame newly identified P-galaczosidase homologs.

29. Full-length PRO239 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encodling polypepirs to in the present application as PROM3. In particular. Applicants have identified Andl isolated cDl4A encoding a PR0239 polypeptide, as disclosed in further detail in dhe Examples below. Using BLAST and FastA sequence alignment computer program, Applicants found that various portions of the PR0239 polypeptide have significant homology with densin proteins. Accordingly, it is presently believed that PR0239 polypeptide disclosed in the present Application is a newly identified member of the densin famnily and possesses cell adhesion and the ability to effect synaptic processes as is typical of the densin family.

Full-lengh PRO257 Polvoetfdes IThe present invention provides newly identified and isolated maclotide sequences encoding polypeptides referred to in die present application as PROM5. In particular, Applicants have identified and isolated cDNA encoding a PR0257 polypeptide, as disclosed in further detail in dhe Examples below. Using BLAST anid FastA sequence alignment computer programs, Applicants found that various portions of the PR0257 polypeptide have significant homology with the ebnerin precursor and ebinerin protein. Accordingly. it is presently believed that PR0257 polypeptide disclosed ith de present application is a newly identified protein member which is related to the boermn protein.

31. Full-lenrth PRO260 Polympftides 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 dietail in the Examples below. Using programs such as BLAST and FastA sequence alignment computer programs. Applicants found that various portions of the PR0260 9 polypeptide have significant homology with the alpha-l-hicosidase precursor. Accordingly, it is presently believed 9.99 tht PRO260 polypeptide disclosed in die present application is a newly identified member of die fucosidase family and possesses enzymatic activity related to fuicose residues typical of the fucosidase family.

32. Fulleneth PR263 Poyneotides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PROM6. In particular, Applicants have identified and isolated cDNA encoding a PR0263 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 PRO263 polypeptide have significant homology with die CD44 artigen 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 antigenis, cancer and H[V marker, cell-cell or cellmatrix interactions, regulating cell traffic. lymph node hocning, tranmiSsion of growth signals, and presentation of ccnokines and growth facors to traveling cells.

33. Fullnt R(Y70 Pollpeptides The present invention provides newly identified and isolated nucleoide sequences encoding polypeptidcs referred to in the present application as PR0270. In particular. Applicants have identified and-isolated cDNA encoding a PR0270 polypeptide, as disclosed in further detail in the Examples below. Using BLAST. FastA and semjaie aligrment comiptter programs, Applicants found that that various portions of the PR0270 polypeptdde have significant homology with various thiorcdoxin proteins. Accordingly, it is presently believed that PR0270 polypeptide disclosed in the present application is a newly identified member of the thioredoxin family and possesses the ability to effect reduction-oxidation (redox) state typical of the diioredoxin, fasmily.

4 &so.34. Full-Ileth PRO271 Polvptides The present invention provides newly identified and isolated nucleotdec sequences encoding polypeptides referred to in the present application as PR0271. In particular. Applicants have identified and isolated cDNA encoding a PR0271 polypeptde. as disclosed in fiurr detal in the Examples below. Using BLAST and FastA sequence alignment computer program. Applicant foundi tat the PR0271 polypeptide has significant homology with various link proteins and precusors thereof. Accordingly, it is presently believed that PR0271 polypetdec disclosed an the present application is a newly identified link protein homolog.

.20 35. Full-length PRO272 Povoenides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides :referred to in the present application as PR0272. In particular. Applicants have identified and isolated cDNA encoding a PR0272 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and PastA #.so sequence alignment computer programs. Applicants found that various portions of the PR0272 polypeptide have significant homology with the human reticulocalbin protein anid its precursors. Applicants have also found that the S DNA encoding the PR0272 polypeptide has significant homology with the mous reticulocalbin precursor protein.

Accordingl, it is presently believed that PR0272 polpeptide disclosed in the present application is a newly identified member of the reticulocalbin family and possesses the ability to bind calcium typical of the reticulocalbin family.

36. FUll-leneth PR0294 Eolvpeptidios The present invention provides newly identified and isolated nucleotide sequences encoding polypptides referred to in the present application as PR0294. In particular, Applicants have identified and isolated cDNA encoding a PR0294 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that various portions of the PR0294 polypeptide have significan homology with the various portions of a nmber 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. FuII-lenith ER029S Pfbtgides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptidcs referred to in the present application as PRO29S. In particular. Applicants have identified and isolated cDNA encoding a PRO295 polypeptid. as disclosed in Further dctail in the Examples below. Using BLAST and FastA sequence lignment computer programs, Applicants found that various portions of the PR0295 polypeptide have significant homology with integrin proteins. Accordingly, it is presenitly believed that PRO295 polypeptide disclosed in the present application is a newly identified nienober of the integrin famnily and possesses cell adhesion typical of the integrin family.

38. nul-enyth PRO793 Pol=Vpeides 71ce present invention provides newly identified and isolated nurcleodde sequences encoding polypeptides referred to in the present application as PR0293. In particular. Applicants have identified and isolated cDNA encoding a PR0293 polypeptide. as disclosed in fiutr detail in dhe Examples below. Using BLAST and FastA sequence aigsmnmt computer progrzos. Applicants found that portions of the PR0293 polypeptide have significant homology with die neuronal leucine rich mepat proteins 1 and 2, (NLRR-l and NLRR-2), particularly NUAR-2.

Accordingly, it is presently believed that PR0293 polypid& disclosed in the present application is a newly identified mer of the neuronal Icucine rich repeat protein family and possesses ligand-ligand binding activity typical Of the NRIL protein family.

39. Full-leneth PR0247 Polvuotdes S20 The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0247. In particular, Applicants have identified and isolated cDNA encoding a PR0247 polypeptide, as disclosed in futher detail in the Examples below. Using BLAST and FastA sequence aliganent computer programs, Applicants found that various portions of the PR0247 polypeptide have significan homology with densin. Applicants have also found that the DNA encoding die PR0247 polypeptide has significant homology with a number of other proteins. including KIAOZ3I. Accordingly. it is presently believed d=a PR0247 polypeptide disclosed in the present application is a newly identified member of the Icucine rich repeat family and possesses ligand binding abilities typical of this family.

FuiWengilh PR0302. PR0303. PR304. PR307 and PRO343 Polnvgtdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred In in h prsn apri-mion as PR0302. PR0303. PRO304. PR0307 and PR0343. In particular, Applicants have identified and isolated cDNA encoding PR0302, PRO3o3, PR0304, PR0307 and PR0343 polypeptides. as disclosed in further detail in the Examaples below. Using BLAST and FastA sequence alignment computer programs.

Applicants found that various portions of the PR0302. PR030. PR0304, PR0307 and PR0343 polypeptides have significant homology with various protease proteins. Accordingly, it is presently believed that the PR0302. PR0303, PR0304, PR0307 and PR0343 polypeptides disclosed in the present application are newly identified proteasc proteins.

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

Further, Applicants found that various portions of the PRO328 polypeptide have significant hcaology with the cysteine rich secretory protein ("CRISP") as identified by BLAST homology [(ECRISP3_1, S68683, and CRS3_HMAN]. Accordingly, it is presently believed that PR0328 polypeptide disclosed in the present application is a newly identified nmnber of the GLIP or CRISP families and possesses transcriptional regulatory activity 15 typical of the GLIP or CRISP families.

42. Full-Length PRD335, PRO331 and PR0326 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present S*0 20 application as PR0335, PRO331 or PRO326. In particular, Applicants have identified and isolated cINA encoding a PRO335, PRO331 or PR0326 polypeptide, as disclosed in further detail in the Examples below. Using .0 BLAST and FastA sequence alignment ccnputer programs, Applicants found that various portions of the PR0335, PRO331 or PR0326 polypeptide have significant hanology with LIG-1, ALS and in the case of PRO331, additionally, decorin. Accordingly, it is presently believed that the PR0335, PR0331 and PRO326 polypeptides disclosed in the present application are newly identified members 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-lAenth P1R332 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0332. In particular, Applicants have identified and isolated cEM encoding PR0332 polypeptides, as disclosed in further detail in the Exanples below. Using BLAST and FastA sequence alignment conputer programs, Applicants found that a full-length native sequence PRO332 (shown in Figure 108 and SEQ ID N):309) has about 30-40% amino acid sequence 80a identity with a series of known proteoglycan sequences, includinig, for exanpie, fibrcmo&ilin and fibrcmtxdulin precursor seuences of various species (RF)D BOVIN, FD_CHICK, FM:)DJW.T FMVDMOJE, R()DJLt1W, PJU36773), ostemiodulin sequences (AB000114_l, AB007848_1), decorin sequences (CFU83141_1, CU03394_i, PR42266, P...R42267, P..R42260, P _R89439), keratan sulfate proteoglycans (MUfl4836L1, AF022890Ll), corneal proteoglycan (AF022256j-), and bxme/cartilage proteoglycans and proteoglycane precursors (PGSI.BWfINE, P=S_MDJSE, IP3S2jKI1IAN).

Accordingly, it is presently believed that PRO)332 disclosed in the present application is a new proteoglycan-type nolecule, and tray play a role in regulating extracellular tratrix, cartilage, anid/or bine function.- 44. Fufltlength PR0334 Poblyldtg The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0334. In particular, Applicants have identified and isolated cDNA encoding a PR0334 polypeptide. as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that various portions of the PR0334 polypetdec have significant homology with fibulin and flbrillin. Accordingly. it is presently believed that PR0334 polypeptide disclosed in Me present application is a newly identified snenir of the epidermnal growth factor family and possesses properties and activities typical of this family.

4S. Full-length PRO346 PolvnMtdes The present invention provides newly identified and isolated nucleotide sequences-encoding polypeptides referred to in the present application as PR0346. In particular. Applicants have identified and isolated cDl4A encoding a PR0346 polypeptide. as disclosed in further detail in die Examples below. Using BLAST and FastA sequence alignment compuie programs, Applicants fouind that a ful-length native sequence PR0346 (shown in Figure 112 and SEQ ID NO:320) has 28 amino acid sequence identity with carcinoerrbryonic antigen. Accordingly. ic is presently believed that PR0346 disclosed in the present application is a newly identified member of the carcinoenibryonic protein family and may be expressed in association with neoplastic tissue disorders. 46. Full-lenyth PR0269 Polvoentdes The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0268. In particular. Applicants have identified and isolated cDNA encoding a PR0269 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs. Applicants found that portions of the PR0268 polypeptide have significant homology with the various protein disulfide isomerase: proteins: Accordingly. it is presently believed that PR0269 polypeptide disclosed in the present application is a hornolog of the protein disulfide isomerase p5 protein.

47. Full-length PR0330 Povuentides 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 FastA sequence alignment computer program. Applicants found that various portions of the PR0330 polypeptide have significant homology with the murine prolyl 4-hydroxylase alpha-Il subunit protein. Accordingly, it is presently believed that PR0330 polypeptide disclosed in the present application is a novel prolyl 4-hydroxytase subunit polypeptide.

48. Full-length PR03 Ln R310 Polypgptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptodes referred to in the presen application as PR0339 and PRO310. In particular. Applicants have identified and isolated 82 cENA encoding a PRO339 polypeptide, as disclosed in further detail in the Examples below. Applicants have also identified and isolated cdA encoding a PRO310 polypeptide, as disclosed in further detail in the Examples below.

Using BLAST and FastA sequence alignment conputer programs, Applicants found that various portions of the PRO339 and PRO310 polypeptides have significant hanology with small secreted proteins fromn C. elegans and are distantly related to fringe. PR0339 also shows hanology to collagen-like polymers. Sequences which were used to identify PRO310, designated herein as ENA40533 and M A42267, also show hczrology to proteins fraom C. elegans.

Accordingly, it is presently believed that the PR0339 and PRO310 polypeptides disclosed in the present application are newly identified meber of the family of proteins involved in developmrent, and which may have regulatory abilities similar to the capability of fringe to regulate serrate.

49. Full-Length PRD244 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding C-type lectins referred to in the present application as PR0244. In particular, applicants have identified and isolated crA encoding PR0244 polypeptides, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment ccnputer programs, Applicants found that a full-length native sequence PRO244 (shown in Figure 122 and SEQ ID ND:376) has 43% amino acid sequence identity with the hepatic lectin gallus gallus (LECH-OICK), 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 renter of the C-lectin superfamily and rny play a role in immune function, apoptosis, or in the pathogenesis of atherosclerosis. In addition, PR0244 may be useful in identifying tunor-associated epitcpes.

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

82a Variations in the native full-length sequence PRO polypeptides or in various darains 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 nay 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 carpared with the native sequence PRO polypeptide. Opticnally the variation is by substitution of at least one amino acid residue with any other amino acid in one or more of the dcmains of the PRO polypeptide. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by carparing the sequence of the PRO polypeptide with that of hamologous kncwn protein molecules and minimizing the number of amino acid sequence changes made in regions of high hamology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, conservative amino acid rpacements. Inserions or deletions may optionally be in the range of I to 5 amioa acids.

The variation allowed may be determined by systematically aking insertions. deletions or'substinztions of amino acids in the sequence and testing the resulting variants for activity in the in uiro assay descuibed in the Examples below.

71-i variations can be made using methods known in the art such as oligomzclootide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed nmagemsis [Carter et al., c. AL&io Rs 13.4331 (1986); Zollcr et al., liucl- Acids e-t s. JQ:6487 (1997)]. cassette mutageneis [Wells et al., fi=L 34:315 (1985)1 resmeon sclection omitagtesas [Wells cial.. Ptulos-Tcins-R.Soc.Iotmdon ~erA, 11:415 (1986)] or othe known techniques can be performed on the cloned DNA to produce the desired PRO polypcpde variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small. neutral amino acids. Such amino acids include alanine, glyciwe serine, and cysteine. Alanine is typically a preferred scanning amilno add among this group because it ellimiaes the side-chain beyond the beta-carbon and is less likely to alter thec main- chaini 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. The rotins, Freeman Co., Chothia, L MoLi. H j-1Q:l (1976)). If alaknie substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

S1. Modifications or-PRO Polvpeptides Covalent miodifications of PRO polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residucs 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. Derivatization with bifunctional agents is useful, for instance, for crosslinking a PRO polypeptide to a water-insoluble support matrix or surface for use in the method for purifing anti-PRO polypeptide antibodies, and vice-versa. Commonly used crosslinking agents include, I ,l-is(diazoacetyl).2-phenylethane, glutaraldehyde, N-bydroxysucinimid esters, for example, esters with 4-azidosalicylic add, homobifuinctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimiylpropionate), bifunctional maleimides such as his-Ninaleimido-l .8-octane and*agents such as methyl-3-((p-azidophcnyrdthio]propioidte.

Other modifications include deamidation of gitminyl and asparaginyt residues to the corresponding glutamyl and aspantyl residues, respectively. hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threnyl residues, inethylation of the ct-amino groups of lysine, arginine, and bistidine side chains (T.E.

Crihtn Pmi Srcture and Molecular Poerts. W.H. Freeman Co.. San Francisco. pp. 79-86 (1983)].

acetylation of the N-termninal amine, and aniidation of any C-terminal carboxyl group.

Another t"p of covalent modification of die PRO polypeptides included w thin the scope of this invention comprises altering the native glycosylation pattern of the polypcptide. '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 mnore glycosylation sites that are not present in the native sequence PRO polypeptide.

Addition of glycosylation sites to the PRO polypqpsdc may be acconplished 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 threanine residues to the native sequence PRO polyetide (for 0-linked glycosyladon sites). The PRO polypeptide amino acid sequence may optionally be altered through changes at the DNA level, particularly by mtutating the DNA encoding the PRO polypptde at preselected bases such that codons are generated tha will transtaj into the desired amino adds.

Another means of increasing the umber of carbohydrate moieties on the PRO polypeptide polypeptide is by chemical or enzymatic coupling of gtycosidcs to the polypcptide. Such methods are described in the art, in WO 87/05330 published 11 Septemnber 1987, and in Aplin and Wniston. CRC Crit. Rev. Biochem.. pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymaticalty or by mutational substitution of codons encoding for amino acid residues that serve as- targets for glycosylatioa Ciemical deglycosyltulon tchniqiies are known in the art and described, for instance, by Hakimuddin.

et al.. Arch, Biochem- Bignhv- W ~:52 (1987) and by Edge et al., Arial. Biochem, 118:131 (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.tmml 111:350 (1987).

Another type of covalent moidification of PRO potypeptides of the invention comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, polyethylene glycol. polypropylene glycol, or polyoxyalkylenes. in the manner set forth in U.S. Paten Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PRO polypeptides of the present invention may also be modified in a way to form a chiimeric molecule comprising a PRO polypeptdec fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimneric molecule comprises-a fuision of the PRO potypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyt- terminus of the PRO polypeptide. The presence: of such epitope-tagged forms of the PRO polypeptide can be detected using an arwibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO polypeptde to be readily purifictl by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epiuTpe tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO polypeptide with an immnmoglobulin or a particular region of an imuanoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgO molecule.

Various tag potypeptides and their respective antibodies are well known in the art. Examples include polyhistidinc (poly-his) or poty-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CAS (Field et at., Mat. Cell. Biol., 1.2159-2165 (1988)]; the c-myc tag and the 8179, 3C0, 61310, G4, B7 and 9E10 antibodies thereto [Evan et at., Molecular and Cellular Biology, 1:3610-3616 (1985)1; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et at., Protein jEngineering, 1(6):547-553 Other tag polypeptides include the Flag-peptide [Hopp et al., Di j~jggjWg, i: 1204-1210 (1988)); the KT3 epitope peptide (Martin et al., Sceie 2:92-194 (1992)1; an a-tubulin epitope peptdec (Skinner et al., J l Ce., 2:15163- 151 6 6 (1991); and the 77geme 10protein peptide tag [Luz-Freyermthct al., Proc. NaI. Acad. Sci. USA. 17:6393- 6397 (1990)].

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

deletions from or insertions or substitutions of. residues within the amino acid sequence shown fothuman PR0317 in Figure 42. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter posttranslational processes of the PRO317. such as changing the number or position of glycosylation sites. Moreover, like most mammalian genes, PRO317 is presumably encoded by multi-exon genes. Alternative mRNA constructs which may be attributed to different mRNA splicing events following ranscription 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 PR0317, 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 PR0317 polypeptide that are preferred locations for mutagenesis is called "alanin scannmig nmtagenesis," as described by Cunningham and Wells. Sience, 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 alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqucous 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 sequeice variation is predetermined, the nature of the mutation per se ed not be predetermined. For example.

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

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

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

Amino acid sequence insertions include amio- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequenc 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 nmbers of residues but this is not required. Examples of terminal insertions include mature PR0317 with an N-terminal mcthionyl residue, an artifact of the direct production of mature PR0317 in recombinant cell culture, and fusion of a heterologous N-terminal signal sequence to the N-terminus of the mature PRO317 molecule to facilitate the secretion of mature PRO317 from recombinant hosts. Such signal sequences may be obtained from, .o and thus homologous to, the intended host cell species, but also may be from other members of the TGFsuperfamily. Suitable sequences include STII or lpp for E. coli. alpha factor for yeast, and viral signals such as herpes gD or the native EBAF-I sequence for mammalian cells.

Other insertional variants of the PRO317 molecule include the fusion to the N- or C-terminus of PR0317 of immunogenic polypeptides, bacterial polypeptides such as beta-lactamase or an enzyme encoded by the E.

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

A third group of variants are amino acid substitution variants. These variants have at least one amino acid residue in the PR0317 molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as the active sitc(s) of PRO317 and sites where the amino acids fumd 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 where the amino acids found in known members of the TGF- superfamily and novel PR0317 are substantially different in terms of side-chain bulk, charge, or hydrophobicity, but where there also is a high degree of sequence identity at the selected site within various animal analogues of such members among all the animal EBAF-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 PRO317 obtained from various species are identical among all animal species of PRO317 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 1 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes.

is thus formed such that oe 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 arc grown, they are plated onto agarose plates and screened using the oligonuclcodde 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 anjppropriate host.

The method described immediately above may be modified such that a homoduplex molecule is created whbrein both strands of the plasmid contain the mutation(s). The modifications arc as follows: The single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three dcoxyribonuclotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP), is combined with a modified hiodcoxyribocytosine called dCTP-(aS) (which can be obtained from the Amersham Corporation). This mixture is added to the template-oligonucotide 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 dCTP-(aS) instead ofdCTP, which serves to protect it from restriction endonuclease digestion.

After the template strand of the double-stranded hcteroduplex is nicked with an appropriate restriction enzyme, the template strand can be digested with Erxo nuclease or another appropriate nuclease past the region that contains the sitc(s) to be mutagenizcd. The reaction is then stopped to leave a molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four dcoxyribomcleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell such as E. coi JMI01. as described above. DNA encoding PR0317 mutants with more than one amino acid to be substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than about ten amino adds), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonuclcotide is generated for each amino acid to be substituted. The oligonuceotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mtants: wild4ype 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 oligonuclotide 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 iniagcticsis is also suitable for makting amino acid variants of PRO3 17. While the following discussion refers to DNA, it is understood that the technique also finds application with RNA. The PCR technique generally refers to the following procedure (see Erlich. PCR Technolgy. (Stockton Press. NY, 1989). the chapter by R. Higuchi, p. 6 1-70): W~hen small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment tha differs from the template sequence only at the positions where the primers differ from 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 sa-and of the plasmid, but this sequence can be located azrwhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 200 nucleotides from that of the first, such that in the end the entire aimplified region of DNA bounded by the primers can be easily sequenced. PCR amplifcation 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 mutagcnesis. is based on the technique described by Wells et al., JA: 315 (1985). The staring material is the plasmid (or other vector) comprising the PR0317 DNA to be muarted. The codon(s) in the PR0317 DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified miutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonuicleotide-mediared inutagenecsis method to introduce them at appropriate locations in the PR0317 DNA. After dhe restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonuclootide encoding the sequence of the DNA between the:. restriction sites but containng 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 oigonucleotide is referred to as the cassette. This cassette is designed to have 3' and 5' ends tha are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasznzd. This plasmid now contains the mutated PR0317 DNA sequence.

Covalent modifications of PR0317 are also inhcled within the scope of this invention. One type of covalent modification inchudes reacting targeted amino acid residues of the PR0317 with an organic denivatizing agent that is capable of reacting wih seleced side hainor the N- or C-terminal residues of the PROM1. Derivasizaton with -bifunctional agents is useful, for instance. for crossliking PR0317 to a water-insoluble support matrix or surface for use in the method for purifying anti-PR0317 antibodiies. and vice-versa. Commonly used crosslinking agents iclude, 1.1-bis(daroacetyl)-2-phenylethane. glutaraldehyde, N-hydroxysucainimidc esters, for example, esters with 4-azidosalicylic acid, homolAuctional iridoesters, including disuccinimidyl esters such as 3.3'-dithiobis- (succinylpropionate), bifunctional malcinsides such as bis-N-maleimido-1.8-octane, and agents such as methyl-3- ((p-azidophenyr)didhio)propioimidate.

Other modifications include deamlidation of glutaminyl and asparaginyl residues to the corresponding glusamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylasion of hydroxyl groups of seryl or threonyl residues, metbylation of the '-anino groups of lysine, arginie. and histidine side chains (T.E.

Creighmon, Proteins- Strcture and Molecular EPresez W.H. Freeman Co.. San Francisco. pp. 79-86 (1983)).

acetyladon of the N-terminal amine. and amidation of any C-terminal carboxyl group.

Another type of covalcrd modification of the PR0317 polypepddc included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. 'Alterit the native glycosylation pattern* is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native-sequenic e PRO polypeptde, and/or adding one or more glycosylation sites that are not present in the native-sequnc PRO polypeptide. be dedued anioaciddsequence ofPRO317 shown in Figure 42 (SEQ ID NO: 114) has one predicted N-linked glycosylation site at residue 160.

Addition of glycosylauion sites to the PR0317 polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made. for example, by the addition of. or substitution by. one or more acrine or direoninc residues to die native-sequecec PR0317 (for 0-linked glycosylation sitei). The PR0317 amino acid 0.0se=Vence may optionally be altered through changes at the DNA evel, particularly by mutating the DNA encoding the PR0317 polypeptide at preselocted bases such that codons are generated that will translate into the desired amino :0 acids.

0Another means of increasing the number of carbohydrate moieties on the PR0317 po4ypptde is by chemical or enymatic coupling ofglycosides to the polypetide. Stich methods are described in the art, in WO 87/05330 published 11 September 1987. and in Aplin and Wriston, CRC Cit. Rev. iocbem-, pp. 259-306 (1981).

Removal of carbohydrate moieties, present on the PR0317 polypeptde may be accomplished chemically or enzymatically or by mnutational substitution of codons encoding amino acid residues that serve as targets for ,o oglycosyhtion. Chemical deglycosylation iechniq=e art known in the art and described, for instance, by Haltimuddin, et al.. Arch, Biochem- Biohys.. M:252 (1987 and by Edge eral.. Anal ioceI~L, :131 (t98 Enzymatic .oo. cleavage of carbohydrate moieties, on polypeptides can be achieved by the use of a variety of endo- and exoglycosidases as described by Thotakura a at., Meth. EnzXnxd.. flJ:350 (1987).

Another type of covalent modification of PR0317 comprises finking the PR0317 polypepuide to owe of a variety of nionproteinaceous polymers. polyethylene glycol. polypropylene glycol, or polyoxyalkylencs, 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 invention may also be moidified in a way to form a chimeric molecule comprising PR0317 fuised to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of the PR0317 with a tag polypeptide which provides an epitope to which an anti-tag at tibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of the PROM1.

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 cpitope tag. Ini an alternative emnbodimrent. the chimeric molecule may comprise a fusion of the PR0317 with an immunoglobulin or a particular reinof an imimunoglobulin. For a bivaleit form of the chimeric molecule, such a fusion could be to dhe Fc region of an IgG molecule.

Various tag polypeptides and their respective antiboies are well known in the art. Examples include polyhistidine (poly-his) or poly-histidine-glycirie (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CM5 (Field et MolA C1L..il. 1:2159-2165 (1988)). the c-myc tag and the 8F9. 307. 6E10. G4. B7. and 9E10 antibodies thereto (Evan er at., Molecular and Cellular Biology. 5 :3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky a al.. Pe in Eginceing, 2(6):547-553 (1990)). Other tag polypeptides include the Flag-peptide (Hopp et al.. BiaLL~ecnoogX. fi:l 204-1210 (1988)); the K73 epuope peptide (Martin Cral., Sci 192-194 (1992)); an -tubulin epitope peptide (Sinnerr a =20: 15163- 15166 (1991)); and the T7 genie 10 protein peptide tag (Lnz-FrcyermLth ea al. Proc. Mai. Acad-Sci. USA, 82:6393- 6397 (1990)).

53. Preparation of PRO Po~lyides; The description below relates primarily to production of PRO polypeptides by culturing cells transformed or amifected with a vector containing the desired PRO polypeptide nucleic acid. It is. of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare the PRO polypeptid e. For instanoc, the PRO polypeptde wq or pomons thereof. may be produced by direct peptide synthesis using solidphase techniques [see. Stewart ct al.. Solid-Phase Pelltide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J- Ain. Chem. Soc., U~:2149-2154 (1963)1. In Wtro protein, synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City. CA) using manfacurer's instructions. Various portions of the desired PRO polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the fulfl-length PRO polypeptide.

20 A. Isolation of DMA Encoding PRO P-oypsntides DNA encoding PRO polypeptides may be obtained from a eDNA library prepared from tissue believed to possess the desired PRO polypeptide mRNA and to express it al a detectable level. Accordingly, human PRO .polypcptide DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. Ile PRO polypeptide-encoding gene may also be obtained from a genornic library or by oligonucleotide synthesis.

Libraries emn be screened with probes (such as antibodies to the desired PRO polypeptide or oligomxcleotides; of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the eDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloninju A Laboratary 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.. supra; Dieffenbach et al.. PCR Primer-A Laborator Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. The oligonucleotde sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. T~he oligonucleotide is preferably labeled such that it emn be detected upon hybridization to DNA in the library being screened. Methods of labeling arm well known in the art, and include the use of radiolabels likce 32P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including mnoderate stringency and high stringency. are provided in Sambrookct al.. W= Sequences identifled in such library screening methods can be compared and aligned to ocher known sequences deposited and available in public databases such as GearBank or other private sequence databases.

Sequence Identity (at either the amino acdd or nucleotide: level) within defined regons of the moeec or across the full-length sequence can be determined through sequence alignment using computer software programs such as BLAST. AUGN. DNAstar. and INHERIT which employ various algorithms to measure homology.

Nucleic acid having protein coding sequence may be obtained by screening selected CDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, If necesary, using* conventional primer extension procoddins as described in Sambrook ci al., Mjmz, to detect precursors and processing intermediates of rnRNA that may not have been reverse-transcribed into cDNA.

B. Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described herein For PRO polypeptide production and cultured in conventional inurient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions. such as media, temperature. pH and the like, can be selected by the sktilled artisan without undue experimentation. In general. principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found un Mammalian -Cell iotechnologv: a-Practical. Aggroach, M. Butler, ed. (IRL Press, 1991) and Sambrook ct al..

:Meto of trnsection are known to the ordinarily skilled artisan. for example, CaPO, and electroporation.

Depending on the host cell used, transformation is performed using standard technique-s appropriate to suc h cells.

The calcium treatmuent employing calcium chloride, as described in Sambrook et al., LV=, or electroporation is generally used for prokaryotes or other clls that contain substantial cell-wall barriers. Infection with Agmobaderiwm fi~~iejiadet; Is used for transformation of certain plant cells, as described by Shaw et al.. G3=n. 22:315 (1983) and ~.WO ar9/05859 published 29 June 1989. For mamomalian cells without such cell walls the calcium phosphate precipitation method of Graham and van der Eb, Yirolomr. 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transformations have been described in U.S. Patent No. 4399,216. Transformations 4*4* into yeast are typically carried out according to the metho of Van Solingens et al., J. Bact., flQ:946 (1977 and Hsiao.

eal.. Pro- Nagl- Acad -Sci. (UIS& 26.3M2 (1979. However, other methods for introducing DNA into cells, such as by nuclear microinjectioa, electroporation, bacterial protoplast fusion with intact cells or polycations, e.g..

polybre polyornithine, may also be used. For various techniques for transforming -mmalian cells, see Keown et al., Methods in Ezmolnev .Jj:527-S37 (1990) and Mansour et al.. Nature, Ui:348-352 (1988). Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prolraryotes include but are not limited to eubacteria. such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coil. Various E. coil strains are publicly available, such as E. wUl K12 strain MM294 (ATCC 31.446); E. coil X1776 (ATCC 31,537); E. ccli strain W31 (ATCC 27.325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriacec such as &cheridzia. E. coil, EnUeroacute, -Erwinia. Kiebsielia, Proteu, Salmonella, SaLbnonella ryphimuliwn, Serra. Seffoaia mwcesrcaz, and Sldge~a. as well as Baciif such as B. subtlih and B. lichenaforis B.

licheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pscudomnonas such as P. aeruginwsa. and S'rrpxonyces. Various E. cvii strains are publicly available. such as E. coil K12 strain MM294 (ATCC 31.446); E.

coli X1776 (ATCC 3.1,537); E. coil strain W31 10 (ATCC 27.325). aM K5 772 (ATCC 53.635). 'These examples are illustativc rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host stwain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal.amounts of proteolytic; enzymes. For example. strain V/3110 may be modified to effect a genetic mutation in the gene encoding proteins endogenous to the host, with examples of such hosts including E. coil W31 10 strain lAl. which has the complete genotype toL4 E. coil W31 10 strain 9E4, which has the complete genotyle tonAl prr3; E. coil W31 10 str-ain 27C7 (ATCC 55.2M4). which has the complete genotype tontA ptr3 phoA EI5 (argF-lac)!69 degP ompTkan'; E. cvii W31 10 strain 37136. which has the complete genotype ronA pr3 phoA EI5 (argF-lac)169 degP onipT ,bs7itvGkad-';E. coil W31 10 strain 40B4. which is strain 37D6 with anon4anamycin resistant degP deletion mutation; and an E. coli strain having mutant periplismic protease disclosed in U.S. Patent No. 4.946,783 issued 7 August 1990. Alternatively, in s'irm methods of cloning. PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokcatyotes. eukaryotic microbes such as filamentous fuingi or yeast arc suitable cloning or expression hosts for PRO polypeptide-encoding vectors. Saceharontyces cerevasiae is a commonly used lower eukaryotic host microorganism. Others include Sddz~oacchiaromyces pombe (Beach and Nurse, N~ature. Z: 140 [19811; EP) 139.383 published 2 May 1985); Kluyvromyces hosts Patent No. 4,943,529; Fleer ea aL, :BiolTehnolory. 2: 968-975 (191)) such as. K. lacris (MW9SC, CBS683. CBS4574; Louvencourt ea!.L, L Bactiol.. 737 11903D. K. fragils (ATCC 12,424), K. bulgari cus (ATCC 16,045). K. wickeramil (ATCC 24,178).

K. waldi (ATCC 56,500). K. drosophilarum (ATCC 36,906; Van den Berg el al., Bio/Technoloey, 1: 135 (IM9)).

K. thennoolerans. and K. marx Jaus: yarvWa (EP 402,226); Pichiapastoris (EP) 183,070; Sreekrishna ei aL, L.

Basic Microbiol., 21. 265-V8 [1988D; Candida, Trichdenna reesia (EP) 244.234); Neurospora crassa (Case et al., ~Proc. Natil Acad- Sci- USA, 2-6: 5259-5263 [1979]); Schwamuomyces such as Schwanniomyces occidentalis (EP) 394,538 published 31 October 1990); and filamentous fungi such as, PNeurospora. Penilwn, Tolypocladium *pot o:(WO 91/00357 published 10 January 1991), and Aspergilu hosts such as A. nidukan (Ballanccer Biochem.

Biophys. Res, Comnm=, ILa. 284-289'(1983]; T'ilburn ei al.. 9m 205-221 (19831; Yekton a al.. Pzoc. ail.

Acd Sc UA.1: 1470-1474 (1984D) and A. niger (Kelly and Hynes. EMBOL1, 4: 475-479 [1985D.

Methylotropic yeasts are suitable herein and include, but arc not limited to, yeast capable of growth on methanol selected from the genera consistin of Hansenula, Candida, Kiockiera. Pichia. Saccharonyces. Tordlopsis. and PRzodotoruda. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony. 33L Biochemistry of MethYlotroobs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO polypeptides are derived from multicellular organisms. Examples of invertebrate Cells include insect cells such as Drosophila S2 and Spodoptera SO9. as Well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells.

More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7. ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al.. LQSzLYk2OL.

6:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO. Urlaub and Chasin, Proc. Natl. Acad Sci. USA, 22:4216 (1980)); mouse scnoli cells (M4. Mathcr. Biol. Rem.. 23:243-251 (1980)); human lung cells (W138. ATCC CCL human liver cells (Hep G2. HB 8065); and mouse mammary tumor (MMT 060562. ATCC CCLS1). The selection of the appropriate host cell is deemed to be within the skill in the art.

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

The vector may, for example, be in the form of a plasmid. cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endouclease site(s)using techniques known in the art. Vector components generally include, but are not limited to. one or more of a signal sequence, an origin of replication, one or more marker genes, an egos*, enhancer lement, 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 polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or It may be a part of the PRO polypeptide DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase.

.penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S. Patent No. 5,010.182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362.179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In

S.

mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypcptides of the same or related species, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 21 plasmid origin is suitable for yeast, and various viral origins (SV40. polyoma, adcnovirus, 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 Bacili.

An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO polypeptide nucleic acid, such as DHFR or thymidine 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. Nat. Acad. Sd. lSA; 2:4216 (1980). A suitable selection gene for use in yeast Is the tuPl gene Present in the yeast plasmid YRp7 [Sdrichwcb ct ad.- N~ature. 2U:39 (1979); Kingsma et al.. QM,. 2:141 (1979); Tacheniper et al.. Q=l. JQ: 157 (1980)1. The tI7l gene provides a selection marker for a nartant strain of yeast lacdn the abiliy to grow in tryptophan. for cxample, ATCC No. 44076 or PEP4- I [Jones, Gerrs 1:2 (1977)].

Expresiion and cloning vectors usually contain a promotcr operably linked to the PRO polypeptide nucleic acid sequzence to direct inRNA synthesis. Promoters meognized by a variety of potential host cells are well known.

Promoters suitable for use with proktaryotic hosts include the P-laeta2Se and lactose promoter systems [Chang et al. Naure. 2U-.615 (1978); Goeddel cc al.. Ntaue, 211:544 (19179)). alkaline phosphatasc. a tryptophari (tip) promoter system (Goeddel, Nuki AidsesL., 1:4057 (1980); EP 36.7761, and hybrid promoters such as the tac promoter (deBoer etal.. Eno Nadi Aad. Sci. USA, IWI 1-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding the desired PRO polypeptide.

fees*:Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3es..phosphoglycerat kinase (Hitrznin at al.. j- Iialhe., 2U-.2073 (19M]~ or othe glycolyi enzymes [Hess ct J -Ady. Enzme Re= 2: 149 (1968); Holland. ~jBiomin 12:4900 (1978)], such as enolase. glyccraldehyde-3- 0004 phosphat dehydrogecase, hexoldose, pyruvate decarboxytase. phosphofructokiflase. glucose-6-phosphate isomerase, 3-posphglyerair mutase, pynivate kinase. trioscphosphate isomerase, phosphoglucose isonerase, and glucokinae.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol de-hydrogenase 2, isocytochrome C. acid S. S phosphatase. degradative enzymes associated with nitrogen metabolism, metaliotbionein. glyceraldcllyde-31)hahte so dehydrogenase. and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for 20 use in yeast expression are further described in EP 73.657.

PRO polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoomr obtained from the genomes of viruses such as polyonxa virus, fowipoX virus (UK 2.211,504 published July 1989). adenovirus (such as Adenovitus bovine papilloma virus, avian sarcoma viru. cytomegaloviru. a retrovirus, hepatitis-B vinis and Simian Virus 40 (SV40). from heserologous mammalian promoters. the actin promoter or an immunaoglobilin promoter. and from heat-shock promoters, provided such promoters are compatible 0 with the host cell systems.

Transcription of a DNA encoding the desired PRO polypeptdec by higher eukaryotes may be increased by inserting an enhancer sequence into t vector. Enhaocers are cis-acting elements of DNA. usually about from to 300 bp. that act on a promoter to increase its transcuiptioni. Many enhancer sequences are now known from mammalian genes (globin. elastase. albumin. a-fetoprotcin. and insulin. Typically, however, one will usc an enhancer from a cukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomcgalovinis early promoter enhancer. the polyonia enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may he spliced into the vector at a position 5' or 3' to the PRO polypeptide coding sequence, but is preferably located at a site 51 from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organism) will also contain sequences necessary for the termination of' transcription arid flor stabilizing the niRNA. Such sequences are cornanly available from the 5' and, occasionally 3, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments trancribed as polyadenylated fragments in the untranslated portion of the rnRNA encoding PRO polypeptides.

Still other methods. vectors, and host cells suitable for adaptation to the synthesis of PRO polypeptides in recombinant vertebrate cell culture &xe described in Gething et al.. Nature, M2:620-625 (1981); Marnei et al., Nature, Z&L:40-.46 (1979); EP 117.060; and EP 117.058.

D. Detecting Gene AmnnlltiripEnression Gene amplification and/or expression may be measured in a sanple directly, for example, by conventional Southern blotting, Northrnbloting to quantitate the transcription of naRNA (Thomas, Proc. Narl. Acad. Sci, UISA, 7:5201-5205 (1980)M. dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe.

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

Geme expression, alternatively. may be measured by immunological methods, such as immunohistochemical .15 staining of cells or tissue sections and assa of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for i sueitdaklstaining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveiniently, the antibodies may be prepared against a native sequenc PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to a PRO polypeptde DNA and encoding a specific antibody epitope.

E. Purification or Eolynentide Forms of PRO polypeptdes 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 enzymiatic 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 cell lysing agents.

It may be desired to purif PRO polypptdes from, reWmiant: cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-cxcbangc columa; ethanol precipitation; reverse phase HPLC. chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; arumoniUM sulfate precipitation; gel filtration using, for example, Sephadex protein A Sepharose columns to remoeve contaminants such as IgG; and metal chelating columns to bind epitopetagged forms of the PRO polypeptide. Various methods of protein purification may be employed and such methods are known in the sit and described for example in Deucscher. Methods in E=zMogyr, IB2 (1990); Scopes, Ewisin Purification, Pinciples and Practice, Springer-Verlag, New York (1982). The purification step(s) -selected will depend, for example, on the nature of the production process used and the particular PRO polypeptidc produced.

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

The full-length native sequence PRO polypeptide-encoding nucleic acid or portions thereof. may be used as hybridization probes for a cDNA library to isolate the fullcngth PRO polypeptide gene or to isolate still other genes (for instance, those encoding naturally-occurring variants of the PRO polypeptide or PRO polypeptides from other species) which have a desired sequence identity to the PRO polypeptide nucleic; acid sequences. Optionally, the length of the probes will be about 2to aou 5 ass. The hybridition probes may be derived from th micleotide sequence of any of dhe DNA molecules disclosed herein or from genomic sequences including promoters.

enhancer elements and auns of native sequesiee PRO polypeptide encoding DNA. By way of example. a screening method will comprise isolating the coding region of the POpolypeptide gene using the known DNA sequence t synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels. including radiomicleotidecs such as orss S. or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling system. Labeled probes having a sequence complementary to that of the specific PRO polypeptide gene of the present invention can be used to screen libraies of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.

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

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

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

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

which are well characterized in the art.

Nucleic acids which encode a PRO polypcptide or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in tmrm are useful in the development and screening of therapcutically useful reagents. A transgenic animal a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, an embryonic sage. A tansgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding a PRO polypeptide of interest can be used to clone genomic DNA encoding the PRO polypeptide in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding the PRO polypcptide. Methods for generating transgenic animals, particularly annmals such as mice or rats, have become conventional in the art and are described.

for example, in U.S. Patent Nos. 4.736.866 and 4.870.009. Typically, particular cells would be targeted for PRO polypeptide transgene incorporation with tissue-specific enhancers. Transgcnic animals that include a copy of a transgene encoding a PRO polypepide inroduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding the PRO polypeptide. Such animal can be used as tester animals for reagents thought to confer protection from. for example, pathological conditions associated with its overexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced 15 incidence of the pathological condition, compared to untreated animals bearing the transgene. would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO polypeptides can be used to construct a PRO polypcptide "knock out" animal which has a defective or altered gene encoding the PRO polypeptide of interest as a result of homologous recombination between the endogenous gene encoding the PRO polypeptide and altered genomic DNA 20 encoding the PRO polypeptide introduced into an embryonic cell of the animal. For example. cDNA encoding a PRO polypeptide can be used to clone genomic DNA encoding the PRO polypeptide in accordance with established techniques. A portion of the genomic DNA encoding a PRO polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see Thomas and Capecchi, Cel, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line by electroporation) and cells in which the introduced DNA has homologously S recombined with the endogenous DNA are selected (see Li ct al., Cell, 6:915 (1992)]. The selected cells are then injected into a blastocyst of an animal a mouse or rat) to form aggregation chimeras (see Bradley, in Teraocardnomar and Embryonic Stem Cells: A Practical Approach, E. J. Robertson. ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the PRO polypeptide.

With regard to the PRO211 and PRO217 polypeptide, therapeutic indications include disorders associated with the preservation and mainteanc of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions C=oneooits. ZoLLinger-ElIiuion syndrome. gastrointestinal ulceration and congenital microvillu atrophy). skin diseases associated with abnormal keratinocr diffecrentiation psoriasis, epitelial cawcr uh sln squamous cell carcinoma, epidermid carcinoma of the vulva and glionas.

Since the PR0232 polypeptide ad ox-leic acid enoodizig it possess sequence homology to a cell surface stem cell antigen and its enicoding nucleic acid, probes based upon the PR0232 nucleotide sequence may be employed to idemi1 other novel stem cell surface antigen proteins. Soluble forms of the PRO232 polypcpide may be employed as antagonists of membrane bound PRO232 activity both in viaro and in Wvo. PR0232 polypeptides may be employed in screening assays designed to identify agonists or antagonists of the native PR0232 polypeptide, wherein such assays may take the form of any conventional cell-ype or biochemical binding assay. Moreover, the PR0232 polypeptide inayserve as a molecular marker for the tissues in which the polypepde is specifically expressed.

With regard to the PR0187 polypeptides disclosed herein, FGF4 has been implicated in cellular differentiation and embryogenesis, including the patterning which appears during limb formation. FGF-8 aiid the PRO 187 molecules of the invention therefore are lkelIy to have potent effects on cell growth and development.

Diseases which relate to cellular growth and differentiation are therefore sutitable targets for therapeutics based on functionality similar to FOP-S. For example, diseases related to growth or survival of nerve cells including Parkinson's disease, Alzheimer's disease, ALS. neuropathics. Additionally, diease 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. Parent 5,654,270 to Ruoslabti 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 propcrties of the fibromodulin described therein including where it is located and with what it binds and does not.

The PR0219 polypeptdecs of the present invention which play a regulatory role in the blood coagulation cascad may be employed in vivo for therapeutic purposes as well as for in vitro purposes. Those of ordinary skill .*in the art will well know how to employ 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, extraceliular domains derived from these PR0246 polypeptides mray be employed therapeutically in iWv for lessening theecffma of viral infection. Those PR0246 polypeptides which serves tmor specific antigens may be exploited as therapeutic targets for and-rumor drugs, and the lie. Those of ordinary skill in the art will well know how to employ PR0246 polypeptides for such uses.

Assays in which connective growth factor and other growth factors ame usually used should be performed with PROM6. An assay to determine whether TOF beta induces PROM.1 Indicating a role in cancer is performed as known in the art. Wound repair and tissue growth assays ame also performed with PR0261.' The results are applied accordingly.

PR0228 polypeptdecs should be used in assays in which E.MRI, CD97 and latrophilin would be used in to determine their relative activities. The results can be applied accordingly. For example, a competitive binding assay with PR0228 and CD97 can be performed with the ligand for CD917, CDSS.

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 kicd, DNA47412. the EST from which PCR oligos were generated to isolate the full length DNA49435-1219. has been observed to map tolIlplS. Sequence bomology toathe II1 locus would indicate that PRO533 may have utility in the treatment of Usher Syndrome or Atrophia areata.

As mentioned previously. fibroblast growth ftan can act upon cells in both a mitogenic and Don-mitogenic warmner. These haors ame mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, Inducing gramiuosa cells. adrenal cortical cells. chreodrocy=e. rnyoblasts. corneal and vascular endothelial cells (bovine or human). vascular smooth mscle cells, lens. retin and prostatic epithelial cells. oligodendocyres, astrocytes, chrondocytes, myoblasts and osteoblasts.

Non-initogenic actions of fibroblast growth factors include promotion of cell migration into a wound area (chemoraxis). initiation of ew blood vessel formuiladon (angiogenesis), modulation of nerve regeneration and survival (ncurotrophism). modulation of endocrine functions, and stimuslation or suppression of specific cellular protein exrsion. extracellular matrix production and cell survival. Baird, A. Bohlen. Handbook of Plwmacol.

2XQ): 369-418 (IM9). 'flcse properties provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healing, nerve repair, collateral blood vessel formation, and the like. For example. fibroblast growth factors. have been suggested to minimize inyocardiumn damnage in heart disease and surgery (U.S.P.

4.379,437).

Since the PR0245 polypeptidc and nucic acid encoding it possess sequence homology to a trarnmembrane protein tyrosine kase protein and its encoding nucleic acid, probes based upon the PRO245 nucleotide sequence way be employed to identify other novel tansmmembrAne tyrosine kinase proteins. Soluble forms of the PR0245 polypeptide may be employed as antagonists of mrn bound PR0245 activity both in Wro and in v~ivo. PR0245 polypeptides may 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 way 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 homology to SLIT and Icucine rich repeat protein. Therefore, these proteins are useful in assays described in the literature, mwzn. wherein the SLIT and leucine rich repeat protein are used. Regarding the SLIT protein. PR0227 ***canl be used in an assay to determine the affect of PR0227 on neurodegenerative disease. Additionally; PR0227 has homology to human glycoprotein V. In the case of PRO22, this polypeptide is used in an assay to deterinen 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 neutrodegenerative diseases or their reversal.

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

PR0287 polypeptides and portions thereof which effect the activity of bone morphogenic Protein "BMPI*/procollagen 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 pornuone thereof may have therapeutic applications in wound healing and tissue repair. Peptides having homology to procollagen C-proemnase enhanoer protein and its precuror may also be used to induce bowe and/or cartilage formation and are therefore of particular interest to the cientific and medical commtis.

Therapeutic indications for PR0214 polypeptides include disorders associated with the preservat .ion and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions (ecg., enterocolitis.

Zollinger-Ellison syndrom, gastrointestinal ulceration and congenital microvillus atrophy). idn diseases associated with abnormal kerarinocyte differentiation psoriasis, epithelial cancers such as lung squamnous cell carcinoma.

cpidermoid carcinoma of the ivulva anti Sliomas.

Soldie on the generation and analysis of mice deficient in members of the TGF- superfamily are reported in Matzuk, Trends in Endocrinol. and Metabol., fi: 120127 (1995).

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

Bloactive compositions comprising PR0317 or agonists or antagonists thereof may be administered in a suitable therapeutic 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 my be complexed with a variety of well established compounds or compositions which enhance *.:stability or pharmacological properties such as half-ife. It is contemplated that the therapeutic, bioactive composition may be delivered by intravenous infusion into the bloodstream or any other effective means which could be used for treating problems of the kidneiy, uterus, endomtiuun, blood vessels, or related tissue, in the heart or genital tract.

Dosages and administration of PRO317'. PR0317 agonist, or PR0317 antagonist in a pharmaceutical composition may be determined by one of ordinary sktill in the art of clincal pharmacology or pharmacoinetics.

See, for example, Mordenti and Rescigno. brada eerh 2:17-25 (199); Moren t a L. Phamaceutical Resarch- 1:1351-1359 (1991); and Mordenti and Chappel. -Tbe use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Dnie Deelopment Yacobi a at. (eds) (Pergamon Press: NY, 1989), pp. 42-96. An effective amount of PROM1, PR0317 agonist, or PRO317 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 the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 10 ngikg to up to 100 mg/kg of the nmnal's body weight or more per day, preferably about I ug/kg/day to 10 mgtkgday. Typically.

the clinician will administer PROMI. PR0317 agonist, or PR0317 antagonist, until a dosage is reached that achieves the desired effect for treatment of the above merntioned disorders.

PRO3 17 or an MR0317 agonist or PR0317 antagonist may be administered alone or in combination with another to achieve dhe 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, pharmnaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8. more preferably6 to 8, although the pH may vary according to die characteristics of the PROM 1. agonist, or antagonist being formulated and the condition to be treated. Characteristics of the treatmrent compounds include solubility of the molecule, half-life, and antigenicity/tmogenicity: these and other characteristics may aid in deffining an effective carrier.

PR0317 or PR0317 agonists or PR0317 antagonists may be delivered by known routes of administration including but not limited to topical -es~i and gels; tr-Ansm ucosal spray and aerosol, tranisdcrmal patch and bandage; injectable, Intravenous, and lavage formulations; and orally administered liquids and pills, particularly formualated to resist stomach acid and enzymes. The particular formullation, exact dosage, and route of admninistration will be determined by the attending physician and will vary according to each specific situation.

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

severity) of the patient, age, weight, gender, diet, time of administration, drug combination, reaction sensitivities.

and tolerance/response to eay. Long-acting treatment compound formulations (such as liposomally encapsulated PR0317 or PliGylated PR0317 or PR0317 polymeric microspheres, such as polylactic acid-based microspheres) might be admninistered every 3 to 4days, every week, or once every two weeks depending on half-life and clearance rate of the particular treatment compound.

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

4,657,760; 5,206,344; or 5,225.212. It is anticipated that different formulations will be effective for different trea= compounds and different disorders, that administration targetin the uterus, for example, my necessitate ****delivery in a manner different from that to another organ or tissue, such as cardiac tissue.

Where sustained-release admin;stration of PR0317 is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder requiring administration of PROM 1. microencapsulation of PR0317 is contemplated. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH). interferon- (rbIN- interleukin-2, and MN rgp 120. Johnson er giL, Nat, Med., 2: 795-799 (1996); Yasuda. Bimcd. 'fler- 22f: 1221-1223 (1993), Hors er al., BioTeQjglev B: 755-758 (1990); Cleland. 'Design and Production of Single Iinmmization Vaccines Using Polylactide Polyglycolide Mcrosphere Systcms," in Vagnii Desdgn Th Suhmit and Ad -uvrant ARonach Powell and Newman, eds. (PleCu Press: New York, 1995), pp. 439-462; WO 97/03692, WO %6/40072. 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 biocompatibilihy 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 degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, "Controlled release of bioactivc agents from lactid/glycolide polymer." in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Dn Delivery Svstems (Marcel Dekker: New York. 1990). pp. 141.

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 w/w PRO317) with the lowest possible initial burst is necessary. A continuous (zeroorder) release of PRO317 from microparicles for 1-2 weeks Is also desirable. In addition, the encapsulated protein to be released should maintain its integrity and stability over the desired release period.

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

15 The PR0317. PR0317 agonist, or PR0317 antagonist may be administered to a mammal with another biologically active agent, either separately or in the same 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-1 may be administered together with an antagonist to PR0317. such as an anti-PRO317 antibody.

20 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 PR0317 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 PR0301 (SEQ ID NO:119) has a Blast score of 246 and 30% homology at residues 24 to 282 of Figure 44 with A33_HUMAN, an A33 antigen precursor. A33 antigen precursor, as explained in the Background is a tumr-specific antigen, and as such, is a recognized marker and therapeutic target for the diagnosis and treatment of colon cancer. The expression of tumor-specific antigens is often associated with the progression of neoplastic tissue disorders. Native PRO301 (SEQ ID NO:119) and A33_HUMAN also show a Blast score of 245 and homology at residues 21 to 282 of Fig. 44 with A33_HUMAN, the variation dependent upon how spaces are inserted into the compared 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 known as cell surface protein HCAR. This region of PR0301 also shows a similar Blast score and homology with HSU90716_1. Expression of such proteins is usually associated with viral infection and therapeutics for the prevention of such infection may be accordingly conceived. As mentioned in the Background, the expression of viral receptors is often associated with neoplastic tumors.

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

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

PRO229 polypcptides can be fused with peptides of interest to determine whether the fusion peptide has an increased half-life over the pptide of interest. The PR0229 polypeptides can be used accordingly to increase the half-life of polypeptides of interest. Portions of PR0229 which cause the increase in half-life are an embodiment of the invention herein.

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

PR0233 polypcptides and portions thereof which have homology to reductase may also be useful for in ivo therapeutic purposes, as well as for various other applications. The identification of novel reductase proteins and 20 related molecules may be relevant to a number of human disorders such as inflammatory disease, organ failure.

atherosclerosis, cardiac injury, infertility, birth defects, premature aging, AIDS, cancer, diabetic complications and Smutations in general. Given that oxygen free radicals and antioxidants appear to play important roles in a number of disease processes, the identification of new reductase proteins and reductase-like molecules is of special importance in that such proteins may serve as potential therapeutics for a variety of different human disorders. Such polypeptides may also play important roles in biotechnological and medical research, as well as various industrial applications.

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

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

PRO235 polypeptides and portions thereof which may be involved in cell adhesion are also useful for in vivo therapeutic purposes, as well as for various in vito applications. In addition, PR0235 polypeptides and portions thereof may have therapeutic applications in disease states which involve cell adhesion. Given the physiological importance of cell adhesion mechanisms in vivo. efforts are currently being under taken to identify new. native proteins which are involved in cell adhesion. Therefore pcptides having homology to plexin are of particular interest to the scientific and medical communities.

Because the PR0236 and PR0262 polypeptides disclosed herein are homologous to various known Pgalactosidasc proteins, the PR0236 and PR0262 polypeptides disclosed herein will find use in conjugates of monoclonal antibodies and the polypeptide for specific kiling of tmor cells by geertation of active drug from a galactosylazed prodrug the generation of 5-fluoridimi fromt the prodrug P-D-galactosyl.S-fluorouridine). The PR0236 and PR0262 polypeptdes disclosed herein may also find various uses both in vivo and in vitro, wherein those uses will be similar or identical to uses for which P-galactosidase: proteins are now employed. Those of ordinary skill in the art will well ktnow how to employ PR0236 and PR0262 polypeptides for such uses.

PR0239 polypeptides and poris thereof which have homology to dcoiin may also be usefu for in viva therapeutic purposcs, as well as for various In tru applications. In addition, PR0239 polypeptides and portions thereof may have theapeutic applications in disease states which inrvolve: synaptic mechanisms, regeneration or cell adliesion.

Given the physiological importance of synaptic processes, regeneration and cell adhesion mechanisms in vivo, efforts arc currenuly being under takrn to identify new, native proteins which are involved in synaptic machinery and cell adhesion. Therefore, pcptdecs having homology to densin are of particular interest to the scientific and medical communities.

OVO.

The PR0260 polypetdecs described herein can be used in assays to determine their relation to fucosidase.

In particular, the PR0260 polypeptides 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 functional or locational 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 PRO263 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 vivo therapeutic purposes, as well as for various in vitro applications. More specifically. PR0270 polypeptides may affect the expression of a large variety of gene thought to be involved in the pathogenesis of AIDS, cancer, :atherosclerosis, diabetic complications and in pathological conditions involving oxidative stress such as stroke and inflammation. In addition, PR0270 polypeptides and portions thereof may affect the expression of a gene which have a role in apotosis. Therefore. peptides having homology to tioredoxin are particularly desirable to the scientific and medical conunities.

*PR0272 polypeptides; and portions thereof which possess the ability to bind calcium may also have numerous 9. in vivo therapeutic uses, as well as various in vifu applications. Therefore. peptides having homology to reticulocalbin are particularly desirable. Those with ordinary SUil 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 usefu for in viva therapeutic pur-poses, as well as for various other applications. The identification of novel collagens 'and collage-like molecules may have relevance to a number of human disorders. Thus, the identification of new collagens and collage-like molecules is of special importance in that such proteins my serve as; potential therapeutics for a variety of different human disorders. Such polypeptides may also play important roles in biotechnological and medical research as well as various industrial applications. Given the large rnumber of uses for collagen, there is substantial interest in polypeptides with homology to the collagen molecule.

107 PRD295 polypeptides and portions thereof which have hoology 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 nodulating the binding or activity of cells of the immune system.

Thus, the identification of new integrins and integrin-like molecules is of special inportance in that such proteins may serve as potential therapeutics for a variety of different human disorders. Such polypeptides may also play important roles in biotechnological and medical research as well as various industrial applications. As a result, there is particular scientific and medical interest in new molecules, such as PRO295.

As the PRO293 polypeptide is clearly a leucine rich repeat polypeptide harologue, the peptide can be used in all applications that the known NLRR-1 and NLRR-2 polypeptides are used. The activity can be 15 carpared between these peptides and thus applied accordingly.

The PRO247 polypeptides described herein can be used in assays in which densin is used to determine the activity of PR0247 relative to densin or these other proteins. The results can be used accordingly in diagnostics and/or therapeutic applications with PR0247.

20 PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides of the present invention which possess protease activity may be enployed both in vivo for therapeutic purposes and in vitro. Those of ordinary skill in the art will well know how to enploy the PRO302, PRO303, PRO304, PRO307 and ao* PR0343 polypeptides of the present invention for such purposes.

PR0328 polypeptides and portions thereof which have honology 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 PR0326 including uses in coapetitive assays with LIG-1, ALS and decorin to determine their relative activities.

The results can be used accordingly. PRO335, PRO331 or PRO326 can also be 107a used in assays where LIG-1 would be used to determine if the same effects are incurred.

PWRO332 contains GAG repeat (GKEK) at amino acid positions 625- 628 in Fig. 108 (SEQ ID M:309). Slippage in such repeats can be associated with human disease. Accordingly, PRO332 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 mrotif.

Other uses of PRO334 include use in assays in which fibrillin or fibulin would be used to determine the relative activity of PRO334 to fibrillin or fibulin. In particular, PR0334 can be used in assays which require the mechanism inparted by epidernal growth factor repeats.

Native PR0346 (SEQ ID N1:319) has a Blast score of 230, corresponding to 27% hanology between amino acid residues 21 to 343 with residues 35 to 1040 CIM6_HUMAN, a carcinoearbryonic antigen cgm6 precursor.

This harmlogy region includes nearly all but 2 N-terminal extracellular danain residues, including an imuunoglobulin superfamily harology at residues 148 to 339 of PRO346 in addition to several transmranbrane.

.o residues (340-343). CArcinemryoaic antigen precursor, as expLdAid in the Background is a tumor-specific antigen, amd as such. is a recognized marker and therapeutic target for the diagnosis and treatmntmof colon cance. The espression Of tumor-specific antigens is often associated with the progression of neoplastic tsime disorders. Native PR0346 (SEQ ID N0320 and P -W06974. a human carcizxnenbryaoic antigen CEA-d have a Blast score of 224 and homology of 28% between residues 2 to 343 and 67 to 342, respectively. This homology includes the entire exmaceliar domain residues of native PR0346. minus the initiator methionine (residues 2 to 18) as well as several transmembrane residues (340-343).

PR0268 polypeptides which have protein disulfide isomerase activity will be useful for many applications where protein disullide isomerase activity is desirable including, for example, for use in promoting proper disulfide bond formation in recombinantly produced proteins so as to increase the yield of correctly folded protein. Those of ordinary skil in the art will readily know how to employ such PRO268 polypeptides for such purposes.

PR0330 polypeptides 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 skilliIn the art will well know how to employ the PR0330 polypetides of the present invention for such purposes.

Anti-PRO Polypentide Antibodies The present invention further provides anti-PRO polypeptide antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispocific, and betcroconjugate antibodies.

A. Polydlonal Antibodies The anti-PRO polypptide artibodies may comprise polyconl antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a marmmal, far example. by one *or more injections of an immunizing agent and, if desired, an adjuvant. Typically. the immunizing agent and/or adjuvant will be injected in the manmal by trultiple subaitancous or imtraperltoneal injections. The immrunizing agent may include the PRO polypeptide or a fusion protein thereof. It my be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammral being immtmized. Examples of such immunogenic proteins iuded but are not limited to keyhole limpet hemocyanin, serun aihumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Frcund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

B. MOnOelonal Antibodie The anti-PRO polyptidd antidies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be preparcd using bybridoma methods, such as those described by Kohler and Milstein, Nature, Ufi495 (1975).

In a hybridoma. method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunnizing agent to elicit lymphocytes tham produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, t lymphocytes mnay be itmmunized in itro.

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

Generally, eitder peripheral blood lymphbocytes C*PBLs') are used if cells Of human origin are desired, or spleen cells or lymph node cells are used if noo-human mammalian sources arc desired. Thec lymphocytes arc then fused with an immortalized cell line using a suitable fasing agent, such as polyethylene glycol. to form a hybridoma cell (Goding.

Monoclonal Antibodies- Principles and Practice. Academic Press, (1986) pp. 59-103). Immortalized cell line are usually transformed mammalian cells. particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse mycloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances diat inhibit the growth or survival of the unfissed, immortalized cells.

For example, if the parenta cells lark the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typcally will include hypoxanthinc, aminopterin. andl thymidine (*HAT medium"), which substances prevent the growth of HC3PRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells. and are sensitive to a mediu such as HAT medium. More preferred immnortalized cell line are murine mycloma linets, which can be obtained, for instance, from the Salk Institute Cell Distribution Center. San Diego. California and the American Type Culture Collection, Rockville, Maryland. Human niyeloma and mouse-human heteromyeloma cell lines also have been described for the production of humn mnonoclonal antibodies (Kozbor, J. Immwwnl., 1fl:3001 (1984); Brodeur et al. Montoclonal Antibody Production Technques and Applications, Marcel, Dekker. Inc.. New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells ame cultured can then be assayed for the presence of :monoclonal antibodies directed against the PRO polypeptide. of interest. Preferably, the binding specificity of monoclonal antibodies produced by the hybridomna cells is determined by immunoprecipitation or by an in itro binding assay, such as innnn.~~y(I)or enzymec-linked immuriotibsorbent assay (ELISA). Such techniques and assays are known in the art. Ile binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem.. J92:220 (1980).

After the desired hybridonia cells are identified, the clones may be subcloncd by Limiting dibition procedures and grown by standard methods (Goding, siwzal. Suitable culture media for this purpose include, for example.

Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells my be grown in vivo as ascites in a mammal.

The monoctonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional irtnmunoglobulint purification procedure such as, for example, protein A-Sepharose.

hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4.816.567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of arasrne: antibodies). The hybridoim cells of the invention serve as a preferred source of such DNA. Owce isolated, the DNA ay be placed into expression vectors, which arc then transtected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or mycloitta Cells that do not otherwise produce inxmunoglobulin protein, to obtain the synthesis of monoclonal antibodies i the recmbinant host cells. The DNA also may be mrodified, for example. by substituting the coding sequence for human heavy andi light chain constant domains in place of the homologous murine sequences Patent No. 4.816,567; Morrison et al., mipza or by covalently joining to the imniunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-imrnunoglobulin polypeptide can be substituted for the constant domains of an antibody of thc invention. or can be substituted for the variable domains of one antigencombining site of an antibody of the invention to create a chimeric bivalen antibody.

Thle antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies arc well known in the art. For example. one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavydainm nted genray a anpintinhe Fcgonso as topretheaychai crosslinking. Alternatively, the relevant cysteine residues ame substituted with another amino acid residue or axe deleted so as to prevent crosslinking. I In itro methods arc also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly. Fab fragments, can be accomplished using routine techniques known in the art.

C. Humanized Antibodies The andi-PRO polypeptide antibodies of the invention may further comprise humanized antibodies or human *antibodies. Humanized forms of non-human murine) antibodies are chimneric imniunoglobulins, iuuglobulin chains or fragments thereof (such as Fv, Fab, Fab'. or other antigen-binding subsequences of antibodies) which contain mninimial sequence derived from non-human immunoglobulin. Humanized antibodies include human inimunoglobulins (recipient antibody) in which residues from a complementary determining region 20 (CDR) of the recipient arc replaced by residuas 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 instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequencs In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human iminunoglobuhn and all or substantially all of the FR regions ame those of a human imimunioglobulin consensus sequencc. Ihe humanized antibody optimally also will comprise at least a portion of an irnumuoglobulin constant region typically that of a human iznmunoglobulin (Jones d al., Nature, 2U: S22-525 (1986); Riechann et al..

Nature, IZ:323-329 (1988); and Presta. Qkir?. op. Srnwt. Blot., 2:593-596 (199)].

Methods for humanizing non-human antibodies am 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. Thbese non-human amino acid residuas are often referred to as "import residues, which are typically taken from an 'import' variable domain.

Humanization can be essentially perfored following the method of Winter and co-workers (Jones er al.. Nature. n21: S22-525 (1986); Riechmann ar al., Nature. Ma:323-327 (1988); Vertioeyen ef at.. Science. 2:1534-1536 (1988)].

by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly.

such 'humanized" antibodies are chitneric antibodies Patent No. 4,816,567), wherein substantially less than an intact humran variable domain has been substituted by the corresponding sequence from a non-human species. In practice. humanized antibodies are typically human antibodies in which some CDR residues aEd Possibly Some FR residues arc substituted by residues from analogous sites in rodent antibodies.

Human antibodics can also be produced using various techniques known in the art. including phage display libraies[Hoogom ad W nter J. Mot. Siol.,W:381 (991); Marks et Mo. Biol,f22S81 The techniques of Cote dtal. and Boemr nc are also available for the preparation of human monoclonal antibodies (cote er aL, monodorza! ibor wii Cancer Therapy, Alan R. Lism, p. 77 (1985) and Boemnr ecial..). Immwtol., IAZM:96-95 (1991)].

D. Bispecifc Antibodies Bispecific antibodies are mnonoclonal. 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 polypeptde, the other one is for any other antigen. and preferably for a cell-surface protein or receptor or receptor subunit.

Methods for nmaking bispecific antibodies am known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-cxpression of two immunoglobulin heavy-chaiui/light-chain pairs, where the two heavy chain have different specificities (Milstein and Cueilo. ture, 3M5:537-539 (1983)]. Because of the random assortment of izumunogbalin heavy and light chains. these hybridomas (quadrtmas) produce a potential mixnzze: often different antibody molecules. of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chroumatography steps. Similar procedures are disclosed in WO 93108829, published 13 May 1993. and in Trauneccer et EMBO IQ:3655-36S9 (1991).

Antibody variable domains with the desiredl binding specificities (antibody-antigen~combining sites) can be fused to inxnunoglobulin constant domain sequences. The fusion preferably is with an inmunoglobulin heavy-chain constant domnain, comprising at least part of the hinge CH2. and CH3 regions. It is preferred to have the first heavychain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.

DNAs encoding the imnmunoglobulin hecavy-chain fusions anid. if desired, the immnunoglobulin fight chain, are inserted into separate expression vectors, and are co-tranisfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Mfetd in Enzymology, j21:210 (1986).

E. Heteroconougate-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 eels to unwanted cells Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known mctb~ds in synthetic protein chemistry. including those involving crosslinking agents. For example, immunotoxins may be constructed using a &isulfide exchange reaction or by brfori a thioedier bond. Examples of suitable reagents for this purpose include irninothiolatc: and methyl-4-mercaptobutyriicate and those disclosed, for example. in U.S.

Patent No. 4,676,980.

S6. Uses for Ante-Pro Polynentlde Antibndies The anti-PRO polypeptide antibodies of the anvcution have various utilties. For example, anti-PRO polypcptdde 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 dhe arn may be used. such as competitive .binding assays, direct or indlirect sandwich assays and immmpreciPitation assays conducted in either heterogeneous or homogeneous phases [Zola. Monoclonal Antibodies- A Manual of Tediniues, CRC Press, Inc: (1987 pp. 147-.158). The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing. either directly or indireedy, a detectable signal. For example, die detectable moiety may be a radioisotope, such as 1H. S. oP" 1. a fluorescent or chemiluminescem compound, such as fluorescein isothiocyanate. rhodamine, or luciferin, or an enzyme. such as alkaline phosphasase, beta-galacosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed. including those methods described by Hunter et Nature, 1A4:945 (1962); David et at.. Biochemistry, 13:1014 (1974); Pain et J. ImmwwoL Met., 40:219 (1981). and Nygren, J.

Hlutochem. and Cyotocem., J: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 imxnobilued antibody then is contacted with a sample containing the PRO polypeptide to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the :PRO polypeptide, which is bound to the immnobilized antibody. Finally, the support is washed with another suitable solvent that will release the PRO polypeptide from the antibody.

With regard to PRO211 and PRO2i?, therapeutic indications include disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions avterocolitis. Zollinger-Bliscas syndrome, gastrointestinal ulceration and congenital microvillus atrophy), skin diseases associated with abnormal keratinocyte differentiation psoriasis, cpithelial cancers such as lung squamoaus cell carcinoma, epidermoid carcinoma of the vulva and gliomas.

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

Native PR0533 is a 216 amino acid polypeptide of which residues 1-22 are the signal sequence. Residues 3 to 216 have a Blast score of 509. corresponding to 53% homnology to fibroblast growth factor. At the muiceotde level, DNA47412, the EST from which PCR otigos were generated to isolate the full length DNA49435-12 19, has been observed to map to lipIS. Sequence homology tod in lpiS locus would indicate that PR0533 may have utility in the treatment of Usher Syndrome or Atrophia areata.

As mentioned previously. fibroblast growth facon can act upon clls in both a mitogewec and rion-mitogenic mnc. These fumtrs ame ritogenic for a wide variety of normal diploid mesoderm-derived and neural crest-dcrived cells,biducing gramilosacells, adrcmal corical cellsdroadrocytes, myoblasts, corneal and vascular endothelial cells (bovine or human), vascular smooth muscle cells, lens, retin and prostatic epithelial cells, oligodendrocytes, astrucy=es chmontlocytes, rnyoblasts and osteoblasts. Antibodies to these factors can be generated to modulate such effects.

Non-mitogenic actions of fibroblast growth factors inelude promotion of cell migration into a wound area (chemotaxis), iniiaion of new blood vessel foimlaijon (angiogenesis), modulation of nerve regeneration and survival (necurotrophism). modulation of endocrine functions, and stimulation or suppression of specific cellular protein expression, extracelfular matrix production and cell survival. Baird, A. Bohlen. Hand book of Eip. PArmacol.

21(l): 369-418 (1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches in 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 heart disease and surgery (U.S.P.

4,378,437). Antibodies to thes factors can be generated to modulate such effects.

Therapeutic indications for PR0214 polypeptdecs include disorders associated with the preservation and maintenance of gastrointestinal =ncosa and the repair of acute and chronic inucosal lesions emterocolitis, Zotlinger-Elison sytndrorre, gastrointestinal ulceration and congenital nierovillus atrophy), skin diseases associated with abnormal keratmocyte differentiation psoriasis. epithelial cancers, such as king squamous cell carcinoma, epidermnoid carcinoma of the vulva and gliomas.

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

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

44 with A33 HUMAN. an A33 antigen precursor. A33 antigen precursor, as explained in the Background is a tumnor-specific antigen, and as such, is a recognized marker and therapeutic target for the diagnosis and treatment of *~9.colon cancer. Thec expression of tumor-specific antigens is often associated with the progression of neoplastic tissue disorders. Native PRO301 (SEQ ID NO:119) and A33 -HUMAN also show a Blast score of 245 and 30% homology at residues 21 to 282 of Fig. 44 with A33 HUMAN, the variation dependent upon how spaces are inserted into the compared sequences. Native PRO301 (SEQ ID NO:119) also has a Blast score of 165 and 29% homology at residues to 255 of Fig. 44 with HS46KDA a human coxsackie and adenovirus receptor protein, also kown as cell surface protein HCAR. This region of PRO301 also shows a similar Blast score and homology with HSU90716_1.

Expression of such proteins is usually associated with viral infection and therapeutics for the prevention of such infection may be accordingly conceived. Accordingly, antibodies to the above identified antigens and receptors have therapeutic potential as diagnostic and treatment techniques.

Therapeutic uses for the PR0234 polypeptides of the invention includes treatments associated with leukocyte homing or the interaction between leukocytes and the cadotheiumn during an inflammatory response. Examples include asthma, rheumatoid arthritis. psoriasis and multiple scrosis.

Cancer-assoW~edJ or specific antigens permit the creation of tmor or cancer specific monoclonal antibodies (mAbs) whuich arc specific to such nir antigens. Such mAbs, which can distinguish between normal and cancerous cells art useful in the diagnosis. prognosis and treatmntmof the disease.

Cancer specific monoclonal antibodies (inAbs) which arm specific to tinoer antigens. Such mAbs. which can distinguish between normal and. cancerous cells art useful in the diagnosis, Prognosis and treatmnt of the disease. Particular antigens are known to be associated with eoPlastic diseases, such as colorcaul and breast cancer.

Since colon .canocr is a widespread disease, early diagnosis and treatment is an important medical goal. Diagnosis and treatment of cancer can be implemented using monoclonal antibodies (mAbs) specific therefore having fluorescent. nuclear magnetic or radioactive tags. Radioactive genes, toxins and/or drug tagged mAbs can be used for treaanen in situ with nnial 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 the present specification an hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were used according to manufacturer's istructions unless otherwise indicated. The source of tdose cells identified in the following examples, and throughout the specification. by ATCC accession murmbers is the Ametrican Type Culture Collection, Rockvyille, Maryland.

EXMPE1: Extracellutar Domain HornologX Screeing to Identify Novel Polvnepdes and cDNA Encoding T1he extraccllua domain (ECD) sequences (includig the secretion signal sequence, if any) from about 950 known screted proteins from dre Swiss-Noit public database were used to search EST databases. The EST databases included public databases Dayboff, Genflank). and proprietary databases LIFEEQr.- Iny Pharmnaceuticals. Palo Alto. CA). The search was performed using the computer program BLAST or BLAST2 (Ahscll and Gish, Methods in Enrymogy 266: 460-80 (1996); http://blast.wuslVedu/blastREADME.hml) as a comparison of the ECD protein sequences to a 6 firame translation of the EST sequences. Those comparisons with a Blast sore of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program 'phrap' (Phil Green. University of Washington. Seattle, WA; (tp:/fbozeman.mb.wasbigoLeduphrap.docs/phrapbu~).

Using this cxtracellular domain homology screen, consensus DNA sequences were assembled relative to the other identified EST sequen=e. in addition, the consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.

Based upon the consensus sequences obtained as described above, oligonucleotides were theni synthesized and used to identify by PCR a eDNA library that contained the sequence of interest and for use ar probes 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 arc often designed to give a PCR product of about 100-1000 bp in lenth. The probe sequences are typically 40-55 bp in length. In sonie cases, additional oligonucleotdes are synthesized when thc consensus sequence is greater than about I-I.skbp. In order to scren several libraries for a ful-length clone. DNA from the libraries was screened by PCR amplification. as per Ausubel et al..,urn ProtocpLh In moleeutir Biolog'v, with the PCR primer pair. A positive library was then used to isolate clones encodin the gene of interest using the probe oligonucleodde and one of the primner pairs.

The cDNA libraries used to isolate the cDNA cloaws were constructed by standard methods using cornmercially available reagents such as those from Invitrogen, San Diego. CA. The cDNA was primed with oligo dT contaiining a Nod site, linked with blunt to Sall hemikinased adaptors, cleaved with :Nod, sized appropriately by gel electrophoresis, arid cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; is a precursor of pRKSD that does not contain the Sfl site; see. Holmes et al., Science. 2.U:l27-128O (1991)) in the unique Xhol and Notd sites.

EXAMPLE.2: Isolation of cD2NA, Clones Encoding PRO211 and PRO217 Consensus DNA sequences were assembled as described in Example 1 above and were designated as DNA28M3 aMi DNA28760. respectively. Based on these consensus sequences, oligonucleotides were synthesized and used to identify by PCR a cDNA library that contained the sequences 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 DNA33094-1 131 were fetal lung libraries.

cDNA clones were sequenced in their entirety. The entire nucleotide sequences of PROZI11 (DNA32292- 1131; UNQI8S) and PR0217 (UJNQ191; DNA33094-l 131) are shown in Figure 1 (SEQ ID NO; 1) and Figure 3 :(SEQ ID respectively. The predicted polypeptides are 353 and 379 amino acid in length, respectively. with respective molecular weights of approximately 3 8.190 and 41t.520 daltons.

The oligonucleotide sequences used in the above procedures were the following: 287 3 0.p (OUI 516) (SEQ ID 5S-AGGGAGCACGGACAGTGTGCAGATGTGGACGATGCATAGA3' 28730.f (OLI 517) (SEQ ID NO:6) 5'-AGAGTGTATCTCTGGCTACGC-3* 29730.r (OLI 5.18) (SEQ ID NO:7) S'-TAAGTCCGGCACATTACAGGTC-3' 28760.p (OLI 617) (SEQ ID NO:8) rGTGTGACCGTr-I-ICAC3 28760.f (OLI 618) (SEQ ID NO:9) 5'-AAAGACOCATCTGCGAGTGTCC-3* 29760.r (OUI 619) (SEQ ID 5:-TGCTGAMTCACACTGCTCTCCC-3' EXAMPLE 3: Isolation of cDNA Clones Encoding Hume PRO230 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 DNA30857. An EST proprietary to Gmensech was employed in the consensus assembly. The -EST is designiated is DNA20088 and has the nucleotide sequence shown in Figur 7 (SEQ ID NO: 13).

Based on the DNA30957 consensus sequence, oligonulootidcs 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 PROM3.

A pair of PCR primers (forward and reverse) were synthesized: forwar PCR *ie 5*-TFCGAGGCCTCTGAGAAGrGGCC-3 (SEQ ID NO:14) reverse PCR 11imer S'-GGCGGTATCcCTGGCcTCC-3 (SEQ ID Additionally. a synthetic oligonrucleotide hybridization probe was constructed from the consensus DNA30857 sequence which had the following nucleotide sequence hybridization probe 5'-TCTCCACAGCAGCTGTGGCATCCGATCGTGTCTCAATCCA-ICTCTGGGY3...(SEQ MD N0416) In order to screti several libraries for a source of a MulIlength 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 PR0230 gene using the probe oigonucleotide and one of the PCR primers.

RNA for construction of the eDNA libraries was isolated from humnan fetal lung tissue. DNAcqxndrg of the clones isolated as described above gave the hili-length 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).

Clowe UNQ204 (DNA33m2-1 136) 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 nucleotide positions 150 1-1503 (Figure SEQ ID NO:l11). The predicted polypeptide precursor is 467 amino acids long (Figure 6).

EAMPLEA4: Isolation of eDNA Clones Encoding 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, ofigonucleotide3 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 PROM3.

A pair of PCR primers (forward and reverse) were synthesized: forard PCR primer 5'-TGCGTGjCTACTCCrGCAAAGCCC..3* (SEQ ID NO:19) reverse PCR Ominer 5'-TGCACAAGTCGGFGjTCACAGCACG.3- (SEQ ID Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30935 sequence which had the folowing nucleotide sequence CCCACTGGG-3- (SEQ ID N0:21)' In order to scree several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0232 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction afthe cDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of tie clones isolated as described above gave the full-length DNA scqucte for PR0232 therein designated as U1NQ206 (DNA34435-1140)J and the derived protein sequence for PROM3.

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

Clon UNQ206 (DNA3443S-1 140) contains a single open reading frame with an apparent translational initiation site at uleotide positions 17-19 and ending at the stop codon at nucleocide positions 359-361 (Fig. 8; SEQ ID NO: 17).

The predicted polypeptide precursor is 114 amino acids long (Fig. Clone UNQ206 (DNA34435-1140) has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit Do. ATCC 209250.

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

EAMPLE.,: Isolaion of cDNA Clones Fncodinj PROI 87 A proprietary expressed sequence tag (EMi DNA databas OIMEEQ. Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST (#843 193) was identified which showed homology to fibroblast growth factor (FGF-8) also known as androgen-induced growth factor. mnRNA was isolated from human fetaluhng tissue wsig reagents and protocols from Invitrogen, San Diego, CA (Fasn Track The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents Invitrogen, San Diego, CA, Life Technologies, Gaithersburg. MD). The cDNA was primed with oligo dT containing a Notd site, linked with blunt to Sall bemilcinased adaptors, cleaved with Notd, sized appropriately by gel elecirophoresis, and cloned in a defined orientation into the cloning vecto pRK5D using reagents and protocols from Wie Technologies, Gaithersburg, MD **20 (Super Script Plasinid Systemn). The double-stranded cDNA was sized to greater than 1000 bp and the SallNotI linhotred eDNA was cloned into Xhol/NotI cleaved vector. pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sf1l restriction enzyme site preceding the Xhot/NotI cDNA cloning sites.

Several libraries from various tissue sources were screened by PCR amplification with the following ofigonueceotide probes: IN843193.f (0L1315) (SEQ ID NO:24) S'-CAGTACGTGAGGGACCAGGGGCCATGA..3' N&43193.r (011 317) (SEQ ID 5-CCGGTGACCTGCACGTGCrrGCCA..3' A positive library was then used to isolate clones encoding the PR01897 gene using one of the above oligonucleotides and the following oligonucleotide probe: 1N843193.p (011 316) (SEQ ID NO:26) 3 A cDNA clanec was sequenced in entirety. The entire nucleotide sequence of PRO0187 (DNA27864-1155) is shown in Figure 10 (SEQ MD NO:22). Clone DNA27864-1 155 contains a single open reading framne with an apparent translational initiation site at nucdeotide position I (Figure 10; SEQ ID NO:22). The predicted polypeptide precursor is 205 amino acids long. Clone DNA27864-1 155 has been deposited with the ATCC (designation: DNA27864-11SS) and is assigned ATCC deposit Do. ATCC 209375.

Based on a BLAST and FastA seqenc a~ign analysis (using the ALIGN computer program) of the fulllength sequenice, the PRO187 polypeptide shows 74% amino acid sequence identity (Blast score 310) to Iniman fibroblast growth factor-S (androgen-induced growth factor).

EXAMLE6: Isolation of eD)NA Clones Encoding PR0265 A consensus DNA sequence was assembled relative to other EST sequences as descn6&1i in Example 1 above using phrap. This consensus sequence is herein designated DMA33679. Based on the DNA33679 consensus sequence. oligomicleocides 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 fuill-length coding sequence for PROW6.

PCR primers (two forward and one reverse) were synthesized: forward PCR inrimer A- S*-CGGFCTACCTGTATGGCAACC.3' (SEQ ID NO.29); fonvard PCR Rrinier B: 5S-GCAGGACACCAGATACC.3' (SEQ ID *reverse PCR mimer S'-ACGCAGA flAGA ~GTC-3* (SEQ ID N031) a.**Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA33679 .***sequence which had the following micleodde sequence hyridizatin Rpmb 3 .(SEQ ID NO:32) In order to screen several libraries for a source of a fulfl-length clone, DNA from the libraries was screened by PCR amplification with PCR primer pairs identified above. A positive library iw then wsed to isolate clones encoding the PR0265 gene using the probe oligoiucleotide and one of the PCR primers.

RN'A for construction of the cDNA libraries was isolated from human a fetal brain library.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0265 (herein designated as UNQ232 (DNA363S0-1 158) (SEQ ID NO:27) and the derived protein sequence for PROM6.

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

Clone UNQ232 (DNA36350-1 158) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 352-354 and ending at the stop codoo at positions 2332-2334 (Figure 12). The predicted polypeptide precursor is 660 amino acids long (Figure 13). Clone UNQ232 (DNA36350-11S8) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209378.

Analysis of the amino acid sequence of the full-length PR0265 polypeptide suggests that portions of it possess significant homology to the fibromodulin and the fibromoxdin precursor, thereby indicating that PR0265 may be a novel member of the leucine rich repeat family, particularly relatd to fibroniedulin.

EXAMPfl 7: Isolatian of cnNA Clones ead ins! umian PRO719 A consensus DNA sequence was assembled relative to other EST sequeneswing pgkap as described in Example 1 above. This consensus sequence is hercin designated DNA28729. Based on the DNA2S79 consensus sequaee. ollgonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of inerest, and 2) for wse as probes to isolate a clone of the full-length coding sequemc for PROM1.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer S'-GTGACCCTGGrrrGrGAATAcTCc-3, (SEQ ID reverse PCR primer S'-ACAGCCATGGTCrATAJ~GCGG-3' (SEQ ID NO:36) Additionally, a synthetic ofigonucleotide hybridization probe was constructed from the consensus DNA29729 sequence wjiich had the following nucleotide sequence hbldfiian rob S*-GCCGTCAGTGTCCGAGGGACACGTGCCCGCAGCGATGGGAG.3 (SEQ ID NO:37) In order lo screen several libraries for a source of a funl-length clone, DNA from (he libraries Was Screened by PCR amplification with the PCR primter pair identified above. A positive library was then used t6 isolate clones encoding the PR0219 gene using the probe oligonucleodde and one of the PCR primrs.

RNA for construction of die eDNA librauies was isolated from human fetal kidney tissue.

**see:DNA sequiencing of the clones isolated as described above gave die MiIngth DNA sequence for PR0219 [hrei designated as UNQ193 (DNA32290-1 164)1 (SEQ ID 140:33) and die derived protein sequence for PROM1.

0 The enire nuclcoide seqimenc of UNQ 193 (DNA32290-1164) is shown in Figures 14A-B (SEQ ID NO:33).

Clone UNQ 193 (D)NA32290-1 164) contains a single open reading frame with an apparent translational initiation site 21MICIeotkle positions 204-206 andl ending at ie stop codon at nucleotide positions 2949-2951 (Figures 14A-B). The predicted polypeptide precursor is 915 amino acids long (Figure 15). Clone UNQ193 CDNA32290-1164) hsbee deposited with ATCC and is assigned ATCC deposit no. ATCC 209384.

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

EXAMPLE 8: Isolation of cDNA Clones Encodimg Human PR0246 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in 0e ~BExmple I above. This consensus sequence is herein designated DNA30955. Based on the DNA30955 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a eDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the ful-length coding sequence for PROM4.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR plrmer 5'-AGGGTCTCCAGGAGAGACrC-3' (SEQ ID reverse PCR Dnnler S-AITGTGGGCCrGCAGACATAGAC.31 (SEQ ID NO:41) Additionally. a synthetic oligonuceotide hybridization probe was constructed from the consensus DNA30955 sequence which had the following nucleotide sequence hybridization robe rCCACrC3(SEQ ID NO:42) In Order to screen several libraas for asource of a full-length clone, DNA from t libraries was screeed by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0246 gene using the probe oligomxicotde and one of the pCR p rmers.

RNA for construction of the eDNA libraries was isolated from human fetal liver tissue. DNA~nring of (he clones isolated as described above gave die full-lngth DNA sequence for PR0246 [herein designated as U1NQ220 (DNA3S639-1 172)] (SEQ ID NO:38) and die derived protein sequence for PROM4.

7he entire nucleotide sequence of [JNQ220 (DNA35630-1 172) is shown in Figure 16 (SEQ ID NO:38).

Clone UNQ220 (DNA35639-l 172) contains a single open reading frme with an apparent translational initiation site at nucleotide positions 126-128 and eoding at the stop codon at nuclootide positions 129671298 (Figue 16). The predicted pojypeptide precursor is 390 amino acids long (Figure 17). Clone UNQ220 (DNA35639-1 172) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209396.

Analysis of the amino acid sequence of the fiu-length PR0246 polypeptide suggests that it possess significant homology to the human cell surface protein HCAR, thereby indicating that PR0246 way be a novel cell surface virus receptor.

EXWj{~l-j 9 holation of cDNA Clones Eending Human PRO228 A consensus DNA sequence was assembled relative to other EST sequences using pbrap as described in ERample 1 above. This consesus sequne is herein designated DNA2S75S. An EST proprietary to Genentech was eniloyed in the consensus assembly. This EST is shown in Figure 20 (SEQ ID NO:50) and is herein designated as DNA219SI.

Based on the DNA28758 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 PR0228.

PCR primers (forward and reverse) were synthesized: *.:forward PrR grimer 5'-GGTAATGAGCTCCATACA..3* (SEQ ID NO:51) forward PcR primec 5'-GGAGTAGAAAGCGCATGG-.3' (SEQ ID NO:S2) forward PCR 11rimer S1-CACCTGATACCATGAATGCAG.3' (SEQ ID NO:53) *reverse PCR primer S'CGAGCTCGAA7TAArCG.3* (SEQ ID NO:54) reverse PCR primer 5*-GGATCTCCTGAGCrAGG-.3' (SEQ ID reverse PCR nrimer 5*-CCTAGTGT)GTATC1TTG.3' (SEQ ID NO:56) Additionally, a synthetic oigonucleotide hybridization probe was constructed from the consensus DNA28758 sequence which had the following nucleotide sequence bvbridization urobe 5'AGGCCCCTAGC~GTACCTAAArTCAT3 MSE M NO:57) In order to screeni several libraries for a source of a full-length cdone, DNA from the libraries was screened by PCR amplification with the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0228 gene using the probe oligonucleoodde and one of the PCR primers.

RNA far constrction of the eDNA libraries was isolated from human fetal Midny tissue.

DNA sequecig of the clones isolated as described above gave the full-length DNA sequel=e for PR0228 (herein designatad as UNQ2G2 (DNA33092-1202)] (SE D NO:48) and the derived protein sequejice for PR0228.

The entire nucleotide sequence of UNQ202 CDNA33092-im0) is shown in Figure 18 (SEQ ID NO:48).

Clone UNQ2O2 (DNA33092-1202) contains a single open reading ftrne with an appaent trnltoa initiation site at nucleotide positions 24-26 of SEQ ID NO:48 and ending at the stop codon after nueleotide position 2093 of SEQ 13D NO:48. The predicted polypeptidc precursor is 69 aminto acids long (Figure 19). Clone UNQ202 (DNA33092- 1202) has been deposited with ATCC and is assigned ATCC dep osit no. ATCC 209420.

Analysis of dhe amino acid sequence of the full-length PR0229 polypeptide suggests that portions of it possess significant homology to the secretin-related proteins CD97 and EMRI as well as the secittin member, latrophilin. thereby indicating that PR0228 may be a new member of the secectin related proteins.

Isolation-of cDNA Clones Fncodfinp Human PR0533 The EST s~queace accession nu--er AF007268. a murine fibroblast growth factor (FGF-1S) was used to search various public EST databases GenBank, Dayhoff, etc.). The search was performed using the computer program BLAST or BLAS72 [Ak~schul et al., Methods On Fn=Moee, 2fl:460-480 (1996); hq:I/bhig.wustd/Lsas EAM~htoil as a comparison of the ECD protein sequences to a 6 frame tranlation of the JEST sequences. The search resulted in a bit with GenBank EST AA220994, which has been identified as stratagenc NT2 neuronal precursor 937230.

Based on the Genbank EST AA220994 sequence. oligonucleotides were synthesized: 1) to identify by PCR a eDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length 15 coding sequnce. Forward and reverse PCR primers may range from 20 to 30 aucleotides (typically about 24), and ame designed to give a PCR product of 100-100 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 ffll-ength clone, DNA from the libraries :was screened by PCR amplification, as per Ausubel etal. Current Protocols in Molecuar Biology, with the PCt primer pair. A positive library was then used to isolate clones encoding the geme 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 amplification with the PCR primter 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 of the CDNA libraries was isolated from human fetal retinai. The cDNA libraries used to isolated the cDNA clones were constructed by standard methods using commercially available reagents Invirrogen San Diego, CA; Clontedi, etc.) The cDNA was primed with oligo dT containing a Nod site, linked with blunt to Sall hemikinased adaptors, cleaved with Nod, sized appropriately by gel electrophoresis, and cloned in a defined orientationt into a suitable cloning vector (such as pRCE or pRKD; pRK5B is a precursor of pRK5D that does riot contain the 5111 site; see, Holmes et al., Science. 1278-1280 (1991)) in dhe unique X(hoI and Notd sites.

A eDNA clone was sequenced in its entirety. The full length nucleotide sequence of PR0533 is shown in Figure 21 (SEQ ID NO:S8). Clone DNA49435-1219 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 459-461 (Figure 21; SEQ ID NO:5 The predicted polypeptide precursor is 216 amino acids long. Clone DNA47412-1219 has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209480.

Based on a BLAST-2 and FastA sequence alignmnt analysis of the fuln-length sequence, PR0533 shows amino acid sequence identity to fibroblast growth factor The oligonucleotide sequences used in the above procedure were the following: FGF1S.forward: 5'-ATCCGCCCAGATGGCrACAATGTGTA.3' (SEQ ID FGFIS.probe: S'-GCCTCCCGGTCTCCCTrGAGCAGTGCCMAACAGCGGCAGTGTA..3' (SEQ ID NO:6 1); FGF1S.reverse: S'-CCAGTCCGGTGACAAGCCCA)JA-3* (SEQ ID NO:62).

EXAMPLE11: Isolation of cDNA Clones Encoding Humane PRO245 A onsensus 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 DNA30954.

Based on the DNA30954 consensus sequiae oligornicletides were synthesized co identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the MIl-length coding sequence far PR0245.

A pair of PCR primers (forward and reverse) were synthesized: forwrd PCR pri S'-ATCGITGTGAA7TAGTGCCC-3' (SEQ ED *reverse PCR primac 5'-ACCTGCGATATCCAACAGAArG-3- (SEQ ID NO:66) Additionally, a synthetic oligoriucleotide hybridization probe was constructed from the consensus DNA30954 sequence which had the following nucleotide sequence hybridizaton probe 5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGI-jCC..3. (SEQ ID NO:67) :In order to screen several libraries for a source of a fuI-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0245 gene using the probe oligonucleotide -and one of the PCR primers.

of RNA for construction of the cDNA Libraries was isolated from human fetal liver tissue. DNA=cm~ng of the clones isolated as described above gave the ful-length DNA sequence for PR0245 [herein designated as UNQ219 (DNA35638-1 141)] and the derived protein sequence for PROM4.

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

25 Clone UNQ219 (DNA35638-1 14 1) contains a single open reading frame 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 predicd polyeptide precursor is 312 amino acids long (Fig. 24). Clone UNQ219 (DNA3563S-l 141) has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no. ATCC 209265.

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

EXAMPLE 12: Isolation of eDNA Clones Encoding Human PR0220, PRO221 and PR0227 PRO22f A consensus DNA sequence was assembled relative to die other identified EST sequences as described in Example I above, wherein the consensus sequence is designated herein as DMA28749. Based on the DNA29749 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 MI-length coding sequence for PR0220.

A pair of PCR primers (forward and reverse) were synthesized: fayr-C 12imr .TCACCTGGAGCC[TATrGGCC..3 *(SEQ ID NO:74) reverse PCR prmer 5S-ATACCAGCTATAACCAGGC~rCG.3- (SEQ ID NO:7S) Additionally, a synthetic oligortucleoddc. hybridization probe was constructed from the consensus DNA29749 sequence which had the following nucleotide sequence: hyrdzto rb M TCCTCAACAAArTAG GG3(SEQ ID NO:76).

In order to screen several libraries for a source of a full-length clone. DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO220 genm using the probe oligomicleotide and one of the PCR primers.

.RNA for construction of the eDNA libraries was isolated from human fetal lung tissue. DNAseqtcming of the clones isolated as described above give the fiu-length DNA sequence for PR0220 [herein designated as UNQ194 (DNA32298-1132) and the derived protein sequence for PR0220.

The entire nucleotide sequence of UNQ194 (DNA32299-1132) is shown in Figure 25 (SEQ ED NO:68).

Clone UNQ194 (DNA32298-1132) contains a single open reading framne with an apparent translational initiation site at nucleotdec positions 480-482 and ending at the stop codon at macleotide positions 2604-2606 (Figure 25). The predicted polypeptide precursor is 708 amino acids long (Figure 26). Clone UNQ194 (DNA32298-1132) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209257.

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

PRO221 A consensus DNA sequence was assembled relative to the other identfied EST sequences as described in Example 1 above, wherein the consensus sequence is designated herein as DNA2856. 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 die MIl-length coding sequence for PR0221.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primier 5'-CCATGTGTCTCCTCCTACAAAG.3* (SEQ ID NO:77) reverse PCR p~rimer 5'GGGAATAGATGTGATCTGrOG-JJ.3. (SEQ ID NO:78) Additionally, a synthetic oligomucleotide hybridization probe was constncted from the consensus DNA28756 sequence which had the following nucleotide sequence: 5'CCTTGATCATTAGAAACAAACICT~G3(E ID NO:79) In order to screwn several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplifcation with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0221 gene using the probe Oligonucleotide and one of the PCR primers.

RNA for cons truction of the cDNA libraries was isolated from hwnan fetal lung tissue. DNAxqrring of the clones isolated as described above gave die full-length DNA sequence for PRCY221 (herein designated as UNQ195 (DNA33089-1 132) and the derived protein sequence for PR0221.

The entire nucleotide sequence of UNQ195 (DNA33089-1132) is shown in Figure 27 (SEQ ID Clone UNQ195 (DNA330&91 132) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 179-181 and ending at the stop codon at nucleotide positions 956-958 (Figure 27). lhe predicted polypeptde precursor is 259 amino acids long (Figure 28). PR0221 is believed to have a transmembrane region at amino acids 206-225. Clone UNQ195 (DNA33089-l 132) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209262.

Analysis of the amin acid sequence of the full-length PR0221 shows it has homology to member of dhe 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 1 above. wherein the consensus sequence is designated herein as DNA29740. Based on the DNA28740 15 consensus sequence, oligonucleotides were synthesized to idenify 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 PR0227.

A pair of PCR primers (forward and reverse) were synthesized: forward PrR MWme 5'-AGCAACCGCCrGAAGCTCACC.3* (SEQ ID .*reverse PCR pirimer 5'-AAGGCGCGGTGAAAGATGTAGACG..3' (SEQ ID NO:81) Additionally, a synthetic ofigonucleotdec hybridization probe was constructed from dhe consensus DNA28740 sequence which had the following nucleotide sequence: ATGG~GCA3(SEQ ID NO:82).

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 PRO227 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. DNAw9rrg of the clones isolated as described above gave the full-length DNA sequence for PR0227 (herein designated a3 UJNQ201 (DNA33786-1 132) and the derived protein sequence for PR0227.

The entire mucleotide sequence of UNQ201 (DNA33786-I 132) is shown in Figure 29 (SEQ ID NO:72).

Clone UNQ201 (DNA33786-1132) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 117-119 and ending at the stop codon at nucleotidie positions 1977-1979 (Figure 29). The predicted polypeptide preursor is 620 amino acids long (Figure 30). PR0227 is believed to have a t==nmembrane region. Clone UNQ201 (DNA33786-1132) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209253.

Analysis of the amino acid sequence of the full-length PR0221 shows it has homlogy to mierber of the Icucuic rich repeat protein superfamily, including dhe platelet glycoprotein V precursor and the human glycoprotein

V.

EAh2LEZf: Isolation of cDNA Clones Fiicnding Hurnan PRO258 A consensus DNA sequence was assemnbled relative to other EST sequences using pra-p as described in Example 1 above. Ibis consensus sequenc is herein designated DNA28746.

Based an the DNA28746 consensus sequence. oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO2S8.

PCR primers (forward and reverse) were synthesized: forward PCR primer 5'-GCTAGGAAITCCACAGAAGCCC-3- (SEQ ID NO:SS) rees PC Srmr 5-AACCrGGAATGTCACCGAGCTG-3* (SEQ ID NO.:6) reverse PCR p~rimer 5'-CCTAGCACAGTGACGAGGGACTrGC;(3- (SEQ ID NO:87) Additionally. synthetic oligoauelcotide hybridization probes were constructed from the consensus DNA29740 sequence which had the following nucleotide sequence: hybtidizatin jrobe 5S-AAGACACAGCCACCCTAArGTCAGTC7CGjGGAGCAGCC-IYAGCC.3.(SEQ ID NO:88) 5SGCCCTGGCAGACGAGGGCGAGTACACCTGCrCAATCCACTJATGCTGYT3. (SEQ ID NO:89) In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened 20 by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0258 gene using the probe oligonucleotide and one of the PCR primers.

.:RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. DNAsegiencirg of the clonies isolated as described above gave the full-lnt DN equence frP05 hri eintda UNQ225 (DNA35918-l 174)1 (SEQ ID NO:83) and the derived protein sequence for PR02j8.

The entire nucleotide sequence of UNQ225 (DNA35918-1174) is shown in Figure 31 (SEQ ID NO:83).

Clone UNQ22S (DNA3S918-1174) contins a single open reading frame with an apparent translational initiation site at nucleotide positions 147-149 of SEQ ID 14O:83 and ending at the stop codon after nucleotide position 1340 of SEQ ID NO:83 (Figure 31). The predicted polypepidec precursor is 398 amnino acids long (Figure 32). Clone UNQ225 (DNA35918-l 174) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209402.

Analysis of the amino acid sequence of the 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 superfamnily.

EAMPEU14: Isolation of cDNA Clones Enodina Human PRO266 An expressed sequence tag database was searched for ESTs having homology to SLIT, resulting in the identification of a single EST sequence: designated herein as T73996. Based on the 173996 EST sequence.

oligonucleotides were synthesized: 1) to identify by PCR a eDNA library that contained the sequence of interest, and 2) fr use as probes to isolate a clone of the ful-length coding sequence for PR0266.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR pri*e S'-GGGATCrGGGCAACAATAAC-3' (SEQ ID NO:92) reverse PCR grimer S'-AUrG1TGTGCAGGCTGAGfTAAG-3Y (SEQ ID) NO:93) Additionally, a synthetic oligorrecleotide hybridization probe was constructed which had the following nucleotdec sequence hybrdization probe S'-GGTGGCTATACATGGATAGCAATACCTGGACACGCGTCCCGGG.3 (SEQ ID NO:94) In order to scree several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR simplification 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 primiers.

RNA for construction of tie eDNA libraries was isolated from human fetal brain tissue. DNAntqrsng of the clones isolated as described above gave the full-length DNA sequence for PR0266 [herein designated as UNQ233 (DNA3715O-1 178)1 (SEQ ID NO:90) and the derived protein sequence for PR0266.

The entire micloodde sequence of UNQ233 (DNA37150-1178) is shown in Figure 33 (SEQ ID Clone UNQ233 (DNA37ISO-1 178) contains a single open reading frame with an apparent translational initiation site atnuclecotide positions 167-169 and ending at the stop codon after nucleotide position 2254 of SEQ ID NO:90. The predicted polypeptide precursor is 696 amino acids long (Figure 34). Clone UNQ233 (DNA3715O-1 178) has been :deposited with ATCC and is assigned ATCC deposit no. ATCC 209401.

*Analysis of the amino acid sequence of the MIl-length PR0266 polypeptide suggests that portions of it possess significant homology to the SLIT protein, thereby indicating that PR0266 may be a novel leucine rich repeat protein.

****EXAMPTLEJ15: Isolation of cDNA Clones Encoding Human PRO269 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 DNA357OS. Based on the DNA35705 consensus sequnce, 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-lengtht coding sequence for PR0269.

Forward and reverse PCR primers were synthesized: forward PCR pdrimr(.fl) 5*-TGGAAGGAGATGCGATt3CCACCTG -3' (SEQ ID NO:97) forward PCR primer 5S-TGACCAGTGGGGMAGGACAG-3* (SEQ ID NO:98) forward PCR grimer (M1) 5'-ACAGAGCAGAGGGTGCCTTG-3' (SEQ ID NO:99) reverse ECR primer S'-TCAGGGACAAGTGGTGTCTCTCCC-3' (SEQ ID NO: 100) reverse PCR primer (4r) 5'-TCAGGGAAGGAGTGTGCAGflCTG-3' (SEQ ID NO: 101) Additionally. a synthetic oligomiceocde hybridization probe was constructed from the Consensus DNA,3S705 sequence which had the following nucleotide sequence: *-ACAGCTCCCGATCTCAGTTACrFGCATCGCGGACGAAATCGGC 1XTCC3 (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 amnplification with the PCR primer pairs identified above. A positive library was then used to isolate clone encoding the PR0269 gene using the probe oliganucleotide anid one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from htuman fetal kidney tissue.

DNA sequencing of the clo=e isolated as described above gave the full-length DNA sequence for OR0269 (herrin designated, as UNQ236 (DNA38260-i 180)) (SEQ ID NO:95) and the derived protein sequence for PR0269.

The entire nucleodde sequence of UNQ236 (DNA3826D-1180) Is shown in lFigure 35 (SEQ ID Clon UNQ236 (DNA38260-118Q) contains a single open reading fiam with an apparent translational initiation site at nucleotide positions 314-316 and ending at the stop codon at nucleotide positions 1784-1786 (Fig. 35, SEQ ID NO:95). Tbr predicted polypeptide precursor is 490 amino acids long (Fig. 36). Clone UNQ236 (DNA38260-1 180) has been deposited with ATCC and is assigne ATCC deposit no. ATCC 20997.

Analysis of the amnom 2id sequence of the full-length PR0269 suggests that portions of it possess significant homcology to the humn thrmbomodulin proteins, thereby indicating that PR0269 may possess one or more thrombomodulin-like domains.

EXAMPLE1 M Isolation of eD)NA Clones ncoding. Human PR287 A consusDNAseecoding PRO2wasassemdrelative tothe other idntfied STsqujence as descibed in Example 1 above, wherein the consensus sequence is designated herein as DNA28 Based on the DNA2MS2 consensus sequence. oligonaiceotides 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 PR0287.

A pair of PCR primers (forward and reverse) were synthesized: forard PcR primer S'CCGATrCATAGACCCGAGACF.3- (SEQ ID NO: 105) reverse PCR DTimer 5'-GTCAAGGAGTCCTCCACAATAC.3* (SEQ ID NO: 106) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA2872 sequence which had the following ancleoude sequence 5*-GTGTACAATGGCCATGCCAATGGCCAGCGCJGCCGCPJ=GT- 3 (SEQ ID NO:107) In order to screeni several libraries for a source of a full-letigth clone, DNA from die libraries was screened by PCR amplifition with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0287 gene using the probe oligonucleotide arnd 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 PRO28 (herein designated as UNQ2SO (DNA39969-1 185). SEQ ID NO: 1031 and the derived protein sequence for PROW8.

The entire nucleoude sequence of UJNQ250 (DNA39969-1195) is shown in Figure 37 (SEQ ID NO: 103).

C3o= UNQ250 (DNA39969-i1895) contains a single open reading frame with an apparent translationa initiation site at nucleotide positions 307-309 and ending at the stop codon at nucleotide positions 1552-1554 (Fig. 37; SEQ tD NO:103). The predicted polypeptide precursor is 415 amino 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 amin acid sequence of die full-length PR0287 suggests that it may possess one or maore procollagen C-proteinase enhancer protein precursor or procollagen C-proteinase enhancer protein-like domains.

Based on a BLAST and FastA sequence alignmnt analysis of the full-length sequence, PR0287 shows nucleic acid sequence identity to procollagen C-proteinase enhancer protein precursor and procollagen C-proteinase enhancer protein (47 and 54%, respectively).

4 ~XMPLEI: 17 hladio ofcNACoes EMcodin Humn P!RQ214 A consensus DNA sequence was assembled using phrap as described in Example I above. This consensus *.DNA sequence is designated herein as DNA1S744. Based on this consensus sequence. oligonucleotides were s)Uhesized: 1) to identify by PCR a eDNA library that contained the sequence of interest, and 2) for use as probes ;:15 to isolate a clone of the full-length coding sequence.

In order to screen several libiaries for a source of n full-length clone, DNA from the libraries was screened by PCR amiplification with the PCR primer pair identified below. A positive library was then used to isolate clones :x :encoding the PR0214 gene using the probe oligonucleotdec and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. A cDNA clone was sequenced in its entirety. The full length nucleotide sequence of DNA32286-1191 is shown in Figure 39 (SEQ ID NO:108). DNA32286-1 191 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 amino acids long (SEQ ID NO: 109).

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

The oligonucleotide sequences used in the above procedure were the following: 28744.p

S.-CCGGCTATCAGCAGGTGGGCCCAGTGTCGAGTGGATAGTTA

3 (SEQ ID NO: 110) 29744.f (0U556) s5-A7IWFGCGTGAACACTGAGGGC.3* (SEQ ID NO: 111) 28744.r (0U1557) 5'-ATCTGCTTGTAGCCCrCGGCAC..3- (SEQ ID NO:112) EXAMPE I R: Isolation of eDNA Clones Egcong Human PRO'417 A consensus DNA sequence was assembled using phrap as described in Example 1 above, wherein the consensus sequence is herein designated as DNA28M2. Based on this consensus sequence. oligonuctcotides were syntheized: 1) to idetify by PCR a cDNA library that contained the sequence of interest, and 2) for use as Probes to isolate a clone of the ful-lngth coding sequence. The forward and reverse PCR primers, respectively. synthesized for this purpose were: (0U4 89) (SEQ ID NO: 115) and (0U490) (SEQ MD NO: 116).

The probe synthesized for this purpose was: 5*-TGTGTGGACATAGACGAGGCCGcccAcCCAGACdc (0U488) (SEQ ID NO: 117) neRNA for construction of the cDNA Libraries was isolated from human fetal kIdney tissue.

In order to srcm several libaries for a source of a full-length clone, DNA from the libraries was screened by PCR amp~lification. as per Ausubel et al. Current 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 oligonuclcotide identified above and one of the PCR primers.

A cDNA done was sequenced in its entirety. Tbr entire nucleotide sequence of DNA33461-1199 (encoding *PR0317) is shown in Figure: 41 (SEQ ID NO: 113). Clone DNA33461-1199 containts a single open reading frame with an apparent translational initiation site at nucleotide positions 68-70 (Fig. 41; SEQ ID NO:113). The predicted polypeptdec precursor is 366 amino acids long. The predicted signal sequence is amino acids 1-18 of Figure 42 (SEQ IDNO:114). There is one predicted N-linked glycosylation site at amino acid residue 160. Clone DNA33461-1 199 has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209367.

Based on BLAST'h and FastA~hi seqluence alignment analysis (using the ALIGN'U computer program) of the full-length PRO317sequence, PR0317 shows the most amino acid sequence identity to EBAF-I The results also demonstrate a significantbhornology between himan PR0317 and mouse LEFTY protein. The C-terminal end of the PR0317 protein contains many conserved sequences consistent with the pattern expected of a memaber of the TUF- superfamily.

I situ expression analysis in human tissues performed as described below evidences that there is distinctly strong expression of the PR0317 polypeptide in pancreatic tissue.

EXAMPLrE 19: Isolation of rDNA clones Encodir Human PRO301 A consensus DNA sequence designated herein as DNA35936 was assemhbled using plirap as described in Example I above. Based on this consensus sequence. oligormcleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence.

In order to 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 dhe probe oligonaicleotide and one of the PCR primers.

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

A cDNA clone was sequenced in its eniirety. Thie full length micleotide sequence of native sequence PRO301 is shown in Figure 43 (ME H) NO: 118). Clone DNA40628-1216 contains a single open reading franie with an apparent translational initiation site at nucleotide positions 52-54 (Fig. 43; SEQ ID 14O:118). The predicted polypeptide precursor is 299 amino acids long with a predicted molecular. weight of 32,583 daltons and pI of 8.29.

Clone DNA40628-1216 has been deposited with ATCC and is assigned ATCC deposit No. ATCC 209432.

Based on a BLAST and FastA sequence alignment analysis of the ful-lengt sequence. PRO301 shows amino add sequec identity to A33 antigen precursor and coxsackic and adenovirus receptor protein The oligonucleotide sequences used in the above procedure were dhe following: 01.12162 (3S936.fl) 5*-TCGCGGAGCTGTGI TGITCCC-31 (SEQ ED NO:120) 0L12163 (35936.pl) 5S.TGATCGCGATGGGACAAAGGCGCAGCCGAGAACT-JYJ1GCC- 3 (SEQ MD NO: 121) 0L12164 (35936.t2) S'-ACACCTGGTI'CAAAGATGGG-3'(SEQ ID NO:122) 012165 (35936.rl) .5S-TAGOAAGAGTGCTGAAGGCACGG-3- (SEQ ID NO:123) 0L12166 (35936.03) 5'-TTGCCflACCAGGTrGCTAC-3* (SEQ MD NO:124) OL012167 (35936.r2) S*-ACTCAGCAGTGGTAGGAAAG-3- (SEQ MD NO: 125) EXAMPELE 20: Isolation of cDNA Clones Encodin Human PRO224 A consensus DNA sequence assembled relative to the other identified EST sequences as described in Exoanmile 1. wherein the consensus sxequwe is designated herein as DNA30845. Based on the DNA30845 consensus sequence. oligoraelotides were synthesized toidentif by PCR a cD)NA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PR0224.

:A pair of PCR primers (forward and reverse) were synthesized: **.forward PCR uprimne 5'-AAGTrCCAGTGCCGCACCAGTGGC-3' (SEQ ID NO: 128) reverse PCR primer 5'-TTGGTrCCACAGCCGAGCTCGTCO..3- (SEQ ID NO: 129) Additionally, a synthetic oligonucleooide hybridization probe was constructed from the consensus DNA30845 :o.osequence which had the following nucleotide sequence S-GAGGAGGAGTGCAGGAGAGCCATGTACCCAGGITGCCACC.3' (SEQ ID NQ:130) In order to screen several libtaries 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 fth PR0224 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolatcd from human fetal liver tissue. DNAw xrkg of the clones isolated as described above gave the full-length DNA sequence for PR0224 [herein designated as UNQ198 (DNA33221-l 133)] and the derived protein sequencie for PROM2.

The entire nucleotide sequence of UNQl 99(DNA33221-1133) is shown in Figure 45 (SEQ ID NO: 126).

Clon UNQ198 (DNA33221-1133) contains a single open reading fram with an apparent translational initiatlin site at nucleotide positions 96-98 and ending at the stop codon at nucleotide positions 942-944 (Figure 45; SEQ ID NO: 126). The start of a transinembrane region begins at nucleotide position 777. The predicted polypeptide precursor is 282 amino acids long (Figure 46). Clone UNQ198 (DNA33221-1133) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 20963.

Analysis of the amino acid sequence of the ful-length PR0224 suggest& that it has homology to very low' density lipoprotein receptors. apolipoprotei E receptor and chicken cocytc receptors P95. Based on a BLAST and FastA sequence aligrnmenh analysis of the full-length sequence. PR0224 has amio 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%.

]EXAMPEB2 1: Isolation of cDNA Clones Encodine Human PRO=2 A consensus DNA sequence was assembled relative to the other identified EST sequences as described in Example 1 above. wherein the consensus sequene is designaed herein as DNA28771. Based on the DNA28771 consensus sequence. oligonucleoties were synthesized to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PR0222.

A pair of PCR primers (forward and reverse) were sydieslzed: fowr PRD'ne -ATCTCCTATCGCTGCTMTCCGG-3' (SEQ ID NO:133) *reverse PCR lmimcr 5'-AGCCAGGATCGCAGTACTC3* (SEQ ID NO:134) see Additionally, a synthetic oligonucleotide hybridiztation probe was constructed from the consensus DNA28771 sequence which had the following nucleotide sequence: hb~ridizattinn probe 9:.:15 5,-AMrAAAcTFGATGGGTCTGcGTATcT JAGTG ACAAAcc1-ATCT-3* (SEQ ID NO:135) In order to scree several libraries for a source of a full-length clone. DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO22 gene using the probe oligonucleodde and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from humnan fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0222 (herein designated as UNQ196 (DNA33107-1135)] and the derived protein sequence for PR0222.

0. The entire nucacotlac sequence of UNQ196 (DNA33107-1135) is shown in Figure 47 (SEQ MD NO: 13 1).

Clone UNQ196 (DNA33 107-1 135) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 159-161 and ending at the stop codon at nucleotide positions 1629-1631 (Fig. 47; SEQ ID NO:131). Thepredictedpolypeptdeprcursois490auno 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 PastA sequence alignment analysis of the full4ength sequence. PR0222 shows amino acid sequence identity to mouse complement factor h precursor complement receptor mouse complement C3b receptor type 2 long form precursor (2.5-47 and hwrnan hypothetical protein kia247 EXAMPLE 22: Isolation of cDNA clones Encodine PR234 A consensus DNA sequence was assembled (DNA30926) using phrap as described in Example I above.

Based on this consensus sequence. oligonucleotides were sytuhesized: 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 ful-length coding sequence.

RNA for the construction of the CDNA libraries was isolated using standard isolation protocols. e.g., Ausubel et at.. Current Protocols In Molecular Biology, from tissue or cell line sources or it was purchased from commercial gources alonteeh). The cDNA libraries wcd to isolate the cDNA clones were constructod by standard methods Ausubel et at.) wing commnercially available reagents Invitrogen). This library was derived from 22 week old feta brain tissue.

A cDNA dlone was sequenced in its entirety. The entire micleotde sequence of PR0234 is shown in Figure 49 (SEQ ID NO:136). The predicted potypeptide precursor is 382 amino acids long and has a calculated miolecular weight of approximately 43.1 Wa.

The oligonucleotide sequences wsed in the above procedure were the following: 3 0 9 2 6.p (0U8S26) (SEQ MD NO: 138): GATGGTGAC=lCGGATrGGGCTCA-3* 3M96.f (0U827) (SEQ ID NO:139): S-AAGCCAAAGAAGCCTGCAGGAGGG-3* 30926.r (0US28) (SEQ MD NO: 140): S'-CAGTCCAAGCATAAAGYGTCCTGGC-31 ~EXAMPLE 23: Isolation of cDNA Clamsi Encodin Huma~n 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 D14A30933. Based on the DNA30933 *15 consensus sequence. aligonucleotides 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 Mil-length coding sequence for PR023 1.

Three PCR primers (tw~o forward and one reverse) were synthesized: forward PCR primer I 5'-CCAACTACCAAAGCTGCTGGAGCC-3- (SEQ ID NO:l43) forar R rier2 5'GrCAGC'CTATAcCACGGGAAGGA-3 (SEQ MD NO.144) reverse PCR nrixner 5*-TCCTCCCGTGGTAATAGAGCTGC-3' (SEQ ID 140:145) Additionally. a synthetic oliganuceotide hybridization probe was constructed from the consensus DNA30933 sequence which had the following nuelcotdec sequence hbidizatinpbe (SEQ ID NO: 146) as:25 In order to screen several libraries for a source of a Mil-length clone. DNA from the libraries was screened by PCI amplification with the PCI primer pairs identified above. A positive library was then used to isolate clones encoding the PR0231 gene using the probe oligonudeotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. DNAwqxmng of the clones isolated as described above gave the full-length DNA sequee for PR0231 [herein designated as UNQ205 (DNA34434-1 139)) and the derived protein sequence for PR023 I.

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

Clone UNQ205 (DNA34434-1139) contains a single open reading fram 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 pobypdde precursor is 428 arnino acids long (Fig. 52). Clone UNQ205 (DNA34434-1 139) has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no. ATCC 209252.

Analysis of the amin acid sequence of the full-length PR0231 suggests that it possesses 30% and 31 amino acid identity with the human and rat prostatic adid phosphatase precursor proteins, respectively.

EXAMELEli: LIoation of cDNA Clones Encndiny Human PR022 A consensus DNA sequence was assembled rciative to other EST sequences using phrap as described in Example I above. This consensus sequence is herein designated DNA28762. BSed on the DNA29762 consensus sequence, oligonucotdes wcre synthesized: 1) to identify by PCR a cDNA librazy that contained the sequence of interest, and 2) for use as probes to isolate a clone of die full-length coding sequence for PR0229.

A pair of PCR primers (forward and reverse) were synthesized: frar CRprm 5'-TFCAGCrCATCACCTrCACCTGCC-3' (SEQ ID NO: 149) reverse PCR mimer 5'-GGCTCATACAAATACCACAGJI.3' (SEQ ID NO: 150) Additionally, a synthetic ofigonucleotide hybridization probe was constructed from dhe consensus DNA28762 sequence-which had the following nucodde sequence 5 SGGOCCTCCACCGrGTGAAGGGCGGGTAGGTGG

CAGGCAT

3 .(SEQ ID NO:151) In order to screen several librries for a source of a full-length clone, DNA from the libraries was screened by PCR amplication with thec PCR priner pair identified above. A positive library was then used to isolate clones 15 encoding the PR0229 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of die cDNA libraries was isolated from hurman fetal liver tissue. DMAsqmd* of the clones isolated as desribed above gave the ful-length DNA sequence for PR0229 [herein designated as UNQ203 (DNA233100-1159)l (SEQ ID NO: 147) and the derived protein sequence for PR0229.

:0.:0The entire nudleotide sequence of UJNQ203 (DNA331OO-1 I59) is shown in Figure 53 (SEQ ID NO: 147).

Clone UNQ203 (DNA33I100-11159) comins a single open reading frame with an apparent translational initiation site at nucleotide positions 98-100 and ending at die stop codon at nucleotide positions 1139-1141 (Figure 53). The *00.0*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 25 possess significant homology to antigen wcl.1, M130 antigen and CD6.

EXAMPLE 25: Isolation of cDN Clones Encodiri, Iunan PRQflR A consensus DNA sequence was assembled relaive to other EST sequences using pbrap as described above in Example 1. This consensus sequence is herein designated DNA3O908. Based on the DNA30908 consensus sequence, olig onucleotides were synthesized: 1) to identify by PCR a eDNA library that contained the sequence of interest, and 2) for wse as probes to isolate a clone of the full-length coding sequence for PROM3.

PCR primers (forward mnd reverse) were synthesized: forward PCR grimcr I 5S-GGTUCAAACTGTGErTGC.

3 (SEQ ID NO:154) forward PCR stimer 2 5'-CAGGGCAAGATGAGCATT-.3' (SEQ 0) NO: 155) reverse PCR primer 5'-TCATACrGTTCCATrCGCACC 3 (SEQ ID NO:1S6) Additionally, a synthetic oligomicleotide hybridization probe was constructed from the consensus DNA30908 sequence which had the following mczlotide sequence hvlridization probe 5'-AATOGTGGGGCCCTAGAAGAGCTCATCAGAGAACTCACCGTTCATGC.3 (SEQ ID NO: 157) In order to screen several librauies for a source of a full-length clone, DNA from the libraries was screenecd by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0238 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from humnan fetal liver tissue. DNAsbqxnjng of the clones isolated as described above gave the full-length DNA sequence for PR0238 and the derived protein sequence for PROM3.

The cadre nucleodde sequence of DNA35600-1162 is shown in Figure 55 (SEQ ID NO: 152). Clone DNA35600-1162 contains a single open reading (minme with an apparent translational initiation site at micleotide positions 134-136 and ending prior to the stop codon at nucleotide positions 1064-1066 (Figure 55). The predicted polypeptide precursor is 310 amino acids long (Figure 56). Clonw DNA356OD-1162 has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209370.

Analysis of the amin acid sequence of the full-length PR0238 polypeptide suggests that portions o f it possess significant homology to reductase, particularly oxidoreductase, thereby indicating that PR0238 may be a novel reductase.

£XAMPLIR 26: lsolation of eDNA Cones Encodsw Humn PRO733 Tbr exnrcdiUlr domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) :databases. The EST database included public EST databases GenBank) and a proprietary EST DNA database

(UPESEQ

TM

Incyte Pharmaceuticals. Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altshul et al.. ehd nFM de 266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6 framec translation of the EST sequence. Those comparisons resulting in a BLAST score of 70 (or 25 in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequeces with the program "phrap (Phil Green, -University of Washington. Seattle. Washington; hup:/Abozeman.mbt.washington.edu/phlap.docs/phrap.hunl).

An expressed sequme tag (EST) was identified by the EST database search and a consensus DNA sequence was assembled relativc to other EST sequences using phrap. This consensus sequence is herein designated DNA30945. Based on the DNA30945 cosensus sequ~ee. oligonuclootides 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 sequece= for PR0233.

Forward and reverse PCR primers were synthesized: forward PCR p'me 5'-GGTGAAGGCAGAAATrGG3AGAT3-3' (SEQ ID NO: 160) reverse PCR mimer 5*-ATCCCATGCATCAGCCTG=rACC.3* (SEQ I'D NO: 161) Additionally. a synthetic oligonueceotide hybridization probe was constructed from the consensus DNA30945 sequence which had the following nicleotide sequence hvbridiinn probe 3 (SEQ ID NO: 162) In order to screen several libraries for a source of a ful-length clone, DNA from the librarie s was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0233 gene using the piobc olig~onucleod RNA for construction of the eDNA libraries was isolated from human fetal brain tissue. DNAieqming of the clones isolated as described above gave the full-length DNA sequence for PR0233 (herein designated as UNQ207 (DNA34436-1238) (SEQ ID NO:I58) and the derived protein sequence for PR0233. The entire macleodde sequence of UNQ2O7 (DNA34436-1238) is shown in Figure 57 (SEQ ID NO: 158).

Clone UNQ2O7 (DNA34436-1238) cntains a single open reading frame with an apparent translational initiation site at nucleotide positions 101-103 and ending at the stop codon at macleotide positions 1001-1003 (Figure S7). The *.*.predicted polypeptide precursor is 300 amino acids long (Figure 58). The full-length PR0233 protein shown in Figure 58 has an estimated molecular weight of about 32.964 daltons; and a pl of about 9.52. Clone UNQ207 15 (DNA34436- 1238) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209523.

Analysis of the amino acid sequence of the full-lenigth PR0233 polypeptide suggests that portions of it possess significant homology to reductase proteins, thereby indicating that PR0233 may be a novel reductase.

EAMPLE 2 Isolation of cDNA Cons Encdinr Human PR223 A consensus DNA sequence was assanbled relative to other EST sequences using phrap as described in Example I above. This consensus sequence is herein designated DNA30836. Based on the DNA30836 consensus sequence. oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of ***.interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0223.

PCR primer pairs (one forward and two reverse) were synthesized: forward PCR primier 5'-TTCCATGCCACCTAAGGGAGACTC3* (SEQ ID NO:165) reverse PCR primer I S*-TGGATGAGGTGTGC.J4TGGCTGGC.3' (SEQ DNO: 166) reverse PCR prnimer 2 S'-AGCFCTCAGAGGCTGGTCATAGGG-3' (SEQ ID NO: 167) Additionally, a synthetic oligonudeotide hybridization probe was constructed from the consensus. DNA30836 sequence which had the following nucleotide sequence I CCGATACTAG~rTCGCTCCC (SEQ ID NO:168) In order to screen several libraries for a source of a full-lngth clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0223 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. DNAie*ciag of the clones isolated as described above gave the full-length DNA sequence for PR0223 (herein designated as UNQ197 (DNA33206-1 165)] (SEQ ID NO:163) and the derived protein sequence for PR0223.

Thc entire nucleotdde sequence of UNQ1g? (DNA33206-I 165) is shown in Figure 59 (SEQ MD NO: 163).

Clowe UNQ197 (DNA33206-1 165) contains a single open reading frame with an apparent tralasional initiation site at nucleotide positions 97-99 and ending at the stop codon at nuclode positions 1525-1527 (Figure 59). The predicted polypeptide precursor is 476 amino acids long (Figure 60). Clone UNQ197 (DNA33206-1 165) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 20972.

Analysis of the amino acid sequence of the full-length PR0223 polypeptide suggests that it possesses significant homology to various seuine carboxypeptidase proteins, thereby indicating that PR0223 ay be a novel serine carboxypeptidase.

EXAMPLE 29: Isolatinn Of enNA Clones Encoding Human PRO235 A consensus DNA sequence was assembled relative to other EST sequences using pbrap as described in' Example I above. iThis consensus sequtioe is herein designated 'DNA30927'. Based on the DNA30927 consensus sequience, oligonudeoddes 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 PROM3.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer S*-TGGAATACCGCCrCCTGCAG-3' (SEQ ID NO: 171) reverse PCR primer 5'-CTCTGCCCTFGGAGAAGATGGC-3' (SEQ ID NO: 172) Additionally, a synthetic oLigonuecotde hybridization probe was constructed from the consensus DNA30927 :sequence which had the following nucleotide sequence ybridization probe 5,-GGACrcACTGGCCCAGGCCIFCAATATCACCAGCCAGGACGAT.3' (SEQ ID NO: 173) In order to schj several libraries for a source of a full-length clone, DNA from the libraries was screened by PRamplification with the PCR primer pair Identified above. A positive library was then used to isolate clones encoding the PR0235 gene using the probe oligonucleodde and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal liver tisse. DNAwqixirg :25 of the clones isolated as described above gave the full-length DNA sequence for PR0235 [herein designattd as UNQ209 (DNA35558-1167)] (SEQ ID NO: 169) and the derived protein sequence for PROM3.

The entire nucleotide sequence of UNQ209 (DNA3S55&1 167) is shown in Figure 61 (SEQ ID NO: 169).

Clone UNQ2O9 (DNA35558-1167) cowntan a single open reding 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 amin acids long (Figure 62). Clone UNQ209 (DNA35558-1167) has been deposited with ATCC and is assigned ATCC deposit no. 209374.

Analysis of the am;n acid sequence of the full-length PR0235 polypeptide suggests that portions Of it possess significanit homology to the human, mouse and Xernops plexin protein. thereby indicating that PRO235 may be a novel plexin protein.

IPXAMPLER.29: Isolation of eDNA Clones ErKcoding Hutman PR0236 and Human pR0262 Consensus DNA sequences were assembled relative to other EiST sequences using phrap as described in Example 1 above. These consensus sequences are herein designated DNA30901 and DNA30947. Based on the DNA30901 and DNA30947 consensus sequence oligomucotide3 were synthesized: 1) to identify by PCR a cDNA library that-contained the sequene of interest, and 2) for use as probes to isolate a clone of the fulfl-length coding sequence for PR0236 and PROM6, respectively.

Based upon the DNA30901 consensus sequence, a pair of PCR primers (forard and reverse) were synthesized: forward PCR primer S'-TGGCTACTCCAAGACCCTGGCATG-3* (SEQ IDNO: 178) reverse PCR primer 5'-TGGACAAATCCCCrrGCTCAGCCC.3' (SEQ ID NO: 179) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30901 sequence which had the following nucleotide sequence yfIdditin jmob 5'-GGGUTrCACCGAAGCAGTGGACCTrnFGACCAC~rGATcJTCCGGG-3* (SEQ ID NO:lS0) Based upon the DNA30847 consensus sequence, a pair of PCR primers (forward and reverse) were synthesized: forward PCR lDrimer Y-CCAGCTATGACTATGATGCACC-3' (SEQ tD NO:181) reverse PCR Vrme 5'-TGGCACCCAGAATGGTGTrGGCTC.3- (SEQ ID NO: 182) :Additionally, a synthetic oigonucotidc hybridization probe was constructed from the consensus DNA30847 *sequence which had the following nucleotide sequence hybridization Rrobe 5'-CGAGATGTCATCAGCAAGTrCCAGGAAGTr'CCrIOGACCT7rACCrCC3 (SEQ ID NO:1183) In order to screen several libraries for a source of full-length Clones, DNA from the libraries was screened by PCR ainplificaznn with the PCR priner pairs identified above. Positive libraries were then used to isolate Clones encoding the PR0236 and PR0262 genes using the probe oligonucleotides and one of the PCR primers.

9:25 RNA for construction of the cDNA libraries was isolated from human fetal lung tissue for PR0236 and human fetal liver tissue for PR0262.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0236 [herein designated as UNQ210 (DNA35599-1 168)] (SEQ MD NO: 174). the derived protein sequece= for PR0236.

the fufl-letgth DNA sequence for PRCY262 (herein designated as UNQ229 (DNA36992-1168)J (SEQ MD NO: 176) and the derived protein sequence for PR0262.

The entire nuclotide sequence of UNQ210 (DNA35599-1 168) is shown in Figure 63 (SEQ ID NO:174).

Clone UNQ210 (DNA35599-1 168) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 69-71 and codling at the stop codon at nucleotide positions 1977-1979 (Figure 63). The predicted polypeptide precursor is 636 amino acids long (Figure 64). Clone UNQ210 (DNA3SS99-l 168) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209373.

The entire nucleotide sequence of UNQ229 (DNA36992-1 168) is shown in Figure 65 (SEQ ID NO: 176).

Clone UNQ229 (DNA36992-1 168) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 240-242 and ending at the stop codon at nucleotide positions 2202-22D4 (Figure 65). Thbe predicted polypeptide precursor is 654 amino acids long (Figure 66). Clone UNQ2 29 (DNA36992-1168) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209382.

Analysis at the amino acid sequence of the fuD-length PR0236 and PR0262 polypeptides suggests that Portions of dhose polypeptidcs possess signifiamd homology to P-galactosidase proteins derived from various sources.

thereby indicating that PR10236 and PR0262 may be novel P-galactosidase homologs.

EXAMU.E.3Il: Isolation of' reNA Clones Eacoriing Hum=n pR0239 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. Ibis consensus sequence is herein designated DNA30909. Based on the DNA30909 consensu sequence. oligonueceotdes were synthesized: 1) to identify by PCR a cDNA library diat contained the sequence of interest, and 2) for use as probes to isolate a clone of the ful-ength coding sequence for PROM3.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer 5'-CCTCCCTCTATTACCCATGTC.3* (SEQ ID 140:186) reverse PCR primer 5'-GACCAACrrCTCGGAG~rjAGGJ.3* (SEQ ID NO: 187) Additionally, a synthetic oligonuclcotide hybridization probe was constructed from the consensus ')NA30909 Sequence which had the following nudeaotide sequee hybrdization probe (SEQ ID NO: 188) In order to screent several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with die PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO239 gene using the probe oligonucleoie and one of the PCR primers.

RNA for construcuion of the eDNA libraries was isolated from hirnan feta lung tissue. DNArqxadng of the clones isolated as described above gave the ful-length DNA sequence for PR10239 (herein designated as UNQ213 (DNA34407-1169)] (SEQ ID 140:184) and the derived protein sequence for PRO02M.

The entire nucleotide sequence of UNQ213 (DNA34407-1 169) is shown in Figure 67 (SEQ ID NO: 184)'.

Clone UNQ213 (DNA34407-1169) contains a single opent reading frame with an apparent translational initiation site at nucleotide positions 72-74 and ending at the stop codon at sucleotide positions 1575-1577 (Figure 67). The predicted polypeptide precursor is 501 amin acids long (Figure 68). aonc UNQ2I3 (DNA34407.l 169) has been deposited with ATCC and is assigned ATCC deposit no.ATCC 209383.

Analysis of the amino acid sequence of the full-length PR0239 polypeptide suggests that portions of it possess significant homology to the detisin protein, thereby indicating that PR10239 may be a novel molecule in die densin family.

EXAMPE31: Isolation of eDNA Clones Encodinp Ruma PRO257 A consensus DNA sequence was assembled relative to other EST scquences using phrap as described in Example I above. This consensus sequence is herein designated DNA28731. Based on the DNA28731 consensus sequence. o~igonucleotides 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 at the full-length coding sequence for PR0257.

A pair of PCR primers (forward and reverse) were synthesized: foirward PCR pd=e 5'-TCTCTATrCCAAACTGTGGCU..3' (SEQ ID NO:191) reverse PCR pri=e S'-TITGATGACGATCGAAGGTGG-.3- (SEQ ID NO:192) Additionally. a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28731 sequence which had the following nudeootide sequence 5'GAGAC7CCACCArrCCAGCCTCGG- (SEQ ID NO: 193)' In order to scr= several libraries for a soumce of a full-length clone. DNA from the libraries was screened by P CR amplification with the PCR Orb=e pair idenitied above. A positive Uity was then used to isolate clones encoding dhe PR0257 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from huiman fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0257 [hereindesignaited as UNQ224 (DNA35841-l 173) (SEQ ID NO:l89) and the derived protein sequence for PR0257.

.The entire zmcleotide sequence of UNQ224 (DNA35S4I-1 173) is shown in Figure 69 (SEQ ID N6:189).

Clone UNQ224 (DNA3SS4l-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 precursor is 607 amino acids long (Figure 70). Clone UNQ224 (DNA3SS4l-l 173) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209403.

Analysis of the amin acid sequence of the full-length PR0257 polypeptide suggests that portions of it posses significant homology to the ebnermn protein. thereby indicating that PR0257 may be a novel protein member related to the ebnermn protein.

EXAMPLE 32: Isolation of cDNA Clones Encoding Humnan PRO260 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example I aborve. This consensus sequence 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 timer- 5'-TGGTTGACCAGGCCAG-FCG3' (SEQ ID NO: 196); reverse PCRitrimerAK 5*-GA11CATCCCAGGAJAAC3' (SEQ ID NO:197); and reverse PCR mimer B 5*AACTrGCAGCATCAGCCACTCrGC.3' (SEQ ID NO:198) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30834 sequence which had the following nuicotide sequene trybridization robe' 5-rCCGTGCCCAGCTrCGGTAGCGAGTGGGGTGGfTAT1xCA- 3 (SEQ ID NO:199) In order to sceeni several libraries for a source of a ful-length clone, DNA from the libraries was screened by PCR amplifiaton with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO260 gene using the probe oligorruclootide and one of fhe PCR primers.

RNA for constiuction of the cDNA libraries was isolated from human fetal kcidney tissue.

DNA sequencing of dic cloe isolated as described above gave the full-length DNA sequence for PR0260 [hertin designated as UNQ227 (DNA33470-1175)] (SEQ ID NO: 194) and die derived protein sequecec for PR0260.

The entire nuceoside sequence of UNQ227 (DNA33470-l 175) is shown in Figure 71 (SEQ ID NO: 194).

Clone U14Q227 (DNA33470-117S) catitains a single open reading frame with an apparent tranislational initiation site at nueleotide positions 67-09 and ending at the stop codon 1468-1470 (see Figure 71). The predicted polypeptide precursor is 467 amino acids long (Figure 72). Clone UNQ227 (D14A33470-l 175) has been deposited with ATCC and is assigned ATCC deposit no. AFCC 209398.

Analysis of the amino acid sequence of tie full-length PR0260 polypeptide suggests that portions- of it posses s significant homoelogy to the alpba-l-fucosidase precursor, thereby indicating that MR0260 may be a novel fiacosidase.

EXAMPLE 33: Isolaiion of cDNA Clones Fncoding Humn PRO263 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA30914. Based on the DNA30914 consensus sequence, oligonueleoddes 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 die IMength coding sequence for PRO263.

PCR primers (tow forward and one reverse) were synthesized: :forward PCR nrimer I: S*-4AGCrrCCATCCAGGTGTCATGC-3' (SEQ ID NO:202); forward PCR primer 2- 5'-GTCAGTGACAGTACCTACTCGG-3' (SEQ ID NO:203); reverse PCR nrier 5'-TGGAGCAOGAGOAGTAGTAOTAGC-3' (SEQ ID 140:204) Additionally, a synthetic oligonueleotide hybridization probe was constructed from the consensus DNA30914 sequence which had the following nueleodde sequetice: hybridization grobe: 5'-AGGAGGCCrGTAGGCrGCTGGGACrAAG7TrrGGCCGGCAAGGACCMAGTr.3' (SEQ ID NO:205) In order to screen seveal libraries for a source of a ful-lngth clone. DNA fronm 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 oligonucleodde and one of the PCR'prm* ers.

RNA for construction of the cDNA libraries was isolated from human fetal liver tissue. DNAseqcm*g of the clones isolated as described above gave the ful-length DNA sequence for PRO263 [herein designated as UNQ230 (DNA34431-1177)J (SEQ ID NO:200) and the derived protein sequence for PROW6.

The entire micleotide sequence of UNQ230 (DNA34431-l 177) is shown in figure 73 (SEQ ID 140:200).

Clone UJNQ230 (DNA34431-I 177 contains a single open reading frame with an apparent translational initiation site at sarcloside positions 16D-162 of SEQ ID N4020 satd ening at dhe stop codon aftr the nucleotide a1 position 1126- 1128 of SEQ ID NO:200 (Figure 73)- The predicted polypeptde precursor is 322 amino acids long (Figure 74).

Clone UNQ230 (DNA3443 1-1 177) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209399.

Analysis of the amino acid sequence at the full-lengt PRC7263 polypeptide suggests that portions of it possess significant homlogy to CD44.antigen. thereby indicating that PR0263 may be a novel cell surface adhesion molecule.

EXAMPIL Isolation of cDMA Clones Eneodina Humain PRO2f A consensus DNA sequence was assembled relative to tbe other identified EST sequences as described in Exwape 1 above, wherein the consensus sequence was designated herein as DNA35712 Based on the DNA3S7 12' consensus sequence, oligosmdeotides were synthesized: 1) to identify by PCR a cDNA librury that contained the sequence of interesc, and 2) for use as probes-to isolate a clow of the full-length coding seque for PROM7.

Forward and. reverse PCR primers were synthesized: forwrd PR V 5'GCMGGATATCGCATGGGCCrAC..3' (SEQ ID NO:208) fomrard PCR primer (12) 5'-TGOAGACAATATCCCTOAGG3' (SEQ ID NO:209) reverse PCR primer 5'-AACAGTTGGCCACAGCATGGCAGG.3* (SEQ ID NO:210) Additionally, a synthetic oligonmcleodde hybridization probe was constructed from the consensus DNA35712 :15 sequence which had the following nucleotide sequence :(SEQ ID N0:211) In aider 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 PR0270 gene using the probe oligonaceotde and one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from lman fetal luing tissue. DNAscxxing of the clones isolated as described above gave the full-lengt DNA sequence for PR0270 (herein designated as UNQ237, DNA39S 10-1 1811 (SEQ ID NO:206) and the derived protein sequence for PR0270.

The entire macleotide sequence of UNQ237, DNA39510-1181 is shown in Figure 75 (SEQ ID N0:206).

Clone UNQ237 (DNA39510-1181) contains; a single open reading hame with an apparent translational initiation site at nucleotide positions 3-5 and ending at the stop codon at nucleotide positions 891-893 (Fig. 75, SEQ ID NO:206).

Thepredidpolypepdeprcinsris 296 aino acids long (Fig. 76). Clone UNQ237 (DNA39S10-l181) has been deposited with ATCC and is assigned ATCC deposit no.. ATCC 209392.

Analysis of the amino acid sequence of the Mlength PR0270 su ggests that portions of it possess significant homology to the thioredoxin-protein, thereby indicating that the PR0270 protein 'may be a novel member of the thioredoxin family.

EXAMPE 351: Isolation of eDNA lones Enroding IHuxn-PR027[ A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in Example 1 above. This consensuts sequence hs herein designated DNA35737. Based on the DNA35737 consensus sequence, oigonrucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use a.s probes to isolate a clone of the fs-length coding sequence for PRO271.

Forward and reverse PCR primers were synthesized: forward PCR p~rimier I 5'-TGCITCGCTACTGCCCTC-3' (SEQ MD NO:214) forward PCR prmer 2 S--CCCrrGTGGGTTGGAG.3- (SEQ ID N02 IS) forward PCR erinier 3 5'-AGGGCTGGAAGCCAGfl.3- (SEQ ID 801216) reverse PCR t~rirner I 5'-AGCCAGTGAGGAAATGCc3 (SEQ MD NO-.217) reverse PCR nrinier 2 S'-TGTCCAAAGTACACACACCTGAGG..3. (SEQ ID N0:218) Additionally. a synthetic oligonneleotd hybridization probe was constructed from die consensus DNA35737 sequence which bad the following macleotide sequence 5S-GATGCCACGATCGCCA 4GGTGACGCC CC0CCTGAA-3 (SEQ ID N01219) In o"der to scramn several libraries for a source of a full-length clone. DNA from the libraries was screened by PCR amplification with the PCR primar pair identified above. A positive library was then used to isolate elones ecncoding the PR0271 geme using the probe oligonucleodde and one of the PCR primers.

RNA for construction of the cDNA libraries was isolmtd from human ftal, brain tissuc. DNXAwseqrnng 0: :15 of the clones isolated as described above gave the fil-length DNA sequence for PR0271 [hrein designated as UNQ238 (DNA39423-l 182)] (SEQ ID NO:1212) and the derived protein sequence for PRO27I.

The entire nuclootide sequence of UNQ238 (DNA39423-1 182) is shown in Figure 77 (SEQ ID 140:212).

Cone UNQ238 (DNA39423-1182) comtins a single open reading fraxne with an apparent translational initiation site at nucleotide positions 101-103 and ending at the stop codon at nucleotide positions 1181-1183 (Figure 77). The predicted polypeptide precursor is 360 amino acids long (Figure 78). Clone UNQ238 (DNA39423-1182) 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 significan homology to the proteoglycan link protean thereby indicating that PR0271 may be a link protein homolog.

EXAMPI 36. Isplarinn of eD2NA Clones Enoding HwniiP2gl A consensus DNA sequence was assembled relative to other EST sequences using pbrap as described in Example I above. This consensus sequence is herein designated DNA36460. Based en the DNA36460 consensus sequence. ofigomicleotides 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 sequene for PROM7.

Forward and reverse PCR primers were synthesized: fowr PCR rimr (.flI) 5'-CGCAGGCCCCATGCCAGG-V' (SEQ ID N0:222) forward PCR gd=e 5'-GAAATCCtGG;GTAATTGrJ.3' (SEQ ID NO:223) reverse PCR unmet 5'-GTGCGCGOTGCTACAG~CATC.3' (SEQ ID, 80:224) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA36460 sequence which had the following nucleotide sequence bdization prob S'CCCCCCTGAGCGACGCTCCCCCATGATGCACGGnGATC- 3 (SEQ MD NO:225) In order to soem several libraries for a source of a full-length clone. DNA from dhe libraries was screened by PCR amplification with the PCR primer pairs identified above. A positive library was then used to isolate Clones encoding the PRO272 gene using the probe oligonuclotide and one of the PCR primer.

RNA for construction of the cDNA libraries was isoand from human fetal -lung tissue. DNAerning of the Clones isolated as described above gave the full-lngth DNA sequerice for PR0272 [herein designated as UNQ239 (DNA40620-l 183)1 (SEQ ID NO:220) and the derived protein sequence for PROM7.

The entire nucleotide sequence of UNQ239 (DNA40620-I 183) is shown in Figure 79 (SEQ ID NO:220).

Cione UNQ239 (DNA40620-l 183) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 35-37 and ending at the stop codon at nucleotide positions 1019-1021 (Figure 79). The predicted polypeptidc precursor is 328 amino acids long (Figure 80). Clone UNQ239 (DNA40620-1183) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 20988.

Analysis of dhe am-o acid sequence of the full-length PR0272 polypetide suggests that portions of it possess significant homology to the human and muse rtticulocalbin proteins, respectively, thereby indicating that PR0272 may be a novel reticulocalbin protein.

EAMPLE 37: Isolation of cDNA Clones Encoding Human PRO294 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 DNA35731. Based on the DNA35731 consensus .*:sequez=. cligonucleotides 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 PR0294.

Forward and reverse PCR primers were synthesized: forward PCR primier S'-TGGTCTCGCACACCGATC-3' (SEQ ID N0:228) forardPCRjaie(12) 5'-CTGCTG(TCCACAGOGGAG-3' (SEQ ID NO:229) forwyard! PCR onr S'-CTGAAGCATACTGCTC-3' (SEQ ID NO:230) forward PCR nrimer(.14) 5-GAGATAGCAAflTCCGCC-3' (SEQ ID NO:23 1) revere PCR Vime (il) Y-TI'CCTCAAGAGGGCAGCC-r' (SEQ 11) NO:232) rrse Cpni~e 5'-CTMGGACCAATGTCCGAGATrr-3' (SEQ ID NO:233) Additionally. a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA3573 1 sequence which had the following nucleodde sequence bybridizain grobc 5-GCTCTGAGGAAGGTGACGCGCGGGGCCFCCGAACCCTTGGCCflG.3* (SEQ ID NO:234) In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR am~plification with tic PCR primer pairs Identified above. A positive library was then used to isolate Clones encoding the PR0294 gene using the probe oligonuceotd& and one of the PCR primners.

MNA for construction of the cDNA libraries was isolated from human fetal brain tissue. D)NASeqmi of the Clones isolated as described above gave the full-lenigth DNA sequerce for PR0294 [herein designated as 144 UtN257 (I40604-1187) (SBEQ ID NO:226) and the derive protein sequence for PR0294.

The entire nucleotide sequence of UNQ257 (lA40604-1187) is shown in Figure 81 (SEQ ID N:226). Clone tUN257 (ENA40604-1187) 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). The predicted polypeptide precursor is 550 amino acids long (Figure 82). Clone tRQ257 (IEA40604-1187) has been deposited with ATCC 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 hamrology to portions of various collagen proteins, thereby indicating that PR0294 ay be collagen-like nmolecule.

E23PIE 38: Isolatien of cMA Clones Encxding Hman A consensus IA sequence was asserbled relative to other EST sequences using phrap as described in Eanple 1 above. This consensus sequence is herein designated IA35814. Based on the ENA35814 consensus *sequence, oligonucleotides were synthesized: 1) to identify by ICR a CIlA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO295.

Forward and reverse PCR primers were synthesized: forward PCR primer(.fl) 5' -3CAGOGA'ICAG (SEQ ID MD: 237) forward CR primer(.f2) 5' -CYTtGCAaIGACGCCIA3G-3' (SEQ ID NO:238) forward PCR prinmer(.f3) 5'-TIGOCAGTCA'IGGAG-3' (SEQ ID NO:239) forward PCR prinmer(.f4) 5' -fCI3XCAAAAA UCAAC-3' (SEQ ID NO:240) reverse PR pri er(.rl) 5' -CIaaII3O2 t (SEQ ID NO:241) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus INA35814 sequence which had the following nucleotide sequence.

hybridization probe 3I1G (SEQ ID NO:242) In order to screen several libraries for a source of a fulllength clone, IUA from the libraries vas screened by PCR 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 prinmers.

RNA for construction of the clEA libraries was isolated froman hman fetal lung tissue. INA sequencing of the clones isolated as 145 described above gave the full-length M~ sequence for PRO295 (herein designated as LM~258 (I1Va38268-1l88) (SE)2 ID N:235) and the derived protein sequne for PlR)295.

The entire nucleotide sequence of UtQ258 (CN.38268-ll88) is shamn in Figure 83 (SEX2 ID N:235). Clone U\IQ258 (EN~A38268-ll88) contains a single open reading frame with an apparent .translational initiation site at nucleotide positions 153-155 and ending at the stop codon at nucleotide positions 1202-1204 (Figure 83). The predicted polypeptide precursor is 350 amino acids long (Figure 84). Clone UNQR258 (EN~A38268-1188) has been deposited with ATIOE and is assigned A=C deposit no. 209421.

Analysis of the amino acid sequence of the full-length PR.0295 polypeptide suggests that portions of it possess significant 1xiio1ogy to the integrin proteins, thereby' indicating that PRO295 nay be a novel integrin.

EXAMLE 39: Isolation of c2tQ Clones Ehoodirag If.man PiP293 The extracellular danain (EMD) sequences (including the secretion signal, if any) of fran about 950 known secreted proteins fran the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases. The EST databases included public EST databases GenBank) and a proprietary EST ENAU database (LIFESDW", Incyte Pharanceuticals, Pal Alto, CA). The search was performed using the ccnputer program BLAST or BLAST2 (Al tshul et al., Methods in Enizymology 266:460-480 (1996)) as a cczrpzarison of the EXI) protein sequences to a 6 frame translation of the EST sequence. Those caTparisons resulting in a BLASr score of 70 (or in sarke cases 90) or greater that did not encode known proteins wer clustered and asseabled into consensus EM, sequences with the program 'phrap" (Phil Green, -University of Vaashingjton, Seattle, Washington; http: //bozeren.nbt .weshinigton .ed/phrap.dcs/phrap .htxnl) Based on an expression tag sequence designated herein as T08294 identified in the above analysis, oligonucleotides were synthesized: 1) to identify byj PC 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 PR0293.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR par 51 -AACAPAAtAA'TGCTCCM-3' (SE)2 ID NO:245) reverse ~pCrir 5 -AAXfTrKICGATAGcrpJc=-3' ID NO: 246) Adi tionally, a synthetic oligonucleotide hybridization probe was constructed from the expression sequience tag which had the following 146 nucleotide sequence.

hybridization probe -A C A GOO-3' (SEQ ID NO:247) 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 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 clNA libraries was isolated from human fetal brain tissue. IEA sequencing of the clones isolated as described above gave the full-length INA sequence for PR0293 [herein designated as UQ256 (DEA37151-1193)] (SEQ ID ND:243) and the derived protein sequence for PR0293.

nThe entire nucleotide sequence of t1U256 (ENA37151-1193) is shown in Figures 85A-B (SEQ ID ND:243). Clone UNQ256 (EA37151-1193) "contains a single open reading frame with an apparent translational initiation site at nucleotide positions 881-883 and ending at the stop codcn after nucleotide position 3019 of SEQ ID N0:243, Figures 85A-B). The predicted polypeptide precursor is 713 amino acids long (Figure 86). Clone 20 UNQ256 (ENA37151-1193) has been deposited with ATCC and is assigned ACC deposit no. ATCC 209393.

Analysis of the amino acid sequence of the full-length PRO293 polypeptide suggests that portions of it possess significant hamology to the NIRR proteins, thereby indicating that PR0293 may be a novel NLRR protein.

4 EXAMPLE 40: Isolation of cdMA Clones Encoding Hnan P1R247 A consensus IEA sequence was assenbled relative to other EST sequences using phrap as described in Ebcanple 1 above. This consensus sequence is herein designated ENA33480. Based an the ENA33480 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cIEA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO247.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR prim 5'-~CACAaIGAGOGCACCAAGC-3' (SEQ ID ND:250) reverse PCR primer 5' -GAlT XGI rG-3' (SEQ ID N0:251) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the [EA33480 expression sequence tag which had the following nucleotide sequence.

147 hybridization probe -CAAMIOCAAGGA (SEQ ID NO:252) 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 primer pair identified above. A positive library was then used to isolate clones encoding the PR0247 gene using the probe oligonucleotide and one of the PCR primers.

RN for construction of the cIEA libraries was isolated from human fetal brain tissue. IEA sequencing of the clones isolated as described above gave the full-length IIA sequence for PRO247 [herein designated as UNQ221 (INA35673-1201) (SEQ ID NO:248) and the derived protein sequence for PRO247.

The entire nucleotide sequence of UN221 (ElA35673-1201) is shown in Figures 89A-B (SEQ ID ND:248). Clone UNQ221 (IEUA35673-1201) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 80-82 of SEQ ID NO:248 and ending at the stop codon after nucleotide position 1717 of SED ID NO:249 (Figures 89A-B). The predicted polypeptide precursor is 546 amino acids long (Figure 88). Clone UNQ221 (ENA35673-1201) has been deposited with A'TCC and is assigned A'T deposit no. 209418.

Analysis of the amino acid sequence of the full-length PR0247 polypeptide suggests that portions of it possess significant hnology to the densin molecule and KIAA0231, thereby indicating that PRO247 mray be a novel leucine rich repeat protein.

EXAMPLE 41: Isolation of cra Clanes Enrcodim Iman P2O302, PRW303, PR0304, PD307 and PRO343 Consensus IWA sequences were assemrbled relative to other ES' sequences using phrap as described in Exanple 1 above. These consensus sequences are herein designated E11NA35953, ENA35955, JJ135958, ENA37160 and INA30895. Based on the EMA35953 consensus sequence, oligonucleotides were synthesized: 1) to identify by CR 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 PRO302.

PCR priirers (forward and reverse) were synthesized: forward PCR prirer 1 5' -GITGCAAGGA'IT'PACITGIT-3' (SED ID NO:263) forward PCR priner 2 5' -GCAAGGIGIXtTAAx IG-3' (SEe ID N3:264) reverse PC primer 5'-ACITAGACCAAGOCAGCPO-3' (SEQ ID 0:265) Also, a synthetic oligonucleotide hybridization probe was constructed fran 148 the consensus ENA35953 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, EIA from 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 PRO302 gene using the probe oligonucleotide and one of the PCR primers.

RNA for construction of the clENA libraries was isolated fromn human fetal kidney tissue (LIB228) ONA sequencing of the clones isolated as described above gave the full-length [NA sequence for PRO302 [herein designated as tUM265 (ENA40370-1217) (SEQ ID NO:253) and the derived protein sequence for PR0302.

15 The entire nucleotide sequence of UNQ265 (EMA40370-1217) is shown in Figure 89 (SEQ ID NO:253) Clone UNQ265 (EIA40370-1217) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 34-36 and ending at the stop codon at nucleotide positions 1390-1392 (Figure 89). The predicted polypeptide precursor is 20 452 amino acids long (Figure 90). Various unique aspects of the PRO302 protein are shown in Figure 90. Clone UNQ265 (INA40370-1217) has been deposited with the ATCC on November 21, 1997 and is assigned ATOC deposit no. ATCC 209485.

Based on the EIA35955 consensus sequence, oligonucleotides 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 PR0303.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR primer 5' -<GAACACCC GACATI=-3' (SEQ ID NO:267) reverse PCR priner 5'-<AA;TGOCGCAAQGCAIAAC7lG-3' (SEQ ID NO:268) .Additionally, a synthetic oligonucleotide hybridization probe was constructed fran the consensus IMA35955 sequence which had the following nucleotide sequence: hybridization probe 5' (SEQ ID NO:269) In order to screen several libraries for a source of a fulllength clone, 1A from 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 PRO303 gene using the probe oligonucleotide and 149 one of the ICR prianers.

RNA for construction of the cEA libraries was isolated frcm hu~man fetal lung tissue 1EA sequencing of the clones isolated as described above gave the full-length =1A sequence for PR0303 [herein designated as UNQ266 (IEA42551-1217) (SE ID ND:255) and the derived protein sequence for PRO303.

The entire nucleotide sequence of U1Q266 (IEA42551-1217) is shown in Figure 91 (SEQ ID N:255). Clone UNQ266 (II42551-1217) contains a single open reading franme with an apparent translational initiation site at nucleotide positions 20-22 and ending at the stop codon at nucleotide positions 962-964 (Figure 91). The predicted polypeptide precursor is 314 amino acids long (Figure 92). Various unique aspects of the PRO303 protein are shown in Figure 92. Clone UNQ266 (IDA42551-1217) has been deposited on 0" 15 Novanber 21, 1997 with the A'Itt and is assigned CC deposit no. ATC 0209483.

Based an the DAN35958 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 20 full-length coding sequence for PR0304.

Pairs of [CR priners (forward and reverse) were synthesized: forward PCR priner 1 5.'-OGGI3AGA A CICAAG3' (SED ID NO:270) forward PCR priner 2 5'-CAGCCC0CIACAIGC-3' (SEQ ID NO):271) forward PCR priner 3 5'-TACIGGGIGCAGAAC-3' (SEQ ID ND:272) reverse PCR prier 5' -0OAAGAOXAG(XGGAGAXCOC-3' (SED ID NO:273) Additionally, a synthetic olignucleotide hybridization probe was constructed franm the consensus ENA35958 sequence which had the following nucleotide sequence hybridization probe 5' GC:.rIA (SE ID NO:274) In order to screen several libraries for a source of a fulllength clone, EA 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 PRO304 gene using the probe olignucleotide and one of the PCR prinrers.

RM for construction of the cENA libraries was isolated froman 22 week human fetal brain tissue (LIB153).

IA sequencing of the clones isolated as described above gave the full-length OlA sequence for P103-4 [herein designated as UQ267 150 (E39520-1217)] (SED ID N:257) and the derived protein sequence for PR0304.

The entire nucleotide sequence of UNQ267 (IA39520-1217) is shown in Figure 93 (SEQ ID NO:257). Clone UND267 (MA39520-1217) 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 UQ267 (ENA39520-1217) has been deposited with A'ICC on November 21, 1997 and is assigned A'IO deposit no.

g. A1CC 209482..

Based on the ENA37160 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 PR0307.

Pairs of PCR priners (forward and reverse) ware synthesized: forward PCR priner 1 5' -GOOCAGGATIUCAGGGCI-3' (SB2 ID NO:275) forward PCR priner 2 5'-G3CIMGACAGCAGGTIC-3' (SEQ ID NO:276) forwarid PCR.prirer 3 5'-7GACAAIMACOGACAGG-3' (SED ID NO:277) reverse PCR priner 5'-G CATC tACAA-3' (SE ID NO:278) Additionally, a synthetic olignucleotide hybridization probe was constructed fran the consensus NA37160 sequence wich had the following

S..

@0c. nucleotide sequence hybridization probe S25 5' (SEQ ID N1:279) In order to screen several libraries for a source of a fulllength clone, A 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 P1RO307 gene using the probe olignucleotide and one of the PCR priners.

RNA for construction of the chA libraries was isolated fran human fetal liver tissue (LIB229).

NA sequencing of the clones isolated as described above gave the full-length A sequence for PRO307 (herein designated as tQ270 (IA41225-1217) (SED ID NO:259) and the derived protein sequence for PR0307.

The entire nucleotide sequence of 1N270 (IM41225-1217) is shown in Figure 95 (SEQ ID ND:259). Clone UN270 (EMA41225-121 7 contains a single open reading frare 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 UNQ270 ([NA41225-1217) has been deposited with AICC on Novenber 21, 1997 and is assigned A'C deposit no.

ATCC 209491.

Based on the IENA30895 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cI1A library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0343.

A pair of PCR primers (forward and reverse) were synthesized: forward PCR prier 5' (SEQ ID ND:280) reverse PCR priner 5'-g A G3A CoTCA'IGOCAGGIGGA-3' (SEQ ID NO:281) 15 Additionally, a synthetic olignucleotide hybridization probe was •constructed from the consensus ENA30895 sequence which had the following nucleotide sequence hybridization probe 5' I TCAOGAC 20 TOGAAGIC GCLAICIT A GCACIGAGGACA'IGC'GT 20 GIOCLGCTACT-3' (SEQ ID NO:282) In order to screen several libraries for a source of a fulllength clone, A 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 PR0343 gene using the probe olignucleotide and one of the PCR primers.

RNA for construction of the cDEA libraries was isolated fran human fetal lung tissue (LIB26).

ENA sequencing of the clones isolated as described above gave 3 0 the full-length EMA sequence for PR0343 [herein designated as UNQ302 (IEA43318-1217) is shown in Figure 97 (SEQ ID N0:261) and derived protein sequence for PRO343.

The entire nucleotide sequence of UNQ302 (ENA43318-1 2 1 7 is shown in Figure 97 (SEQ ID NO:261). Clone UNQ302 (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 PR0343 protein are shown in Figure 98. Clone tRM302 (IA43318-1217) has been 152 deposited with ATCC on November 21, 1997 and is assigned ATC deposit no.

ATCC 209481.

EXAMPLE 42: Isolation of cEma Clones Enxoding Bman PRO328 A consensus IA sequence was asserbled relative to other EST sequences using phrap as described in Exanple 1 above. This consensus sequence is herein designated WEA35615. Based on the IEA 35615 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO328.

Forward and reverse PCR primers were synthesized: forward PCR primer CCIGCAGITIt'CA'ITGC-3' (SEQ ID N0:285) reverse PCR primer 5-AAT (SEQ ID N0:286) Additionally, a synthetic olignucleotide hybridization probe was constructed fran the consensus ENA35615 sequence which had the following nucleotide sequence.

hybridization probe -A GAAAGTIC -3 (SEQ ID NO:287) In order to screen several libraries for a source of a fulllength clone, IEA frman the libraries was screened by PCR anplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PRO328 gene using the probe olignucleotide and one of the PCR primers.

RNA for construction of the cENA libraries was isolated fram human fetal kidney tissue.

S" EMNA sequencing of the clones isolated as described above gave the full-length ENA sequence for PRO328 (herein designated as UNQ289 (ENA40587-12131) (SEQ ID N0:284) and the derived protein sequence for PRO328.

The entire nucleotide sequence of UNQ289 (ENA40587-1231) is shown in Figure 99 (SEQ ID NO:283). Clone UNQ289 (EA40587-1231) contains a single open reading frame 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 (ENA40587-1231) has been deposited with ATCC and is assigned AQC deposit no. ATCC 209438.

Analysis of the amino acid sequence of the full-length PRO328 polypeptide suggests that portions of it possess significantly hcomlogy to the human glioblastama protein and to the cysteine rich secretory protein 153 thereby indicating that PR)328 nay be a novel glioblastara protein or cysteine rich secretory protein.

EWWIZ 43: Iaolatian of crwa dicnen Fzxoding Bman PR335, PRO331 or PRO326 A consensus mA sequence as assembled relative..to other E sequences using phrap as described in Exanpie 1 above. This consensus sequence is herein designated EX36685. Based on the EM6685 consensus sequence, and Incyte EST sequence no. 2228990, oligonucleotides were synthesized: 1) to identify by CR a cIA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0335, PR0331 or PR0326.

Forwad and reverse FCR primers were synthesized for the determination of PRO335: forward PCR primer 5' -GGAACCGAA ITCA(XPTA-3' (SE1 ID NM:294) for CRprm 5' -TAAAC7AAC'rCA L-3' (SBQ ID N:295) forward prier 5' -IGTGCAGACACflANXT-3' (SE2 ID N.:296) forward P pricw± 5'--AGC AICAACA1 3' (SEQ ID NO:297) reverse IR primer 5' -3ATICAGI'AAAATr1W-3' (SE) ID ND:298) reverse PCR primer 5' QGI7IruIiGMrAAGAG3-3' (SEX ID N3:299) reverse PCR priner 5' -G-XcITTA CC~tA~dG3-3' (SE) ID NO:300) Aditionally, a synthetic oligonucleotide hybridization probe was constructed for the determination of PR0335 wich had the following nucleotide sequence.

hybridization probe AGAAACAGCCG=A-3' (SD2 ID NO:301) Forward and reverse PCR printers were synthesized for the determination of PR0331: forward FOR pri.z 5, -O~ITACAA rrCAm3Acr~mo.:-3' (SE2 ID ND:302) reverse P priur 5' -O31CMUI~1tC; tGAIUIttC-3' (SE) ID N.3:303) Aditionally, a synthetic oligonucleotide hybrization probe was constructed for the determination of PR0331 %Aich had the following nucleotide sequence hybridization probe 51 3 (SE3 ID NO:304) Forward and reverse ECR printrs were synthesiied for the determination of PR0326: forward PCX primer 5' -ACCAAGGAAXTCGATtCIC-3' (SD2 ID NO:305) reverse primer 5' -Tr 3CA GM0 ocC-3' (SE)2 ID NO:306) Additionally, a synthetic olignuleotide hybrization probe was constructed 154 o *0 e .o .9 0 00* 0 *09e* e 09 9 0 *0* oo 0o 0.

9* 0 00 0 00 o o o o• o *o °eoo *e oo* *eo Ooo go for the determination of PRO331 which had the following nucleotide sequence hybridization probe -GCICl UIGAOCA (SEQ ID NO:307) 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 PR0335, PRO331 or PR0326 gene using the probe olignucleotide and one of the PCR priners.

RNA for construction of the cErA libraries was isolated from human fetal kidney tissue (PR0335 and PRO326) and human fetal brain (PRO331). NA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0335, PRO331 or PRO326 (herein designated as SEQ ID NOS:288, 290 and 292, respectively; see Figure 103A-B, 105 and 107, respectively], and the derived protein sequence for PR0335, PR0331 or PRO326 (see figures 104, 106 and 108, respectively; and the derived protein sequence for PRO335, PRO331 or PR0326 (see Figures 104, 106 and 108, respectively; SBQ ID NOS:289, 291 and 293, respectively).

The entire nucleotide sequences are shown in Figures 103A-B, 20 105 and 107, deposited with the A'I on June 2, 1998, November 7, 1997 and Noventber 21, 1997, respectively.

Analysis of the amino acid sequence of the full-length PRO335, PRO331 or PR0326 polypeptide suggests that portions of it possess significant honology to the LIG-1 protein, thereby indicating that PRO335, 25 PR0331 and PR0326 may be a novel LIG-1-related protein.

EXAMPLE 44: Isolation of c[M Clones Enooding Bman PR0332 Based upon an ECD harology search performed as described in Exanple 1 above, a consensus ENA sequence designated herein as ENA36688 was 3 0 assembled. Based on the E1A36688 consensus sequence, oligonucleotides were synthesized to identify by PCR a cEA 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 primers (forward and reverse) were synthesized: 5'GGAGA (SQ ID NO:310) 5'-<GCCGG-3 (SEQ ID N: 311) A probe was also synthesized: AAC A (SQ ID NO:312) In order to screen several libraries for a source of a full- 155 length clone, IWA from 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 PR0332 gene using the probe olignucleotide and one of the PCR priners.

RA for construction of the cI'A libraries was isolated from a human fetal liver library (LIB229).

MA sequencing of the clones isolated as described above gave the full-length IA sequence for E1M40982-1235 and the derived protein sequence for PRO332.

The entire nucleotide sequence of 1A40982-1235 is shown in Figures 109A-B (SEQ ID ND:308). Clone IIA40982-1235 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 nmolecular weight of 72,067 (pI: 6 6 0 Clone IN40982-1235 has been **deposited with ATCC and is assigned ATCC deposit no. ATTe 209433.

Based on a BLAST and FastA sequence alignment analysis of the full-length sequence, PRO332 shows about 30-40% amino acid sequence *,identity with a series of know proteoglycan sequences, including, for 20 exanple, fibrcndulin and fibramodulin precursor sequence of various species (FMLDBOVIN, FfDCHICK, FEDRAT, FIDM-KUSE, EDLHUMN, PJ36773), osteaedulin sequences (AB000114_1, AB0078481), decorin sequences (CEU831411, OCU03394_1, PR42266, PR42267, PR42260, PR89439), keratan sulfate proteoglycans (BU483601, AF0228901), corneal proteoglycan (AF0222561), and bone/cartilage proteoglycans and proteoglycane precursors (FGS1BON, PGS2PMSE, EGS2JiUMAN).

EXAMPIE 45: Isolation of catU Calones Encoding Hman PRO334 A consensus IA sequence was asserbled relative to other EST sequences using phrap as described in Exanple 1 above. Based on the consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cWA 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 primers were synthesized for the determination of PRO334: forward PR prier 5' -GA P1t X A r (sEQ ID ND: 315) reverse PCR priner 5' -TIPCACTIrPAGGCAL AOG-3' (SEQ ID NO:316) Additionally, a synthetic olignucleotide hybridization probe was constructed for the determination of PR0334 which had the following 156 nucleotide sequence.

hybridization probe -CTC AG'AGATIITG AA (SEQ ID ND: 317) In order to screen several libraries for a source of a fulllength clone, mNA fran the libraries was screened by PCR anplification with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0334 gene using the probe oligonucleotide and one of the PCR primers.

Human fetal kidney cDNA libraries used to isolate the c1NA clones were constructed by standard methods using ccamercially available reagents such as those frm Invitrogen, San Diego, CA.

DNA sequencing of the clones isolated as described above gave the full-length ENA sequence for PRO334 (herein designated as IA41379- 1236] (SEQ ID NO:313) and the derived protein sequence for PRO334.

The entire nucleotide sequence of ENA41379-1236 (also referred •to as UNQ295) is shown in Figure 109 (SEQ ID N3:313). Clone 11A41379-1236 contains a single open reading frane 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 ATCC deposit no.

A'CC 209488.

Analysis of the amino acid sequence of the full-length PR0334 polypeptide suggests that portions of it possess significant honology to the fibulin and fibrillin proteins, thereby indicating that PR0334 may be a novel manber of the BEGF protein family.

EXAMPLE 46: Isolation of cUA Clones Eooding Human PRO346 A consensus DMA sequence was identified using phrap as described in Exanple 1 above. Specifically, this consensus sequence is herein designated [NA38240. Based on the EUA38240 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cDMA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length PRO346 coding sequence.

3 5 RNA for construction of the czNA libraries was isolated fran human fetal liver. The cENA libraries used to isolate the cENA clones were constructed by standard nethods using commercially available reagents Invirtogen, San Diego, CA; Clontech, etc). The cNA was primed with oligo dr containing a NotI site, linked with blunt to Saill hanikinased 157 adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRK5D that does not contain the SfiI site; see, Holmes et al., Science, 253:1278-1 2 8 0 (1991)) in the unique XhoI and NotI sites.

A cEIA clone was sequenced in entirety. The entire nucleotide sequence of IAI44167-1243 is shown in Figure 111 (SEa ID NO:318). Clone ENA44167-1243 contains a single open reading frame with an apparent translational initiation site-at nucleotide positions 64-66 (Fig. 113; SE2 ID ND:318). The predicted polypeptide precursor is 450 amino acids long.

Clone MA44167-1243 has been deposited with ATOC and is assigned AIVC deposit no. MIC 209434 (designation IIA44167-1243).

Based on a BLAST, BALS-2 and FastA sequence alignment analysis (using the ALIGN ccrplter program) of the full-length sequence, PR346 15 shows amino acid sequence identity to carcinonbrynic antigen 'The oliganucleotide sequences used in the above procedure were the following: OLI2691 (38240.f1) S2 5' -IrVirrrbGCrvI3 3' (SED ID NO:320) OLI2693 (38240.rl) 5' -CACAAIACCCA 3' (SEQ ID NO:321) OLI2692 (38240.pl) OIC IT (SEQ ID NO:322) 00 EXAMPLE 47: Isolation of cEM Clones Enodig IHmnan PRO268 A consensus I sequence was asserbled relative to other EST sequences using phrap as described in Exarple 1 above. This consensus sequence is herein designated DA35698. Based on the UNA35698 consensus sequence, olignucleotides 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 PRO268.

Forward and reverse PCR primers were synthesized: forward PCR primer 1 3AO3l3AG Q3AAA'l(-3' (SEQ ID NO:325) forward PCRK prirer 2 5' -TA'IIGGACAGGA7IUO-3' (SEQ ID NO:326) forward ICR primrer. 3 5' -cX 'CGt1A TG3' (SEQ ID NO: 327) reverse PCR priner -T AA(AANA t" (SEQ ID ND:328) Additionally, a synthetic olignucleotide hybridization probe was constructed frn the consensus ENA35698 sequence which had the following nucleotide sequence 157a b* 0*

O.

0 0 .r 0*0 0

B

hybridization probe 3' (SEQ ID ND:329) In order to screen several libraries for a source of a fulllength clone, [A 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 PRO268 gene using the probe olignucleotide and one of the PCR priners.

RNA for construction of the ciEA libraries was isolated froan humtran fetal lung tissue. ENA sequencing of the clones isolated as described above gave the full-length NA sequence for PR0268 (herein designated as U=2235 (IN39427-1179)] (S2EQ ID N:323) and the derived protein sequence for PRO268.

The entire nucleotide sequence of tlN235 (UP39427-1179) is shown in Figure 113 (SEC ID NO: 323). Clone UN235 (ENA39427-1179) contains a single open reading frane 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 (Ir39427-1179) has been deposited with ACt and is assigned AOC deposit no. TW 209395.

Analysis of the amino acid sequence of the full-length PR0268 polypeptide suggests that it possess significant hcology to protein disulfide isarrerase, thereby indicating that PRO268 emay be a novel protein disulfide iscaerase.

25 EAMPLE 48: Isolation of cEM Clones Eaooding IHman P330 A consensus EM9 sequence was assenbled relative to other EST sequences using phrap as described in Ecanple 1 above. This consensus sequence is herein designated IE35730. Based on the INA35730 consensus sequence, olignucleotides were synthesized: 1) to identify by PCR a cUlA 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' CCAGP ATIICCAGLA-3' (SEQ ID NO:332) forward PCR priner 2 5' GAC C 3' (SEQ ID ND:333) forward PCR priner 3 S'-GICCAAGAAC'ItIWC-3' (SE2 ID ND:334) reverse PCR primer 5'--ACACICACATltID~X'r1cIK-3' (SE2 ID 1N:335) Additionally, a synthetic olignucleotide hybridization probe was constructed fran the consensus which had the following nucleotide sequence 157b hybridization probe AIGACIATITA 3' (SEQ ID ND:336) In order to screen several libraries for a source of a fulllength clone, IEA fran the libraries was screened by PC anplification with the PCR priner pair identified above. A positive library was then used to isolate clones encoding the PR330 gene using the probe olignucleotide.and one of the PCR priners.

IM for construction of the clEA libraries was isolated fran human fetal lung tissue. IIA sequencing of the clones isolated as described above gave the full-length IMA sequence for PRO330 (herein designated as UJ290 (UlA40603-1232)] (SE) ID ND:330) and the derived 6 0 protein sequence for PR0330.

The entire nucleotide sequence of 11290 (I40603-1232) is shown in Figure 115 (S2EQ ID NO:330). Clone UNQ290 (I40603-1232) contains 15 a single open reading frane with an apparent translational initiation site at nucleotide positions 167-169 and ending at the stop codon at nucleotide positions 1766-1768 (Figure 115). The predicted polypeptide precursor is 533 amino acids long (Figure 116). Clone UNQ2290 (E140603-1232) has been deposited with ATCC and is assigned AIC deposit no. ATCC 209486 on 60,20 November 21, 1997.

Analysis of the amino acid sequence of the full-length PR0330 polypeptide suggests that portions of it possess significant harology to mouse prolyl 4-hydroxylase alpha subunit protein, thereby indicating that PR0330 amy be a novel prolyl 4-hydroxylase alpha subunit polypeptide.

40*00: SSEXAMPLE 49: Isolation of cEA Clones dner0im Hnmn PRO310 A consensus D1L sequence was asserbled relative to other EST sequences using phrap as described in EaTrple 1 above. This consensus sequence is herein designated 40553. Based on the LXM40553 consensus sequence, olignucleotides were sythesized: 1) to identify by PCR a cIA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence. for PR0310.

Forward and reverse PCR printers were synthesized: forward PCR priner 1 5' -ICCAAGt0IAWPAOAM-G-3 (SEQ ID N::341) forward PR priner 2 5' -CLTiGriCI'I..A23' (SEQ ID ND:342) reverse ICR priner -GCtAAA ICLYrAAGGCOGTATACtCC-3' (SEQ ID EJ: 343) Additionally, a synthetic olignucleotide hybridization probe was constructed fran the consensus which had the following nucleotide sequence I 157c hybridization probe -3GIGITCNICACTA'IOCTAGGAC-3' (SE* ID ND:344) In order to screen several libraries for a source of a fulllength clone, IA fraom the libraries was screened by PCR anplification with the PCR primrer pair identified above. A positive library was then used to isolate clones encoding the PRO310 gene using the probe olignucleotide and one of the PCR primers.

RA for construction of the cEM libraries was isolated franom human fetal liver tissue. [A sequencing of the clones isolated as described above gave the full-length A sequence for PRO310 [herein designated as (1l43046-1225)] (SEQ ID 1N:339) and the derived protein ***sequence for PRO310 (SEQ ID 10:340).

The entire nucleotide sequence of (1=943046-1225) is shown in Figure 119 (SEQ ID N0:339). Clone (IMA43046-1225) contains a single open 15 reading frame with an apparent translational -initiation site at nucleotide positions 81-83 and ending at .the stop codon at nucleotide positions 1035- 1037 (Figure 119). The predicted polypeptide precursor is 318 amino acids long (Figure 120) and has a calculated mlecular weight of approximately 36,382 daltons. Clone (ENA43046-1225) has been deposited with ATCC and is assigned AC deposit no. AICC 209484.

Analysis of the amino acid sequence of the full-length PRO310 polypeptide suggests that portions of it possess hcrology to C. elegans *proteins and to fringe, thereby indicating that PRO310 may be involved in developrment.

EAMPWIE 50: Isolation of cIEM clones Einoding minan PRO339 An expressed sequence tag (EST) ENA database (LIFESEr, Incyte Pharmaceuticals, Palo Alto, CA) was searched and ESs were identified. An assaebly of Incyte clones and a consensus sequence was fonrmed using phrap as described in Eafnple 1 above.

Forward and revers PCR prinrers waere synthesized based upon the assembly-created consensus sequence: forward PCR primer 1 5' '1 03' (SEQ ID NO:345) forward PCR primer 2 5'-<CIA7Gv.ACCGOC 3 IC-3' (SEQ ID NO:346) forward PCR priner 3 5'-GIIACQACPOZOXGI'GC-3' (SEQ ID NO:43) forward PCR primer 4 5' =AC CAG lI 3 (S ID 1:44) reverse PCR prier 1 5' 3' (SEQ ID 110:45) reverse PCR priiner 2 5' -Cl IItC 1t (SED ID NO:46) Additionally, a synthetic olignucleotide hybridization probe was N:1krotlKtepspectl93178-98*2.doc 18108100 157d constructed fran the consensus sequence which had the following nucleotide sequence.

hybridization probe <X AOGALO'l'L'I' ATCCAAA -3 (SEQ ID N: 47) 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 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 cME libraries was isolated from human fetal liver tissue. A clNA clone was sequenced in entirety. The entire nucleotide sequence of ENA43466-1225 is shown in Figure 117 (SEQ ID N0:337). Clone ENA43466-1225 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 333-335 and 15 ending at the stop codon found at nucleotide positions 2649-2651 (Figure 117; SEQ ID N0:337). The predicted polypeptide precursor is 772 amino acids long and has a calculated molecular weight of approximately 86,226 daltons. Clone EIA43466-1225 has been deposited with A=CC and is assigned ATCC deposit no. ATCC 209490.

20 Based on a BLAST and FastA sequence alignment analysis (using the ALIGN canputer program) of the full-length sequence, PR0339 has homology to C. elegans proteins and collagen-like polymer sequences as well as to fringe, thereby indicating that PRO339 may be involved in development o or tissue growth.

EXAMPLE 51: Isolation of cEM1 Clones Encoding H nan PR0244 A consensus ENA sequence was assembled relative to other EST sequences using phrap as described in Exanple 1 above. Based on this consensus sequence, oligonucleotides were synthesized to identify by PCR a cENA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PR0244.

A pair of PCR primers (forward and reverse) were synthesized: TcTC'G GTAGG-3' (30923.fl) (SEQ ID NO:377) 3' (30923.rl) (SEQ ID NO:378) A probe was also synthesized: TI-3' (30923.pl) (SEQ ID NO:379) In order to screen several libraries for a source of a full-length clone, EA fran the libraries was screened by PCR arplification 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 clNA libraries was isolated from human fetal kidney library. ENA sequencing of the clones isolated as described above gave the full-length DM sequence and the derived protein sequence for PRO244.

The entire nucleotide sequence of PRO244 is shown in Figure 121 (SEQ ID NO:375). Clone CNA35668-1171 contains a single open reading frane 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 ATC (designated as ENIIA35663-1171) and is assigned ATCC deposit no. AT'C 209371. The protein has a cytoplasmic domain (aa 1-20), a transmabrane domain (aa 21-46), and an extracellular damain (aa 47-219), with e *oo a C-lectin domain at aa 55-206.

Based on a BLAST and FastA sequence alignment analysis of the tUl-lcngth sequence. PR0244 shows zwable amin acid sequence identity to hepatic lectin gallus pillus HIC bpl.2O-binding C-type lectin macroptiage lczin 2 (HUMHML2-l. 41 and sequence PR32188 (44%) EXAMPLE 52: Use of PRO Pob esidc-Encoding Nucleic Acid as Hybridization Probes The following method &escribes use of a mucleotide sequence encoding a PRO polypeptide as a hybridization probe.

DNA comprising the coding sequence of of a PRO polypeptide of interest as disclosed herein may be employed as a probe or used as a basis from which topar probes to screen for homologous DNAs (such as those encoding naturally-occurring variants of the PRO polypetide) in hutman dtisse cDNA libraries or humnan tissue genomic libraries.

Hybridization and washing of filters containing either library D14As is performed under the following high stringency conditions. Hybridization of radiolabeled PRO polypeptide-encoding nucleic adid-derived probe to the filters is performed in a solution of 50 formamide, 5x SSC. 0. 1% SDS. 0. 1% sodium pyrophosphate, 50 mM phosphate, pH 6.8. 2x Denhardtis solution, and 10% dextrmn sulfate at 4?C for 20 hours. Washing of dhe filters is performed inan aqueous solution of 0.lIx SSC and 0. 1% SDS at 42C.

DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO polypeptide can then be identified using standard techniques known in the art.

*20 EXAMPLE 53: Expression of PRO PoLvoentides in E. coi This example illustrates preparation of an unglycosylared form of a desired PRO polypeptide by recombinant expression in E. coli.

The DNA sequence encoing the desired PRO polypeptide is initially amplified using selected PCR primers.

The pimers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. COil; see Bolivar ct al.. flz=. 2:95 (1977)) which contains gene for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequecs are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistac gene, a trp promoter, a polyhis leader (including t first six M~l codons, polyhis sequence. and enterolcinase cleavage site). the specific PRO polypeptide coding region, Lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to tranform a selected E. coi strin using the methods described in Sambrook et aI.. m=p. Tratisformants arc identified by their ability to grow on LB platecs and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated andi conlifmed by restiction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such as LB broth stipplemented 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 dhe cells for several more hours, tic cells can be harvested by centrifugation. The cell pellet obtained by the centifugation can be solubilized using various agents known in the art, and the solubilized PRO polypeptide cau then be purified using a metal chelaing column wnder conditions diat allow tight binding of the protein.

PROI1S7, PROW.7 PRO301, PR0224 arid PR0238 were successfully expressed in E. co/ i n a poly-His tagged form, using the following procedure. The DNA encoding PROI187, PROM1, ORO3OI, PR0224 or PR0238 was initially amplified using selected PCR primers. The pruners contained restiction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequece providing for efficient and reliable utrasation initiation, rapid purification on a metal chelation column. and proteolytc removal with enterokinase. The PCR-amplifled. poly-His tagged sequences were then ligated into an expression vector, whichwas used to transform an E. coli host based oncuin S2 (W3 110 fuhA(tonA) Ion galE rpopts(hipitts) clpP(lacla).

Transformams were first grown in LB containing 50 rug/ml carbenicillin at 30'C with shaking until ia 0.D.600 of' was seached. Cultures were then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 -g (NHASO 4 0.71 g sodium citrate'2H20, 1.07 g KCI. 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 MIL water, as well as 110 mM MPOS, pH 7.3, 0.55 glucose arnd 7 mM MgSO.) 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 in 7 M guanidine. 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.lM and 0.02 M. respectively, and the solution was stirred overnight at 40C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution was centrifuged at 40.000 rpm m a Beckman Uluracemifuge for 30 mmr. The supernatant was diluted with 3-5 volumes of metal chelate colunn buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filter 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 (Calbiocheni, Utrol grade), pH 7.4. The protein was eluted with buffer containing 250 mM imidazole. Fractions contininig the desired protein were pooled and stored at VC. Protein concentration was estimated by its absorbance at 280 am using the~ calculated extinction coefficient based on its amino acid sequence.

The proteins were refolded by diluting sample slowly into freshly prepared refolding buffer consisting or: mM Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine, 20 mM glycizie and I mM EDTA. Refolding volumies were chosen so that the final protein concentmaton was between 50 to 100 micrograms/nI. The refolding solution was stirred gently at VC for 12-36 hours. The refolding reaction was quenched by the addition otTFA to a final concentration of 0.4% (pH of approximately Before further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to 2-10% final concentration. The refolded protein was ehromatograplied on a Poros RI/H reversed phase column using a mobile buffer of 0.1 TFA with elution with a gradient of actonitrilc from 10 to 80%. Aliquots of fractions with A280 absorbance were analyzed on SDS polyaciylamide gels and fractions containing homogeneous refolded protein were pooled. Generally, the properly refolded species of most proteins are eluted at dhe lowest concentr-ations of actonitril since those species are the most coapact with their hydrophobic inutriors shieled from interaction with dhe reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving inisfolded forms of proteins from the desired form. the reversed phase step also removes endotoxin from the samples.

Fractions containing the desired folded PR0187. PROM,7 PRO3O1. PPR0224 and PR0238 proteins.

respectively. were pooled and the acetonjurile remoeved wing a gentle stream of nitrogen directed at the solution.

Pxnteinwere formulatedinto 20mM Hepes, pH 6.8Swith 0.14 Msodium chloridc and 4% iannitolby dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulaton buffer and sterile filtered.

EXAMPLE 54: Exnression of PRO Povpennde, in Mammalian Cells This example illustrates preparation of a glycotsylatmd form of a desired PRO polypeptide by recombinant expression in mammali1an cells.

The vector, pRK5 (see EP 307,247. published March 15. 1989). is employed as the expression vector.

Optionally. the PRO polypeptide-eneoding DNA is ligated int pRKS with selected restriction enzymes to allow insertion of the PRO polyeptide DNA using ligation methods such as described in Sambrookc t al., The resulting vector is called pRXS-PRO polypeptide.

In one embodiment. the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal -calf serum and optionally, nutrient components and/or antibiotics. About 10 jig pRK5-PRO polypeptide DNA is mixed with about I pg DNA encoding the VA RNA gene pThimmappaya et al., C&1, 2k.543 (1982)) and dissolved in 500 p 1 I of I1mM Tnis-HCI. 0. 1 mM EDTA, 0.227 M CaClt. To this mixtur is added, dropwise, 500 I 1 of 50 mM HEPES (pH 7.35), 280 mM NaCI, 1.5 mM NaPO 4 and a precipitate is allowed to form for 10 minutes at 250C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 3MC The culture medium is asisatedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are ahe washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.

Approximately 24 hours after the tranfections. the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ,iCi/ml "S-cysteinc and 200,uCi/m -uS-methionine. After a 12 hour incubation the conditioned medittr is collected, concentrated on a spin filter, and loaded onto a 15 SDS gel.- The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO polyptdec. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.

In an alternative technique PRO polypeptide may be introduced into 293 cells trnsiently using te dextra sulfate method described by Somparyrac et al., Emc aniLACad. S, 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 pg pRK5-PRO polypcptide DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextra precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 pug/ml bovine insulin and 0.1 pg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remnove cells and debris. The sample containing expressed PRO polypeptide can then be concentrated and purified by any selected method. such as dialysis and/or column chromatography.

In another embodimnent. PRO polypeptides can be expressed in CHIO cells. The pRK5-PRO polypepcide can be transfamtd into CHO cells using known reagents such as CaPO, or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with cualtre medium (aloune) or medium containing a radiolabel such as IS-methionine. After determining the presence of PRO polypeptide, the culture medium may be replaced with serum free medium- Preferably, the cultures are incubated for about 6 days. and then the conditioned medium is harvested. The mdiumn containing the expressed PRO polypptide can then be concentrated and purified by any selected method.

Epitope-tagged PRO polypeptide may also be expressed in host CHO cells. The PRO polypeptide may be subclojied out of the pRKS vector. The subclone insert can uindergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovinis expression vector. The poly-his tagged PRO polypeptide insert can then be subeloned into a SV40 driven vector containing a selection marker, such as DHFR for selection of stable clones.

Finally, the CHO cells can be transfeeted (as described above) with the SV40 driven vector. Labeling may be performied, as deserlb)cd 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 2 *-chelate affinity chromatography.

PRO21I1, PR0217. PR0230. PRO2I9. PR0245. PRO22I. PR0258, PR0301. PR0224, PRO2, PR0234.

PR0229, PRO22, PR0328 and PRO332 were successfully expressed in CHO cells by both a transient and a stable expression procedure. In addition, PR0232, PR0265, PR0246, PR0228. PR0227. PR0220, PR0266. PR0269, PRO287, PR0214, PRO231, PR0233. PRO238. PR0244, PR0235. PR0236, PR0262, PR0239. PRO257, PR0260, PRO263, PR0270, PR0271, PRO272, PR0294. PRO29S, PRO293, PR0247. PR0303 and PRO268 were successfully transiently expressed in CHO cells.

Stable expression in CH0 cells was perfomed using the following procedure. The proteins were expressed as an lgG construct (mmunoadhesin), in which the coding sequences for the soluble forms extracellular domains) of the respective proteins were fused to an IgG I constant region sequence containing the hinge, CH2 and CH2 domjains and/or is a poly-His tagged form.

Following PCR ampilification. the respective DNAS were subcloned in a CHO expression vector using standard techniques as described in Ausubel et al.. Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997. CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used expression in CHO cells is as describedin LuAcas ai al.. Nud. Adds Res. 7: 9 (1774-1779 (1996). and uses the SV40 early promoter/enhancer to drive expression of the eDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.

Twelve micrograms of the desired plasmid DNA were intoduced into approximately 10 million CHO cells wing commrcially available trnfection reagemi Superfee (Quiagen). Dosper* or Pugewe (Boehringer Mannheim).

The cells were grown and described in Lucas a al.. supra. Approximately 3 x l(Y' Cells are frozen inan alcl for further growth and production as described below.

The ampules containing the plasmid DNA were thawed by placement into water bath a maixed by vortexing. Illi contents were pipette into a centrifuge tube containing 10 wn-s of media amd centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0.2 p~m Filtered PS20 with 5 0.2 pin diafiltered fetal bovine serumr). The cells were then aliquoted into a 100 mL spinner containing 90 mL of selective medi. After 1-2 days. the cells were transferred into a 250 mL spinner filled with 150 mnLseectiv growth medium and incubated at 37*C. After another 2-3. days. a 250 nit, 500 niL and 2000 mLspinners were seeded with 3 x 105 cells/mL. T'he cell media was exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed. a production medium described in US Patent No. 5, 122,469, issued June 16. 1992 was actually used. 3L production spirner is seeded at 1.2 x 10' cells/mL On day 0. the cell number pH were determined. On day 1, the spinncr was sampled and sparging with filtered air was commeine. On day 2, the spirner was sampled, the tempeatur shifted to 330C, and.

mL of 500 giL glucose and 0.6 mL of 10% antifoamn 35% polydimethybloxane emulsion. Dow Corning 365 Medical Grade Emulsion). Thiroughout the production. pH was adjuted as necessary to keep at around 7.2.

After 10 days. or until viability dropped below 70%. the cell culture was harvested by eentrifugtion and filtering through a 0.22 filter. The filtrate was either stored at VC or immediately loaded onto columns for purification.

For the poly-His tagged constructs, the proteins were purified using a Ni-TA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 nil Ni-NTA cohmrm 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 mlhmin. at 4*C. After loading. the column was washed with additional equilibration buffer and the protein chited with equilibration buffer containing 0.25 M imidazole. The highly purified protein was subsequcnly desaled into astorage buffer containing 10 mM Hcpes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -809C.

a. Immunoadhesin (Fc containing) constructs of were purified from the conditioned media as follows. Thec conditioned mediiumn was pumped onto aS m l Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, PH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by colleting 1 ml fractions into Mkbs containing 275 p&L of I. M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity was assessed by SDS polyactylamide gets and by N-terminal amino acid sequencing by Edman degradation.

PRO211, PROW1, PRO230. PR0232. PROWS, PR0265, PRO219. PROW4, PR0228. PR0533, PR0245.

PR0221, PR0227. PR0220. PR0258, PR0266, PR0269, PR0287. PR0214, PR0317, PRO3OI, PR0224, PR0222. PR0234, PR0231, PR0229, PR0233, PR0238. PR0223. PROW3, PR0236. PR0262, PR0239.

PR0257. PR0260, PR0263. PR0270, PR0271, PR0272, PR0294. PR0295, PR0293, PROW4, PR0304.

PR0302, PR0307. PR0303. PR0343. PR0328. PR0326, PR0331. PR0332, PR0334, PROW,6 PR0268, PR0330. PRO310 and PR0339 were also successfully traniently expressed in COS cells.

EXAMPEP. 5: PEressimw of PiRp Polvpenddes in Yeast The following method describes recombinant expression of a desired PRO polypeptide in yeast.

First. yeast expression vectors ame constructed for intraceltular production or secretion of PRO polypeptides from the ADH2/GAPDH promoter. DNA encoding a desired PRO polypeptide. a selected signal peptide and the promoter isinerted into Suitable restriction enzyme sites in the selected plasmid to direct intracellular expres Sion of t PRO polypeptide. For secretion, DNA encoding die PRO polypeptide can be cloned into the selected plasmid.

together with DNA encoding the ADH21GAPDH promoter, the yeast alpha-factor secretory uignal/leader sequence.

andi linker sequences (if needed) for expression of die PRO polypeptide.

Yeast cells, such as yeast strain ABI 10. can then be transformed. with the expression plasruids described above and cultured in selected fermentation media. The transfortned yeast supernatants can be analyzed by precipitation with 10 trlduloroacezic acid and separation by SDS-PAGE, followed by Staining -of the gets with Coomassie Blue stain.

Recombinant PRO polypeptide can subsequently be isolated and purified by removing the yeast cells from the fermntiauion medium by centrifugation and then concentrating the mnedium using selected cartridge filters. The concentrate containing the PRO polypeptide may further be puhified using selected column chromatography resins.

EAMPLE.56: FpEttsion of PRO Polvpeddes in BhculovirsInfected Insect Cells The following method describes reombinant expression of PRO polypeptides in Baculovurus-infected insect cells.

fIr desired PRO polypeptide is fused upstream of an epitope tag contained with a baculovirus expressionI vector. Such epitope tags include poly-his tags and immunoglobuhin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, dhe PRO polypeptide or the desired portion of the PRO polypeptide (such as the sequence encoding the extracellular domain of a transinmbrane 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 subeloted into the expression vector.

Recombinant baculovizus is generated by co-transfecting the above plasmid and BaculoGoldr m virus DNA (Phaxmningen) into Spodapcerafntgiperda 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 infection and protein expression is performed as described by O'Reilley et al., Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford University Press (1994).

Expressed poly-bis tagged PRO polypeptide can then be purified, for example. by Ni'-chelate affinity chromatography as follows. Extracts are prepared from recombicant virus-inected Sf9 cells as described by Rupert c Nawue, 2: 175-179 (199). Briefly. Sf9 cells are washed, resuspended in sonication buffer C25 niL Hepes, pH 7.9; 12.5 mM MgC 5 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 MIKCI). and sonicated twice for seconds on ice. The sonicates are cleared by entrifugation. and the supernatant is diluted 50-fold in loading buffer mM phosphate, 300 mM NaCI. 10% Glycerol. pH 7.8) and filtered through a 0.45 pm filter. A Ni-NTA agarose colurmn (commercially available from Qiagen) is prepared with a bed volume of 5 niL, washed with 25 MiL of water and ilibrated with 25 mnL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per -inwe. Tbc column is washed to baseline with loading buffer. at which point fraction collection is started.

Next, the column is washed with a secondary wash buffer (SO0MM phosphate. 300 mnM Had,. 10% Glycerol, pH which elutes nonspecifically bound protein. After reaching A0%,baseline again, the column is developed with a 0 to 500 mM Imidazolc gradient-in the secondary wash buffer. One rnL fractions are collected and analyzed by SDS-PAGE and silver staining or western blot with Ni'*flTA-conjugaced to -alkaline phospfiatase (Qiagen).

Fractions containing dhe eluted His~d-tagged PRO polypeptide: are pooled and dialyzed against loading buffer.

Alternatively. purification of the IgG tagged (or Pc tagged) PRO polypeptide can be performed using known chromatography techniques, including for instance. Protein A or protein G column chromatography.

PRO2I1, PR0217. PROM.0 PROIS97. PR0265, PROM4, PR0228, PR0533. PROM4. PR0221. PR0220.

PR0258, PR0266. PROM6, PROM. PR0214. PRO3OI, PR0224, PR0222, PR0234. PR023 1. PR0229, PROM3. PROM3. PR0257. FROM.2 PR0294. PR0295. PR0328. PROM2, PR0331, PR0334. PR0346 and PR0310 were successfully expressed in baculovims infected Sf9 or highS insoc cells. While the expression was actually performed in, a 0.5-2 L scale, it can be readily scaled up for larger 8 L) preparations. The proteins were expressed as an lgG construct Cimmnoadhesin). in which the protein extracellular region was fused to an IgG1 constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms.

Following PCR amplification, the respective coding sequences were subcloned into a baculovirus expression vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins), and the vector arnd BaculogoldO baculovirus DNA (Pharmingen) were co-transfected into 105 Spodopterafnrgiperda *SI9') cells (ATCC CRL 1711), using Lipofectin (Gibco BIL). pb.PH.IgG and pb.PH.His are modifications of the conmmercially available baculovinis expression vector pVL1393 (Phariingen), with moodified polylinker regions to include the His or Fc tag sequences. The cells were grown in Hink's TNM-FH medium supplemented with 10 PBS (Hyclone). Cells were A incubated for 5 days at 2gC. The supernatant was harvested and subsequently used for the first viral amplification by infecting SM cells in Hinks3 TNM-FH medium supplemented with 10% PBS at an approximate multiplicity of infection (MOI) of 10. Cells were incubated for 3 days at W8C. The supernatant was harvested and the expression of the constructs in the baculovirus expression vector was determined by batch binding of I ml of supernatant to 25 of Ni-1[TA eads QIAGEN)i for nistidine tagged proteins or I-LotinU-A Sepharose C-Bbeads (Pamca for JgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.

Mic first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MO! of 0.1. Cells were incubated for 3 days at 28*C. The supernatan was harvested and filtered. Batch binding and SDS-PAGE analysis was repeated. as necessary, until expression of the spinner culture was confirmed.

The conditioned medium from the transfected cells (0.5 to 3 Q) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For die poly-His tagged construcs. the protein construct were purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration oif 5 mM. The conditioned media were pumped onto a 6 ml Ni-NTA column equilibrated in 20 mnM Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM irnidazole at a flow rate of 4-5 nullmin. at VC. After loading. the cohumn was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M irnidazole. Mrt highly purified protein was subsequently desalted into a storage buffer containing mM Hepes, 0.14 MI NaCl and 4% manitol, pH 6.8. with a 25 nal 025 Superfine (Pharmacia) column and stored at -S0*C.

Innnunadltsin (Fe containing) conitets of proteins were purified from the conditioned media as follows.

7kt conditioned media were pumped onto a 5 ml Protein A column (Pbarinacia) which had been eqiltibrated in mM Na phosphate buffer. PH 6.8. After loading, the colurnm was washed extensively with equilibration bruffer before ehition with 100 mM citric ad. pH 3.5. let eluted protein was immediately neutralized by collecting- 1 ml fractions into tubes containing 275 mL of 1 MI Tris buffer. PH 9. T7he highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of die proteins vas verified by SDS polyacrylasnide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.

EXAM PUL 5: Emgaration of Antibodies that Bind to PRO Povedpdes This example illustrates preparation of monoclonal antibodies which can specifically bind- to a PRO polypeptide.

Techniques for producing the monoclonal antibodies are known in the art and are described. for instance.

~in Goding. lInnunogens that may be employed include purified PRO polypeptide, fusion proteins- containin the PRO polypetide, and cells expressing recominant PRO polypeptide on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.

Mice, such as Balb/c, are imimmnizod with the PRO poblyppde immunogen emulsified in complete Frend's 20 adjuvant and injected subcutaneously or intraperitoneally in an amount firom I-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi ImwohmclResearch. Hamilton. Ml') and-injected into the animal's hind foot Pads. The immnized mice are then boosted 10 to 12 days later with additional iinmunogen emulsified in the selected adjuvant- Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orhital bleeding for testing in EUISA assays to detect anti-PRO polypeptide antibodies.

After asuitable antibody titer has been detected, tbe animals 'positive" for antibodies can be injected with a final intravenous hijoction of PRO polypeptide. Three to four days later. the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35 polyethylene glycol) to a selected maurine mycloma cell lioe such as P3X63AgU.l1. available frm ATCC, No. CR1. 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT Ozypoxandhine, aminoptern. and thynnidine) mediwnm to inhibit proliferation of non-fused cells, mycloma hybrids, andl spleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity against the PRO polypeptide.

Determiination of 'Positive* hybridoina cells sectie desired monoclonal antibodies against the PRO polypeptide is within the skill in the art.

The Positive hybidama cells can be i~cted intraperitoneafly into syngeneic Bab/c mice to produce ascites cntaining the adi-PRO pobwpptde monoclonial antibodies. Alternatively. the hybridomn cells can be grown in tissue culture flasks or roller bottles. Puification of the mnonoclonal antibodies produced in the ascites can be accomrplished using ammnonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding Of Antibody to protein A or protein G can be employed.

Chimeric PRO Polynentdes PRO polypcptides may be cxprcssd as chimeric proteins with one or more additional jpolypeptide domains added to facilitate protein purification. Such purification facilitating domains include. but are ntiimited to, metal chelating peptides such as histidine-tiyptophan modules that allow purification on immobilized mtetals. protein A domains that allow purification on immobilized imnninoglobulin. and the domain utilized in the FLAGS'h eXlension/affunity purification system (Inmumex Corp.. 'Seattle Wash.). 7bli inclusion of a cleavable linker sequence such as Factor XA or enterokinase (invitrogen, San Diego Calif.) between die purification domain and the PRO polypeptde sequ~ence may be useful to facilitate expression of DNA encoding the PRO polypeptide.

EAMPLE..59: Purification of PRO-Polni.,tides Usine Snecific Antibggdies Native or recombinant PRO polypeptides; may be purified by a variety Of standard techniques in die art of protein purification. For example. pro-PRO polypeptide, ware PRO polypeptide, or pre-PRO polypeptide is Purified by imnmoaffinity chromnatograpy Using antibodies specific for the PRO polypeptide of ineet. in general, an inmmoaffinity columen is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin.

PblYdlonal imnamoglobulins ame prepared from izmim sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharanacia LKB Biotechnology, Piscataway, Likewise, .20 monoclonal antibodies ame prepared from mouse ascites; fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified imniroglobulin is covalcndy attached to a chromatographic resin such :as Cri~r-activated SEPHAROSEm (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufactures instructions.

Such an immunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subciular fraction obtained via differential centrifugation by the addition of detergent or by other 9 izmethods well known in the art. Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells arm grown.

A soluble PRO polypeptide-containing preparation is passed over the immunoaffinity column, and the cohawn is washed under conditions that allow the preferetial absorbance of PRO polypeptide high ionic strength bUffers in the prece Of detee). Then, the column is eluted under conditions that disrupt antibodylPRO polypetide binding a low PH buffer such as approximately pH 2-3. or a high concentration of a chaotrope such as urea or thiocyanate ion). and PRO polypeptide is collected.

EXAMPLE 6: Drg Screnig This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drug screening techniques. The PRO polypeptide or fragment employed in such a test may either be free in solution. affixed to a solid support, borne on a cell, surface. or located intraceliuly.

One method of drug screening utilizes eukaryotic or prokaryatic bost cells which ame stably transformed with recominant zcleic acids expesing the PRO polypeptide or fragment. Drugs are screened against such trans~fortned cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. Ommay measure, for example. the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively, one can examine the duinimtion in complex formation between the PRO polypeptide and its target cell or taget receptors caused by dhe agent being =Wte.

This, dhe present invention provides methods of screening for drugs or any other agents which can affect a PRO poiyeptde-associated disease or disorder. These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying for the presence of a complex between the agent and the PRO polypeptide or fragracnt, or Qu for the prsenc of a complex between the PRO polypeptide or fragment and the cell, e 6by methods well kznwn in the at In such competitive binding assays, the PRO polyeptide or fragment is typically labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present ian bound form, and the amount of free or uzuomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell coruplex.

Another technique for drug screening provides high throghput screening for comunds having suitable .:ewe binding affinity to a polypeptide and is described in detail in WO 84/03564. published on September 13. 1984.

Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PRO polypeptde, the peptide test compounds are reacted with PRO po4lyppde and washed. Bound PRO polypeptide is detected by methods well known i the art. Purified PRO 20 polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. in addition, non-neutralizing antibodies can be used to capture the peptide and immoilize it on the solid support.

:This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies 'capable of binding PRO polypeptide specifically compete with a test compound for binding to PRO polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptdec which shares one or more antigenic determinants with PRO polypeptide.

EAMPELE 61 Rational Drug Desigm The goal of ratinal 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 arc more active or stable forms of the PRO polypeptide or which enhance: or interfere: with the function of the PRO polyeptide in viw (c.f Hodgson, BiotlechnologY. 2: 19-21 (1991)).

In oae approach, the threc-dinmrsional structure of the PRO polypeptide, or of an PRO polypeptide-inhibitor complex. is determined by x-ray crystallography, by computer modeling or, most typically, by a combination Of the two approaches. Both the shape and charges of the PRO polypptde must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often. useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant wtucnnal information is used to design analogous PRO polypeptide-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. Biochomiiu. Uj:7796-7801 (1992) or which act as inhibitors, agonists. or antagonists of native peptides as shown by Athaud a ef at.l. LB1r2G= 113:742-746 (1993).

hsis also possible to isolate a target-specific antibody, selected by functional assay. as described above, and then to solve its crystal structure. ibis approach, in principle, yields a-pharmacore upon which-subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies *(arznids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site *of the andi-ids would be expected to be an analog of tie original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act asthe pharmacore.

**see:By virtue of the present invention, suifficient amounts of the PRO polypeptde may be made available to V6909perform stich analytical studies as X-ray crystaflography. In addition, knowledge of dhe PRO polypepride amino acid seqluence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.

0 EXAMPELE 62: Diapnostic Test Ulsing PR0317 Polvoentide-Sneefic Antibodies Particular anti-PR0317 polypeptide antibodies are usefujl for the diagnosis of prepathologic conditions, and chronic or acute diseases such as gynecological diseases or ischetuic diseases which ame characterized by differences in thec anuat or distribution of PROM 1. PR0317 has been found to be expressed in human kidney and is thus likely .20 to be associated with abnormalities or pathologies which affect this organ. Further, since it is so closely related to EBAF-1. it is likely to affect the endometium and other genital tissues. Further. due to library sources of certain ESTs. it appears that PR0317 may be involved as well in forming blood vessels and hence to be a modulator of 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 tissues. The polypeptide and antibodies of the present invention may be used with or without modification. Frequently, the polypeptide and antibodies will be labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature.

Suitable labels inchud radionuclides. enzyme&, substrates, cofactors, Inhibitors, fluorescent agents, chemailuminescent agents, magnetic particles, and the like. Patent teaching the use of such labels include U.S. Pat. Nos. 3.817.837.

3.850.752; 3.939.350; 3,996,345; 4.277,437; 4,275,149; and 4,366,241. Also, recombinant iznuunoglobulins may be produiced as shown in U.S. Pat. No. 4,816,567.

A variety of protocols for measuring soluble or tmembrane-bound PRo3l7. using either polyclonal or monoclonal antibodies specific for that PRO3 17. are knrown in the art. Examples include enzyme-linked inunosorbent assay (EUISA). radioimnowssay radioreceptor assay and fluorescent activated cell sorting (FACS). A two-site monoclonal-based imnmunoassay utilizing mnonoclonal antibodies reactive to two non-interfering epitopes; on PR0317 is preferred, but a competitive binding assay may be employed. These assays are described. among other ptace. in Maddox et al. I ExIIMed., m:121 1 (1983).

&EXbl.E..U: Identification of PRO3 17 Receptors Purified, PR0317 is useful for characterization and purification of specific cell surface receptors and other binding molecules. Cells which respond to PR0317 by metabolic changes or other specific responses arc likly to express a receptor for P10317. Such receptors include. but tire rnot limnited to. receptors associated with and activated by tyrosine and serine/threonine kinases. See Kolodziejczyk and Hall, up re, for a review on known receptor for the TGF- superfamily. Candidate receptors for this superfamily [all into two primary groups, termed tyeI and ty" U receptors. Both types are serineAhirconine: knases. Upon activation by the appropriate ligandI type I and type 11 receptors physically interact to form hetero-oligowers and subsequently activate: intratcellular signaling cascades. ultimately regulating gene transcription and expression. In addition, TOF- binds to a thitreceptor class.

typ 1, amembrane-anchored proteoglycan lacing the kinase activicy typical of signal transducing molecules.

PR0317 receptors or other PR0317-binding molecues; may be identified by interaction with radiolabeled *:..PR0317. Radioactive labels may be incorporated into FRO3 17 by various methods known in the art. A preferred embodien is the labeling of primary amino groups in PRO3 17 with 1251 Bolton-Hunter reagent (Bolton and Hunter.

Bicen J.,fl:529 (1973)), which has been used to label other polypeptides without concomitant loss of-biological activity (Hebert a al.. LBDnL..Ch=m. 2M:18989 (1991). MvcColl a al Imianol.. 121:4550-4555 (1993)).

Receptor-bearing cells arc 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 imiber and affinity of receptors.

Labeled PR0317 is useful as a reagent for purification of ts specific receptor. In one embodiment of affinity purification, PR0317 is covalently coupled to a chromatography column. Receptor-bearing cells arc extracted, and the extract is passed over the column. The receptor binds to die column by virtue of its biological affinity for PRO3 17. The receptor is recovered from the colunand ua sbcted to N-terminal protein sequencing. This amino acd sequence is then used to design degenerate oligonucloodde probes for cloning the receptor gene.

*00:25 In an altenative method, mRNA is obtained from receptor-bearing cells and nade into a eDNA library. The lbary is tansfiected into a population of cells. and thise clls expressing the receptor are selected using fluOrescently labeled PR03O1. The receptor is identified by recovering and sequencing recombinant DNA from highly labeled cells.

In another alteniative method, amtiodies are raised against the surface of receptor bearing cells, specifically snonockal antibodies;. The monoclonal antdies are screened to identify those which inhibit the binding of labeled PR0317. 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 appopriate materials derived from the uterus. After incubation, PR0317 complexes larger than the size of purified PR0317 are identified by a sizing technique such as size-exclusion chromatography or density gradient centrifugation and are purified by methods known in the an. The soluble receptors or binding protein(s) are subjected to N-terinal sequencing to obtain information sufficient for database identification. if the soluble protein is known, or for cloning, if the soluble protein is unknown.

EXihfPLF 6: Detination of PROI 17-induced Cellular Rr==ns The biological activity of PR0317 is measured. for examp le, by binding of an PR0317 of the invention to an PR0317 receptor. A tettcooipwzu is sawened as an antagonist for its ablty to block binding of PR0317 to the receptor. A test compound is screened as an agonist of the PR0317 for its ability to bind an PR0317 receptor and influence diame physiological events as PR0317 using. for example. the KIRA-EUISA assay described by Sadick et al. Analvtical Bochemista- 22:207-214 (1996) in which activation ofta receptor tyrosin kinase is monitored by immuno-capture of thc activated receptor and quanuitation of the evel or ligand-induced phosphorylation. The assay may be adapted to monitor PR03174izaucod receptor activation through t use of an PR0317 receptor-specific antibody to capture the activated rereptor. These techniques are also applicable to other PRO polypeptides described herein.

00 XAMPLE..65: Use of PRO724 for Screeng Compouds PR0224 is expressed in a cell. stripped of membrane proteints and capable of expressing PR0224. Low Sdensity lipoproteis having a dctoctale 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 bourd to the membrane and within the cell after cell lysis.

0 0 15 Detection of the low density lipoproteins within the cell determines that PR0224 is within the family of low density -lipoprotein receptor proteins. Members found within this family are then used for screening compounds which affect these receptors, mid particularly the uptake of cholesterol via these receptors.

EXAMPLE 66: Ability of PRO Polnetidet to Inhibit Vascular Endotheial Growth Factor VEGFMStimula ted- Proliferation of Endothelial Cell Growth The ability of various PRO polypeptides to inhibit VEGF stimulated proliferation of endothelial cells was tested. Specifically. bovine adrenal cortical capillary endothelial (ACE) cells (from primary culture, maximum 12-14 passages) were plated on 96-well midcrotiter plates (Amersham Life Science) at a density of 500 cellstwell. per 1&L in low glucose DMEM, 10% calf serum, 2 mM glutarmin, x pen/strept and fungizone. supplemented with 3 ng/mL VEGF. Controls were plated the sam way but some did not include VEGF. A test sample of the PRO polypeptide of interest was added in a 100 ;ul volume for a 200 ;L1 final volume. Cells were incubated for 6-7 days at 37*C. The media was aspirated and the cells washed Ix with PBS. An acid phosphatase reaction mixtur (100 1 zl, 0. IM sodium acetate, pH 5.5. 0.1 Triton-100, 10 mM p-nltrophenyl phosphate) was added. After incubation for 2houzrs al 37*C. the reaction was stopped by addition of 10 Id IN NaOH. OD was measured on mnicrotiter plate reader at405 un.L Controls were no cells. cells alone, cells FGF (5ngniL). cells VEGF (3 ng/sL), cells VEGF (3 ng/rnl) TGOP (I ng/rnl) and cells VEGF (3ng/mL) UF (S ng/mL). (TGF-3 at a I ng/mI concentration is known to block 70-90% of VEOF stimulated cell proliferation.) The results were assessed by calculating the percentage inhibition of VEGF (3 ng/mi) stimulated cells proliferation, determined by measuring acid phosphatase activity al 0D405 rn, relative to cells without stimulation, and relative to the reference TGFP 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 tuhmor angiogenesis. The numnerical values (relative kiibition) shown in Table 2 are determined by calculating the percent inhbion of VEGF stimulated proliferation by the PRO polypepdde relative to cells without stimulation and then dividing dat percentage into the percent inhibition obtained by TGF-P at I ng/ml which is kown to block of VEGF stimulated ccli proliferation.

Iable2 a PRO Ncame PRO2Ii PRO211 PRO211 PRO211 PRo211 PRO211 PRO217 PRO217 PRO217 PROzl7 PRO217 PRO217 PRO187 PRO187 PRO187 PRO219 PRO219 PRO219 PRO246 25 PRo246 PRO246 PRO228 PRO228 PRO228 30 PRO228 PRO228 PRO228 PRO228 PRO228 35 PRO228 PRO245 PRO245 PRO245 PRO245 PRO245 PRO245 PRO221 PRO221 PRO221 PRo221 PRO221 PRO221 PRO258 PRO258 PR2m PRO258 PRO258 PRO258 PRO Concentration 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% 0.01% 0.1% 1.0% 2.5 pM 25 nM 250 nM 0.01% 0.1% 1.0% 5.7 pM 57 mM 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 aM 48 nM 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% Relative Inhibition 99.0 1.09 0.95 67.0 0.27 20.0 1.06 0.84 -0.39 0.2 0.88 0.58 0.91 0.82 0.44 0.61 1.09 0.97 1.04 0.49 0.99 0.93 0.57 0.95 0.98 0.77 0.88 0.16 0.48 0.76 0.35 0.11 1.03 0.95 0.49 1.03 1.06 0.82 0.93 0.31 0.43 0.98 1.06 0.95 1.02 0.6 0.69 Tabe cnt

PR

PRO301 PRO301 PRO301 PRO301 PR0301 PRO301 PR0224 PR0224 PR0224 PR0272 PRO272 PRO272 PR0328 PR0328 PR0328 PR0331 PR0331 PR0331 PRO Conc~entration 7.0Ogd 70 pLM 700 puM 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% 1.02 0.8 0.44 0.92 0.85 0.68 101.0 65.0 23.0 0.95 0.57 0.18 0.98 0.96 0.6 0.88 0.82 0.56 .0 O* EXAMPLE 67: Retinal Neuron Survival This example demonstrates that PR0220 polypeptides have efficacy in enhancing the survival of retinal neuron cells.

Sprague Dawley rat pups at postnatal day 7 (mixed population: glia and retinal neuronal types) are killed by decapitation following CO., anesthesia and the eyes are removed under sterile conditions. The neual 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 Can, Mg2*-free PBS. The retinas are incubated at 37*C for 7-10 minutes 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 withou the specific test PRO polypeptide. Cells for all experiments are grown at 37*C in a water saturated atnosphere of 5% CO.. After 2-3 days in culture, cells are stained with calcein AM then fixed using 4% pamaformaldehyde and stained with DAPI for determination of total cell count. The total cells (fluorescerk) are quantified at 20X objective magnification using CCD camera and NIH image software for Mactntosh. Fields in the well are chosen at random.

The effect of various concentration of PRO220 polytides are reported in Table 3 below where percent survival is calculated by dividing the total number of calcein AM positive cells at 2-3 days in culture by the total number of DAPI-labeled cells at 2-3 days in culture. Anything above 30% survival is considered positive.

PRO iName PR0220 PR0220 PR0220 PR0220 PR0220 PRO220 PRO Concentratin 0.01% 0.01% 0.1% 0.1% 1.0% 1.0% Percent Survival 2.4% 4.1% 3.0%9 3.1% 72.4% 42.1% EXAMPL&K 6: Rod Phntorece 1 ,mr Survival This example demonstrates that PR0220 polypeptides have efficacy in enhancing the survival of rod photoreceptor cells.

Sprague Dawlcy rat pups at 7 day postnatal (meixed populatiom: glia and retinal neuronal cell typm) ame killed by decapitation following CO, anesthesis and the eyes are removed under sterile conditions. The neural retin is dissected away form the pigment pepiluim and other ocular tissue and then dissociated into a single cell suspension using 0.25 trypsin in Ca 2 mg'-free PBS. The retinas are incubated at 37C for 7-10 minutes after which the trypsin is inactivated by adding I ml soy'bean "rpin inhtibitor. The cels 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 experiments are grown at 37'C in a water saturated atmosphere of 5 CO2. After 2-3 days in culture, cells are fixed using 4% paraforuadehyde. and then staine using CellTracker Green CMFDA. Rho 4D2 (ascites or IgG 1: 100). a unxxclonal antibody directed towards the visual pigmn rhodopsin is used to detect rod photoreceptor cells by indirect immofluorescence. Thte results are reported as survival: total number of ealceinlCdllrrackcer rhodopsin positive cells at 2-3 days in culture, divided by the total mnber of rhodopsin positive Cells at time 2-3 days in culture. The total cells (fluorescent) are quantified at 20x objective magnification using a CCD camera and NIH' imlage software for Macintosh. Fields in the weillam chosen at random.

The effect of various concentration of PR0220 polypptdes are reported in Table 4 below. Anythin above survival is considered positive..

Table 4 PRO Nme R ocnrto Pecent Survival *.:PR0220 0.01% 0.0% PR0220 0.1% 0.0% *PR0220 2.0% 0.0% PR0220 10% 0.0% PRO=2 20% 66.9% PR0220 1.0% 56.9% EXAMPLE 69: Induction of Endothelial Cell Aotosis The ability of PR0228 polypeptides to induce apoptosis in endothelial cells was tested in hum venous umbilical vein enidothelia] cells (HUVEC. Cell Systems), using a 96-well format, in 0% serum media supplemented with 100 ng/nml 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 and the cellswashed once withB P S miloflIx rypsin was added to thecells in a T-175 flask. and the cells were allowed to stand until they were released from the plate (about 5-10 minutes).

Trypsinization was stopped by adding 5 ml of growth media. The cells were spun at 1000 rpm for 5 minutes at VC.

The media was aspirated and the cells were resuspended in 10.1o of 10% serum complemented medium (Cell Systems). 1 x penn/strep.

The cells were platcd on 96-well microriter plates (Amersham Wfe Science. cytostar-T scintillating snicroplate. RPNQI6O. sterile, tissue-culture treated. indtividually wrapped), in 10% serum (CSG-medium. Cell Systems). aa density of 2x I0'cellsper wellin atotal volume of100 i. Ile PRO229 polypeptide was addedmi triiea tliluons oft1%. 0.33% and 0.11%. Wells wtoutelwre usedaa blanandwells withells only as a negative control. As a positive control 1:3 serial dilutions of 50 Ad of a 3x stock of staurosporine Were used.

The ability of the PRO=2 polypeptide to iftw apoptosis was determined using Annexin V. a member of the calcium and phospholipid binding proteins, to detwc apoptosis.

0.2 mlAnneiV Y- Biotinstock sltion (100 pg/ml) weiluted in 46nil2x C 2 binding buffer and 2.S% BSA (1:25 dilution). 50 phl of the diluted Annexin V Bioti solution were added to each well (except controls) to a final concentration of 1.0 pgomL Ihe samples were incubated for 10-15 minutes with Annexin-Biotin prior to direct addition of "S-Streptavidin. '"S-Strepcavidin was diluted in 2x Ca* binding buffer. 2.5 BSA and was added to all wells at afinal contrauionof3 x 10'cpmlwell. Ile plates were then sealed, centrifuged at 1000 rpm for IS minutes and pLaced on orbital shaker for 2 hours. The analysis was peirformed on 1450 Microbeta Trilux (Willac). Thte results am sdown in Table 5 below where percent above background represents the percentage amount of counts per minute above the negative controls. Percents greater than or equal to 30% above background are considered positive.

*15 Tab1l PRO Name PR ocnr~nPercent Above Backeround PR0228 01%0.7% PR0228 0.11% 47.6% PR0228 0.33% 92.2% PRO=2 0.33% 123.7% *PR0228 1.0% 51.4% PR0228 1.0% 95.3% EAMPE.g PDB12 Cell hbition This example demonstrates that various PRO polypeptides have efficacy in inhibiting protein production by PDB12 pancreatic ductal cells.

PDBI2 pancreatic: ductal cells are plated on fibronectin coated 96 well plates at 1.5xl0' cells per well in 100 pUI180 ItL of growth media. 100 jd.. of growth media with the PRO polypeptidc 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 inactive in this assay. Cells arc incubated for 4 days at 370C. 20 il- of Alaflar Blue Dye (AB) is then added to each well andi ft flourescent reading is measured at 4 hours post addition of AB. on a inicrotiter plate reader at 530 oma excitatio and 590 em emission. The standard employed is cells without Bovinc Pituitary Extract (BPE) and with various concentrations of BPE. Buffer or CM controls from unknowns are run 2 times on each 96 well plate.

The results from these assays are shown in Table 6 below wherein percent decrease in protein production is calculated by comparing the Alama Blue Dye calculated protein concentration produced by the PRO polyetidetreated cells with the Alarnar Blue Dye calculated protein concentration produced by the negative control cells.- A percent decrease in protein production of greater than or equal to 25% as compared to the negative control cells is considered positive.

Tabl PRO NaMe PRO21 1 PRO211I PRO211 PR0297 PRO2W PRO28 PRO2W PRO28 PROW8 PRO301 PRO301 PRO301 PR0293 PR0293 PR0293 PRQCoMenttio 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 Produtn 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 Hleart Hvtrtronhy This assay is designed to measure the ability of various PRO polypeptides to stimulate hypertrophy of adult 20 hear.

Ventricular myocytes freshly isolated from adult (250g) Sprague Dawley rats arc plated at 2000 cell/well in 180 jul volume. Cells are isolated and plated on day 1, the PRO polypeptide-containing test samples or growth medium only (negative control) (20ju1 volume) is added on day 2 and the cells are then fxed and stained on After staining, cell size is visualized wherein cells showing no growth enhancement as compared to control cells are 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 cclii showing large growth enhancement as compared to control cells are given a value of Anty degree of growth enhancement as compared to the negative control cells is considered positive for the assay.

The results are shown in Table 7 below.

30 PRQam PRO2W PR0287 PR0301 PRO301 PR0293 PRO293 PR0303 PRO303 Table PRO Concentrion 20% 20% 20% 20% 20% 20% 20% 20% Growth Enhancement Score EXAMPLEI72: PDBI 2 Cell Proliferation This example demntrates; tha varions PRO polypeptides have efficacy in inducing proliferation of PDB12 pancreatic ductal cells.

PDBI2 pancatic ducta cells are plated on fibronoctin coated 96 well plates at l.5x10' cells per well in 100 p4dl8O puL of growth media. 100 juL 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 Controls contain growth medium contaizing a protein shown to be inactive in this assay. Cclis are incubated for 4 days at 37'C. 20 pL of Alamar Blue Dye (AB) is then added to each well and the flourescent reading is measured at 4 hours post addition of AB. on a microtiter plate reader at 530 nm excitation and 590 nm emission. The standard employed is cells without Bovine Pituitary Extract (BPE) and with various concentrations of BPE. Buffer or growth medium only controls from unknowns are run 2 times on each 96 wel plate.

The results from these assays are shown in Table 8 below wherein percent increase in protein production is calculated by comparing the Alamar Blue Dye calculated protein concentration produced by the PRO polypeptidetreated cells with the Alamar Blue Dye calculated protein concentration produced by the negative control cells. A percent increase in protein production of greater than or equal to 25% as compared to the negative control cells is **10 considered positive.

Table 8 QPRO Nam PRO Concentration Percent Increase in Protein Production PRO301 2.0% 44.0% 15 PRO301l 10% 67.4% SPRO301 50% 185.8% PR0303 2.0% 27.9% PR0303 10% 174.9% PR0303 50% 193.1% ***.,EXAMPLE 73: Enhancement of Heart Neonatal H3vpermphv Induced by PR0224 This assay is designed to measure the ability of PR0224 polypeptides to stinmlate hypertrophy of neonatal heart.

*h Cardiac myocytes from 1-day old Harlan Sprague Dawley rats were obtained. Cells (180 pl at 7.5 x 10 4 /mal, 25 sennrum freshly isolated) are added onday 1 o 96-wcll plates previously coated with DMEM/F12 4% FCS.

Test samples containing the test PR0224 polypeptide or growth medium only (hegative control) (20 el/well) arc added directly to the wells on day 1. PGF (20 il/well) is then added on day 2 at final concentration of 10 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 showinga large increase in size as compared to negative controls are scored 2.0. The results are shown in Table 9 below.

Table 9 PRO Name PRO Concentration Growth Enhancement Score PR0224 0.01% 0.0 PR0224 0.1% 0.0 PRO0224 1.0% EXAMPLE 74. In situ Hybridization In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression.

anabyze the dssue distnbxin of transcription. itify and localize vira infection. follow changes in specific m.RNA synthesis and aid in chromosome mapping.

In stu hybridization was performed following an optimized version of the protocol by Lu and Giliet. Ci YlaiM 1:169-176 (1994). using PCR-generated "P-labeled riboprobes. Briefly. formalin-fixed. paraffin-cmbedded human tisses were sectioned. deparaffinized. depruteinated in protcinase K (20 g/ml) for 15 miinutes at 37'C. and.

further processed for in situ hybridization as described by Lii and Gilett. supra. A UTP-labeled antiscnsc ribopzobe was generated from a PCR product and hybridized at 55C overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

uP-Plbonrobe synthesis pl (125 mCi) of 3 P-UTP (Amersham BF 1002. SA<2M0 Ci/mmol) were speed vac dried. To each 10 tube containing dried ''P-UTP. the following ingredients were added: Id Sx. transcription buffer p1 DTr (100 mM) plIT mix (2.SmM :1IL; each of10 mM GTP.CTP &AT7P+ 10pl HO) 1.0 As UTP (SO PM) 1.0 Id Rnasin p1 DNA template (1Lpg) 1.0'"1 H'O Id RNA polymerase (for PCR products D3 AS, T7 S, usually) The tubes were Incubated at 37*C for one hour. 1.0 pl 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 was pipetted ont DEBI paper. The remaining solution was loaded in a Microcon-SO ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes).

After the final recovery spin. 100 p1I TB were added. 1 id of the final product was pipetted on DEBI paper and counted in 6 ml of Biofluor 11.

The probe was run on aTBEurea gel. 1-3 pl of the probe or 5 plof RNA Mrk Il were added to 3 P of loading buffer. After heating on a 95*C heat block for thre mintes. the gel was inmmediately placed on ice. The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR film with'an intensifying screen in -70*C freezer one hour to overnight.

s' M-bridization A. Pretreatment of frozen sections 'The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for minues. The trays were placed in 55*C incubator for five minutes to reduce condensation. The srles were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes. at room temperature (25 ml 20 x SSC 975 ml SQ 1120). After deproteination in 0.5 pg/mI proceinase K for 10 minutes at 37*C (12.5 jul of 10 mg/mI stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%. 100% ethanol. 2 minutes each.

178 r r a. Pretreatmt of p=arffin-aedded sectian The slides were deparaffinized, place in SQ H 2 0, and rinsed twice in 2 x SSC at roomn tenperature, for 5 minutes each time. The sections were deproteinated in 20 /V/ml proteinase K (500 pl of 10 ng/nml in 250 mi RNase-free RNase buffer; 379C, 15 minutes) human erbryo, or 8 x proteinase K (100 p1 in 250 ml RNase buffer,, 379C, 30 minute;) formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were perforned as described above.

C. Prelybrdization The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% fonamide) saturated filter paper. The tissue was covered with 50 pl of hybridization buffer (3.75g Dextran Sulfate 6 ml SQ H0), vortexed and heated in the microwave for 2 minutes with the cap loosened.

After cooling on ice, 18.75 ml formnamide, 3.75 ml 20 x S9C 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. Iybidization x 10' qmcp probe and 1.0 pl tRNA (50 rrg/ml stock) per slide were heated at 95 0 C for 3 minutes. The slides were cooled an ice, and 48 pl hybridization buffer were added per slide. After vortexing, 50pl 33 P mix were added to 50 pl prehybridization on slide. The slides were incubated overnight at 55 0

C.

E. Washes Washing was done 2 x 10 minutes with 2 x SSC, EDrA at roan 25 tarperature (400 ml 20 x SSC 16 mil 0.25M EDTA, V=4L), followed by RNaseA treatment at 37 0 C for 30 minutes (500 pl of 10 ng/ml in 250 ml RNase buffer 20 pj/ml). The slides were washed 2 x 10 minutes with 2 x SSC, EDIA at roan tenperature. The stringency wash conditions were as follows: 2 hours at 55 0 C, 0.1 x SSC, EDPA (20 ml 20-x SSC 16 ml EDrA, Vf-4L).

F. Oliganucleotides In situ analysis was performed on a variety of MA sequences disclosed herein. The oligonucleotides earployed for these analyses are as follows: IM33094-1131 (PRO217) p1 5* -CATI3AATAGAI (SEQ ID NO: 347) p2 5' -CA-AATTAACC'IATAAG IC 3 (SEB ID ND:348) ENR33223-136 (PED230) pl 5' TIAATA CICT (SEQ ID NO:349) 179 p2 5' -3 (SEX2 ID NO: 350) (3) p3.

p 2 (4) p1 p2 EN34435-1140 (PRO232) 5' -3 (SEQ 5' -3 (SFJQ ID NO: 351) ID NO: 352) (6) p1 p2 (7) 20 p 1 p2 (a) p1 p 2 EtqA35639-1172 (PIO246) 5' TA~T~1~rrU~r-3' ENMh.9435-12219 (PRO533) LGCG IvG3 5' 3' M135638-2141 (PRO245) -3$ 5' -3A''AAd ENA33O89- M32 (PR0221) 5' 3' 5' 3# Eta35918-174 (P1O258) 5' QGacrrwtrtG-3' 5' 3# EMA2286-U9l (PIKO214) A nxItrcrccG c-3' -3 ENA33-221-2133 (PRO224) 5' 3ATL1mATA..

5' 3' ENAh35557-2.137 (1RO34) 5' -3 5' -rAA TA 1tmAcA Ak'utAGt;3' (SEQ ID NO:357) (SEL2 ID NO:358) (SEp ID NO: 353) (SEQ ID NO: 354) (SEQ ID NO: 355) (SEYQ ID NO:356) (SEQ ID (SB2ID NO: 359) NO: 360)

(SEQ~

(SEp ID NO: 361) ID NO:362) (SEQ2 (SB2 (SE2 (SE2 ID NO:363) ID NO:364) MD NO:365) MD NO: 366) p 1 p2 (2.1) p 1 p2 (Ss2 ID NO:367) (sE2 ID NO:368) (12) EM33100-1159 (PRO229) 180 pi 5' -SATITAATAACAAT 3' (SEQ ID 1ND:369) p2 S' -CTAGAAATrCAAC-3' (SEQ ID tOD:370) (13) IMA34431-1177 (P1o263) pl 5' -AATIAAATAATXACIC ACTATTIAAAI 3' (SDEQ ID NO:371) p2 5'-CTA-3AAATTAACICAC (SED ID 1NO:372) (14) IW38268-1188 (PRO295) pl 5' TITIAATA-CICAAACT 3' (SEQ ID 10:373) p 2 5' cr3nAAA ACIACAGGGGI' (SEQ ID 1NO:374) G. Results Sets In situ analysis was perforned on a variety of EA sequences disclosed herein. The results frcn these analyses are as follows.

15 INa33094-1131 (PRO217) ~Highly distinctive expressian pattern, that does not indicate an obvious biological function. In the humnan erbryo it was expressed in outer srooth nuscle layer of the GI tract, respiratory cartilage, branching respiratory epithelium, osteoblasts, tendons, gonad, in the optic nerve head and developing dermis. In the adult expressian was observed in the epidernal pegs of the chinp tongue, 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 5555 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.

Eln fetal tissues examined (E12-E16 weeks) include: Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus, stomach, small intestine, spleen, thynus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower lirb.

Adult inaan tissues examined: Kidney (normal and end-stage), adrenal, mnocardium, aorta, spleen, lynph node, gall bladder, pancreas, lung, skin, eye (inc. retina), prostate, bladder, liver (norral, cirrhotic, acute failure).

Non-bmaan primate tissues examined: Chiirp Tissues: Salivary gland, stcrach, thyroid, parathyroid, skin, thyrrus, ovary, lynph node Rhesus Monkey Tissues: Cerebral cortex, hippocanpus, cerebellum, penis.

180a EM~33223-MG3 Sections show an intense signal associated with arterial and venous vessels in the fetus. In arteries the signal appeared to be conf ined to szwooth-uscle/pericytic cells. The signal is also seen in capillary vessels and in glcmeruli. It is not clear whkether or not endothelial cells are expressing this niRM. 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 f ibroblast-like cells that exress HG;F uncertain histogenesis. In the adult, there was no evidence of expression and the wall of the aorta and vaost vessels appear to be negative. However, expression was seen over vascular channels in the normal prostate and in the epitheliumn lirung the gallbladder. Insurers expression was seen in the vessels of the sof t-tissue sarcoma and a renal cell carcinarra. In sunrxy, this is a molecule that shows relatively specific vascular expression in the fetus as well as in same adult organs. Expression was also observed in the fetal lens and the adult gallbladder.

In a secondary screen, vascular expression was observed. similar to that observed above, seen in feta blocks. Expression is on vascular smooth muscle. rather than endotheliun. Expriession also seen in smooth muscle of the developing csophagus. so as reported previously. this molecule is Dot vascular specific. Expression was examined inr4 lung and 4 breast carcinomas. Substantial expression was seen in vascular smooth muscle of at least 3/4 lung cancrs and 2/4 breast cancess. In addition, in one breast carcinomna, expression was observed in periturnoral stromiiI cells of uricertain histogenesis (possibly myofibroblasts). No endtothelial cell expression was observed in this study.

DlNA443i-I1140 (PRO232) Strong expression in prostatic cpithetiurn and bladder epitheliumn, lower level of expression in bronchial epthelium. High background low level expression seen in a number of sites, including among others. bone, blood.

choroszuarcoma, adult heart and fetal liver. It is felt that this level of signal represents background, partly because signal at ibis level was seen over the blood. All other tissues negative.

Human fetal tissues examined MEl2-016 weeks)~ 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, testis and lower limb.

Adult hw=a tissues examined: Kiduney (normal arid end-stage), adrenal, spleen. lymph node, pancreas, lung, eye (inc.

retina), bladder. liver (normal, cirrhotic. acute failur).

Nan-human aRinatc tissues examined: 20 Chi=z T.issues: adrenal Rhesus Monkey Tissues: Cerebral cortex, hippocampus In a secondary screen, expression was observed in tie epitheliumn of the prostate, the superficial layers of the urethelium of the urinary bladder, the urethelium, lining the renal pelvis and the urethelium of the ureter 0I out of 2 experiments). The urethra of a rhesus monkey was negative; it is unclear whether this represents a true lack of 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 bladder are similar to those previously described using an isotopic detection technique. Expresion of the mRNA for this antigen is NOT prostate epithelial specific. The antigen may serve as a useful marker for uretlelial derived tissues. Expresson in the superficial, post-mnitotic cells, of the urinary tract epithelium also suggest that it is unlikely to represnt a specific stem cell marker, as this would be expected to be expressed specifically in basal epitheliumn.

DNA35639-172 (PR0246) Strongly expressed in fetal vascular endotheium, including tissues of the CNS. Lower level of expression in adult vasculature, including the CNS. Not obviously expressed at higher evels in tmor vascular endothelium.

Signal also seen over bone matrix arnd adult spleen. not obviously cell associated, probably related to non-specific background at these sites.

Human fetal tissues exaMined (EME-16 weks) include: Placenta, umbilical cord, liver, kidney. adrenals. thyroid, linmp. heumi great vessels. csophagus. stomach, smnall utstine. spleen, thymus. pancreas, brain. eye, spinal cord, body wall, pelvis. testis and lower limb.

Adalt hwoa -isues examined: Kidney (normal an m u iazg). adrenal spleen. lymph node. pancrea, lung. eye (Mn.

retina), bladder, liver (normal, cirrhotic, acute Waiue).

Non-human primate tissuei examined: Chimp.ITisue: adrenal Rhiesus Monkey Tissues: Cerebral cortex, hippocampus Dt4A49435-1219 (PROM33 Moderate expresson over cortical neuronecs in the fetal brain- Expression over the inner aspect of the fetal retina, possible expression in the develoiping lens. Expression over fetal skin). cartilag e, small intestine, placental villi and umbilical cord. In adult tissues there is an extremely high level of expression over the gallbladder epithelium.

Moderat expression over the adult kidney, gastric and! colonic epithelia. 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 fera tissues examined (EI-2-E16 weeks) include:- Placenta, umbilical cord, liver, kidney. adrenals, thyroid, *hirngs, heart, great vessels, csophagus, stomach, small intestine, spleen. thymus, pancreas, brain, eye, spinal cord, body wall. pelvis, testis and lower limb.

Adult human tissues examined: Kidney (normal and! end-stage), adrenal, spleen, lymph node, pancreas, lung eye (inc.

:retina), bladder, liver (normal, cirrhotic, acute failure).

Non-human rimate tissues examined: Chimp isung: adreial Rhesus Monkey Tissues: Cerebral cortex. hippocampus, cerebellum.

.0.0 DNA3563R-1 14 (P-RO245) Expression observeod in the endothelium lining a subset of fetal and placental vessels. Endothelial expression was confined to these tissue blocks. Expression als observed over intermediate trophoblast cells of placenta. All other tissues negative.

etal tismue gairrined fE12-E 16 weeks) includg. Placenta. umbilical cord, liver, kidney, adrenals, thyroid. lungs, heart, great vessels, csophagus. stomach small intestine, spleen, thymus. panicreas. 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 (tin), hippocampus(rnx). cercbelluxn(sm), penis, eye, bladder, stomnach, gastric carcinoma, colon, colonic carcinoma, thyroid (chimp), parathyroid (chimp) ovary (chimp) and chondrosarcoma.

Acetomninophen induced liver injury and hepatic cirrhosis DNA33099-1 132 (PRO2) Specific expression over fetal cerebral white and grey manor, as well as over neuronecs in the spinal cord.

Probe appears to cross react with rat. Low level of expression over cerebellar neutrotics in adult reu brain. All other tissues negative.

Fetal tissues examnined (E12-EI6 weeksa) inckide: 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 tissues examined: Liver, kidney, adrenal. myocardium, aorta, spleen, lymph node, pancea, lung. skin. cerebral cortex bippocarnps(rmn), cerebellum(rm). penis, eye, bladder, stomach, gastric earauxiwa colon, colonic carcinoma and chondrosarcoma. Acetominophen induced liver injury and hepatic cirrhosis DNA35919-1 174 (PRO259) Strong expresion in the, nervous system. In dhe rheus monkey brain expression is observed I cortical, hippocainpal and cerebellar neurones. Epression over spinal neurones in the fetal spinal cord, the developing brain and die 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 over the developing hind brain and spinal cord. Strong expresion over interstitial cells in the placental vill All other tissues were negative.

Fetal tissues examined ME2-EI6 weeks) include: Placenta. umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus, stomach.- small intestine,' spleen, tymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult tissues examined: Liver, kidney, renal cell carcinoma, adrenal, aorta, spleen, lymph niode, pancreas, hing, myocardiami, skin, cerebral cortex bippocampus~rm), cerebelium(rm), bladder, prostate, stomach, gastric carcinoma, colon, colonic carcinoma, thyroid (chimp). parathyroid (chimp) ovary (chimp) and chondrosarcoma. Acetorninophen induced liver injury and hepatic cirrhosis.

DNA32296-1 191 (PR0214) Fetal tissue: Low level throughout mesenchyrne. Moderate expression in placental stromal cells in membranous tissues and in thyroid. Low level expression in cortical neurones. Adult tissue: all negative.

Fetal tissues examined (E12-EI6 weeks) include: Placenta, umnbilical cord, liver, kidney, adrenals, thyroid, lungs, heart great vessels. oesopha stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adut tsses xaine ichxe:Liver, kidney, adrenal, myocardium., aorta, spleen, lymph node, pancreas, lung and skin.

(10) DNA33221-nn3 (PR0224 Expression limited so vascular endothelium in fetal spleen, adult spleen, fetal liver, adult thyroid and adult lymph node (chimp). Additional site of expression is die developing spinal ganlia. All other tissues negative.

Humazn fetal tissues examined (IE12-E16 weeks) include: Placenta, umbilical cord, liver, lddricy. adrenals. thyroid.

lungs, heart, great vessels, oesophagus, stomach, small'intestine, spleen, thymus. pancreas, brain, eye, spinal cord, body wall. pelvis and lower limb.

Adult humnan rismes examined: Kidney (normal and end-stage), adrenal, miyocardiunm, aorta, splcen, lymph node.

pancreas, lung. sin, eye (inc. retina), bladder, liver (normal, cirrhotic. acute failure).

Non-human rprimate tissues examined: issjiues: Salivary gland, stomach, thyroid. parathyroid, sin, thymus. ovary, lymph node.

Rhesus Monkey Tissues: Cerebral cortex, bippocanps, cerebellum, penis.

(11) DNA35557-1 137 (PR0234) Specific expression over developing motor neurones in ventral aspect of the fetal spinal cord (will develop into ventral horns of spinal cord). AlD other tissues negative. Possible role in growth, differentiation and/or ~~~~development of spinal mnotor neurons. nld:Paetublclcrliekdearnl~hrilns Fetal tissues examined (P12-Fl6 weeks clue lcnasmiialcrlvr dn drnl,-hrilns heart, great vessels, otsophagus. stomach, small intestine, spleen, thymus. pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult tissues examined: Liver, kidney, adrenal, mnyocardium, aorta, spleen, lymph node, Sucreas. lung, skin., cerebral cortex OWn. bippocanqpus(rm), cercbellum(rm), penis, eye, bladder, stomach, gastric .:carcinoma, colon, colonic carcinoma and ehondrosucoma. Acetomninophen induced liver injury and hepatic cirrhosis (12) DNA33100-1 159 (PR02291 Strikin expression in mononuclear phagocytes (macrophages) of fetal and adult spleen, liver, lymph node and adult thymus (in tingible body macrophages). The highest expression is in the spleen. .All other tissues negative.

Localisation and homology are entrely consistent with a role as a scavenger receptor for cells of the reticuloendothelial System. Expression also observed in placenal mononuclear cells.

HUMa fetal tssues examiined (E12-EI6 weeks) incude: Placenta, umbilcal cord, liver, idney, adrenals, thyroid, lungs, heart, great vessels, oesophagus, stomach, smafl intestine, spleen. thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult human tissues examined: Kidney (normal and end-stage), adrenal, myocardium, aorta, spleen lymph node, gall bladder, pancreas, lung, sin, eye (inc. retina), prostate, bladder, liver (normal, cirrhotic, acute failure).

Non-human rimate tissues examined: Chft Thisses Salivary gland, stomach, thyroid, parathyroid. skin, thymus. ovary, lymph node.

Rhesus Monke Tissues: Cerebral cortex, bippocampus, cerebellum, penis.

(13) DNA34431-1 177 (PR0263) Widepread expression in human fetal tissues and placenta over mononuclear cells, probably nia=rPhages +-lymphocytes. 'Me cellular disorlbtion follows a perivascular pattern in many tissues. Strong expression als0 seen in cpithelial ells of the fetal adrenal cortex. All adult tissues were negative.

Fetal tissue examined (1312-1116 weeks) include: Placenta, umbilical cord. liver, kidney. adrenals, thromid, lungs.

heart, great vessels. csophagus. stomach, small intestine. spleen. thymus, pancreas, brain, eyc. spinal cord, body wall. pelvis and lower limb.

Adult tissues exa&Mind: Liver, kidney, adrenal. spleen. lymph node. pancreas. lung, skin, cerebral cortex (ru), hippocampus(rr). cerebellum(rm), bladder, stomach, colon and colonic carcinoma. Acetorninoplien induced liver injury and hepatic cirrhosis.

A secondary screen evidenced expression over strosnal mononuclear cells probably histiocyces.

(14) DNA38269-1 198 (PR0295) Hfighx expression over ganglion cells in human fetal spinal ganglia and over large neurones in the anterior horns of the developing spinal cord. In the adult there is expression in dhe chimp adrenal medulla (neural), neurones of the dicu monkey brain fippocanpus (-+1and cerba cotx an eroe in gagi in th ora adult human prostate (dhe only section that contains gan~glion cells. je expression in this cell type is presumed NOT to be confined to t prostate). All other tissues negative.

Hnmn fetal ismes eaminedl (El7-Et6 weeks) innludc: Placenta, umbilical cord, liver, kidney, adrenals. thyroid, hmpg, great vessels, stomach. small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis.

testis and lower limb.

Adult human tissues examined: Kidney (ormal ard end-tage), adrenal, spleen. lymph node, pancreas, lung, eye (inc.

retina), bladder, liver (normal, cirrhotic, acuite failure).

Non-human Vmte tissues examined: Chi= issues: adrenal Rhesus Monke Tissues: Cerebral cortex, hippocampus, cerebellum.

Denosit of Material The following materials have been deposited with tie American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD. USA (ATCC): Material AICC 0 o o tDt 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 DNA27864-1 155 ATCC 209375 October 16. 1997 DNA36350-1158 ATCC 209378 October 16, 1997 DNA322WI- 164 ATCC 209384 October 16, 1997 DNA35639-172 ATCC 209396 October 17, 1997 DNA33092-1202 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 209262 September 16. 1997 DNA33786-1 132 ATCC 209253 September 16, 19917 DNA35918-1 174 ATCC 209402 October 17, 1997 186 DNA3 7150-1178 DNA3 8260-1180 DNA39969-1185 DNA3 2286-1191 DNA3 3461-1199 DNA40628- 12 16 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 DNA35841-1173 DNA3 3470-1175.

DNA3 4431-1177 DNA3 9510-1181 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 094 01 ATCC2 093 97 ATCC2 094 00 ATCC2 09385 ATCC2093 67 ATCC2 094 32 ATCC2 092 63 ATCC2 092 51 ATCC2 092 55 ATCC2 09252 ATCC2 093 77 ATCC2 093 70 ATCC2 095 23 ATCC2093 72 ATCC209374 ATCC2 093 73 ATCC2 093 82 ATCC2 09383 ATCC2 094 03 ATCC2 093 98 ATCC2 09399 ATCC2 09392.

ATCC2 093 87 ATCC2 09388 ATCC2 093 94 ATCC2 09421 ATCC2 093 93 ATCC2 094 18 ATCC2 094 85 ATCC209483- ATCC209492 ATCC209491 ATCC2 09481 ATCC209438 ATCC209927 October 17, 1997 October 17, 1997 October 17, 1997 October 16, 1997 October 15, 1997 November 7, 1997 September 16, 1997 September 16. 1997 September 16, 1997 September 16, 1997 October 16, 1997 October 16, 1997 December 10, 1997 October 16, 1997 October 16, 1997 October 16, 1997 October 16, 1997 October 16, 1997 October 17, 1997 October 17, 1997 October 17, 1997 October 17, 1997 October 17, 1997 October 17, 1997 October 17, 1997 October 28, 1997 October 17, 1997 October 28, 1997 November 21, 1997 November 21, 1997 November 21, 1997 November 21, 1997 November 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 5 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 15 availability of the progeny to one determined a I by the U.S. Commissioner of Patents and Trademarks to be entitled thereto, according to 35 Usc 122 and dhe Comumissioner's rules pursuant theret (including 37 CFR 1. 14 with particular reference to 88600G638).

Tle assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost ordestroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Xvailability of the deposited material is not to be consmiwd as a license to practice t invention in contravention of the righits granted under the authoity of any government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice thc invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constuts that art funoctionafly equivalent are within the scope of this invenuion. The -deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable die practice of any aspect of the invetidon, inchidin t best mode thereof, nor is it so be construed as limiting the scope of the claim to the specific ilustriations that it represents. Indeed. various nxdikations of the invention in addition to those shown and described heenwill become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims (15)

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 44 (SEQ ID NO:119).

2. A nucleic acid molecule according to Claim 1, wherein said nucleotide sequence comprises a nucleotide sequence as shown in Figure 43 (SEQ ID NO:118), 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 43 (SEQ ID NO:118), 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 209432.

5. A vector comprising a nucleic acid according to any one of Claims 1 to 4. 4 A vector according to Claim 5, operably linked to 25 control sequences vector. recognized by a host cell transformed with the

7. A host cell comprising a vector according to Claim or Claim 6.

8. A host cell according to Claim 7, wherein said cell is a CHO cell.

9. A host cell according to Claim 7, wherein said cell is an E. coli. A host cell according to Claim 7, wherein said cell is a yeast 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 44 (SEQ ID NO:119).

13. Isolated PRO polypeptide having at least 80% sequence identity to the amino acid sequence encoded by the nucleotide deposited under accession number ATCC 209432.

14. A chimeric molecule comprising a polypeptide according to Claim 12 or Claim 13, fused to a heterologous amino acid sequence. A chimeric molecule according to Claim 14, wherein 20 said heterologous amino acid sequence is an epitope tag sequence.

16. A chimeric molecule according to Claim 14, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.

17. 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; or c) a chimeric molecule according to any one of Claims 14 to 16, together with a pharmaceutically-acceptable carrier.

18. A nucleic acid according to Claim 1 substantially as hereinbefore described with reference to any one of the examples. 190 19 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. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia .*o