EP1037979A2

EP1037979A2 - Polypeptides and nucleic acids encoding the same

Info

Publication number: EP1037979A2
Application number: EP98960440A
Authority: EP
Inventors: William I. Wood; Audrey Goddard; Austin L. Gurney; Jean Yuan; Kevin P. Baker; Jian Chen
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 1997-12-03
Filing date: 1998-12-01
Publication date: 2000-09-27
Also published as: WO1999028462A9; CA2311640A1; WO1999028462A3; NZ527898A; IL178210A0; WO1999028462A2; AU1602999A; NZ504548A; JP2002505850A; NZ527899A; IL136216A; NZ527900A; AU736506B2; KR20010032719A; KR100386046B1; IL197834A0; JP2003116587A; JP2003116588A; IL136216A0; JP3786909B2

Abstract

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

Description

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

Extracellular proteins play an important role in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, e.g., 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. Secreted proteins have various industrial applications, including pharmaceuticals, diagnostics, biosensors and bioreactors. Most protein drugs available at present, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secretory proteins. Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents. Efforts are being undertaken by both industry and academia to identify new, native secreted proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted proteins. Examples of screening methods and techniques are 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)].

Membrane-bound proteins and receptors can play an important role in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, e.g., 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. Such membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. For instance, transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of 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. Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents. Receptor immunoadhesins, for instance, can be employed as therapeutic agents to block receptor-ligand interaction. The membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. Efforts are being undertaken by both industry and academia to identify new, native receptor proteins. Many 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 and characterization of novel secreted and transmembrane polypeptides and novel nucleic acids encoding those polypeptides.

1. PRQ241 Cartilage is a specialized connective tissue with a large extracellular matrix containing a dense network of collagen fibers and a high content of proteoglycan. While the majority of the proteoglycan in cartilage is aggrecan, which contains many chondroitin sulphate and keratin sulphate chains and forms multimolecular aggregates by binding with link protein to hyaluronan, cartilage also contains a number of smaller molecular weight proteoglycans. One of these smaller molecular weight proteoglycans is a protein called biglycan, a proteoglycan which is widely distributed in the extracellular matrix of various other connective tissues including tendon, sclera, skin, and the like. Biglycan is known to possess leucine-rich repeat sequences and two chondroitin sulphate/dermatan sulphate chains and functions to bind to the cell-binding domain of fibronectin so as to inhibit cellular attachment thereto. It is speculated that the small molecular weight proteoglycans such as biglycan may play important roles in the growth and/or repair of cartilage and in degenrative diseases such as arthritis. As such, there is an interest in identifying and characterizing novel polypeptides having homology to biglycan protein.

We herein describe the identification and characterization of novel polypeptides having homology to the biglycan protein, wherein those polypeptides are herein designated PR0241 polypeptides.

2. PRQ243 Chordin (Xenopus, Xchd) is a soluble factor secreted by the Spemann organizer which has potent dorsalizing activity (Sasai et al., Cell 79: 779-90 (1994); Sasai et al, Nature 376: 333-36 (1995). Other dorsalizing factors secreted by the organizer are noggin (Smith and Harlan, Cell 70: 829-840 (1992); Lamb et al, Science 262: 713-718 (1993) and follistatin (Hemmanti-Brivanlou et al. , Cell 77: 283-295 (1994). Chordin subdivides primitive ectoderm into neural versus non-neural domains, and induces notochord and muscle formation by the dorsalization of the mesoderm. It does this by functioning as an antagonist of the ventralizing BMP-4 signals. This inhibition is mediated by direct binding of chordin to BMP-4 in the extracellular space, thereby preventing BMP-4 receptor activation by BMP-4 (Piccolo et al , Develop. Biol. 182: 5-20 (1996).

BMP-4 is expressed in a gradient from the ventral side of the embryo, while chordin is expressed in a gradient complementary to that of BMP-4. Chordin antagonizes BMP-4 to establish the low end of the BMP-4 gradient. Thus, the balance between the signal from chordin and other organizer-derived factors versus the BMP signal provides the ectodermal germ layer with its dorsal-ventral positional information. Chordin may also be involved in the dorsal-ventral patterning of the central nervous system (Sasai et al. Cell 79: 779-90 (1994). It also induces exclusively anterior neural tissues (forebrain-type), thereby anteriorizing the neural type (Sasai et al, Cell 79: 779-90 (1997). Given its role in neuronal induction and patterning, chordin may prove useful in the treatment of neurodegenerative disorders and neural damage, e.g., due to trauma or after chemotherapy.

We herein describe the identification and characterization of novel polypeptides having homology to the chordin protein, wherein those polypeptides are herein designated PR0243 polypeptides.

3. PRQ299 The notch proteins are involved in signaling during development. They may effect asymmetric development potential and may signal expression of other proteins involved in development. [See Robey , E. , Curr. Opin. Genet. Dev.. 7{4):551 (1997), Simpson, P., Curr. Opin. Genet. Dev.. 7{4):537 (1997), Blobel, CP., Cell, 90(41:589 (1997)], Nakayama, H. et al., Dev. Genet.. 21(1 :21 (1997), Nakayama, H. et al., Dev. Genet.. 21(11:21 (1997), Sullivan, S.A. et al. , Dev. Genet.. 20(31:208 (1997) and Hayashi, H. et al., Int. J. Dev. Biol.. 40(61:1089(19961.] Serrate-mediated activation of notch has been observed in the dorsal compartment of the Drosophila wing imaginal disc. Fleming et al., Development. 124(15):2973 (1997). Notch is of interest for both its role in development as well as its signaling abilities. Also of interest are novel polypeptides which may have a role in development and/or signaling.

We herein describe the identification and characterization of novel polypeptides having homology to the notch protein, wherein those polypeptides are herein designated PR0299 polypeptides.

4. PRQ323

Dipeptidases are enzymatic proteins which function to cleave a large variety of different dipeptides and which are involved in an enormous number of very important biological processes in mammalian and non-mammalian organisms. Numerous different dipeptidase enzymes from a variety of different mammalian and non-mammalian organisms have been both identified and characterized. The mammalian dipeptidase enzymes play important roles in many different biological processes including, for example, protein digestion, activation, inactivation, or modulation of dipeptide hormone activity, and alteration of die physical properties of proteins and enzymes.

In light of the important physiological roles played by dipeptidase enzymes, efforts are being undertaken by both industry and academia to identify new, native dipeptidase homologs. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane -bound receptor proteins. Examples of screening methods and techniques are described in the literature

[see, for example, Klein et al., Proc. Natl. Acad. Sci.. 93:7108-7113 (1996); U.S. Patent No. 5,536,637)].

We herein describe the identification and characterization of novel polypeptides having homology to various dipeptidase enzymes, designated herein as PR0323 polypeptides.

5. PRQ327

The anterior pituitary hormone prolactin is encoded by a member of the growth hormone/prolactin/placental lactogen gene family. In mammals, prolactin is primarily responsible for the development of the mammary gland and lactation. Prolactin functions to stimulate the expression of milk protein genes by increasing both gene transcription and mRNA half-life.

The physiological effects of the prolactin protein are mediated through the ability of prolactin to bind to a cell surface prolactin receptor. The prolactin receptor is found in a variety of different cell types, has a molecular mass of approximately 40,000 and is apparendy not linked by disulfide bonds to itself or to other subunits. Prolactin receptor levels are differentially regulated depending upon the tissue studied.

Given the important physiological roles played by cell surface receptor molecules in vivo, efforts are currently being undertaken by both industry and academia to identify new, native membrane -bound receptor proteins, including those which share sequence homology with the prolactin receptor. Many of these efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel membrane-bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Natl. Acad. Sci.. 93:7108-7113 (1996); U.S. Patent No. 5,536,637)].

We herein describe the identification and characterization of novel polypeptides having significant homology to the prolactin receptor protein, designated herein as PR0327 polypeptides.

6. PRQ233

Studies have reported that 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 tissue necrosis, organ failure, atherosclerosis, infertility, birth defects, premature aging, mutations and malignancy. Thus, the control of oxidation and reduction is important for a number of reasons, including the control and prevention of strokes, heart attacks, oxidative stress and hypertension.

Oxygen free radicals and antioxidants appear to play an important role in the central nervous system after cerebral ischemia and reperfusion. Moreover, cardiac injury, related to ischaemia and reperfusion has been reported to be caused by die action of free radicals. In this regard, reductases, and particularly, oxidoreductases, are of interest. In addition, the transcription factors, NF-kappa B and AP-1, are known to be regulated by redox state and to affect the expression of a large variety of genes thought to be involved in the pathogenesis of AIDS, cancer, atherosclerosis and diabetic complications. Publications further describing this subject matter include Kelsey et al., Br. J. Cancer. 76(7):852-854 (1997); Friedrich and Weiss, J. Theor. Biol.. 187(4): 529-540 (1997) and Pieulle et al., J. Bacteriol.. 179(18):5684-5692 (1997). Given the physiological importance of redox reactions in vivo, efforts are currently being under taken to identify new, native proteins which are involved in redox reactions. We describe herein the identification and characterization of novel polypeptides which have homology to reductase, designated herein as PR0233 polypeptides.

7. PRQ344

The complement proteins comprise a large group of serum proteins some of which act in an enzymatic cascade, producing effector molecules involved in inflammation. The complement proteins are of particular physiological importance in regulating movement and function of cells involved in inflammation. Given the physiological importance of inflammation and related mechanisms in vivo, efforts are currently being under taken to identify new, native proteins which are involved in inflamation. We describe herein the identification and characterization of novel polypeptides which have homology to complement proteins, wherein those polypeptides are herein designated as PR0344 polypeptides.

8. PRQ347

Cysteine-rich proteins are generally proteins which have intricate three-dimensional structures and/or exist in multimeric forms due to the presence of numerous cysteine residues which are capable of forming disulfide bridges. One well known cysteine-rich protein is the mannose receptor which is expressed in, among other tissues, liver where it serves to bind to mannose and transport it into liver cells. Other cysteine-rich proteins are known to play important roles in many o er physiological and biochemical processes. As such, there is an interest in identifying novel cysteine-rich proteins. In this regard, Applicants describe herein the identification and characterization of novel cysteine-rich polypeptides that has significant sequence homology to the cysteine-rich secretory protein-3, designated herein as PR0347 polypeptides.

9. PRQ354

Inter-alpha-trypsin inhibitor (ITI) is a large (Mr approximately 240,000) circulating protease inhibitor found in the plasma of many mammalian species. The intact inhibitor is a glycoprotein and consists of three glycosylated subunits that interact through a strong glycosanunoglycan linkage. The anti-trypsin activity of ITI is located on the smallest subunit (i.e., the light chain) of the complex, wherein that light chain is now known as the protein bikunin. The mature light chain consists of a 21-amino acid N-terminal sequence, glycosylated at Ser-10, followed by two tandem Kunitz-type domains, the first of which is glycosylated at Asn-45 and die second of which is capable of inhibiting trypsin, chymotrypsin and plasmin. The remaining two chains of the ITI complex are heavy chains which function to interact with the enzymatically active light chain of the complex.

Efforts are being undertaken by both industry and academia to identify new, native proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane -bound receptor proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Natl. Acad. Sci.. 93:7108-7113 (1996); U.S. Patent No. 5,536,637)]. We herein describe the identification and characterization of novel polypeptides having significant homology to the ITI heavy chain, designated in the present application as PR0354 polypeptides.

10. PRQ355

Cytotoxic or regulatory T cell associated molecule or "CRTAM" protein is structurally related to the immunoglobulin superfamily. The CRTAM protein should be capable of mediating various immune responses. Antibodies typically bind to CRTAM proteins with high affinity. Zlotnik, A., Faseb. 10(6): A1037, Abr. 216, June 1996. Given the physiological importance of T cell antigens and immune processes in vivo, efforts are currently being under taken to identify new, native proteins which are involved in immune responses. See also Kennedy et al., U.S. Pat. No. 5,686,257 (1997). We describe herein the identification and characterization of novel polypeptides which have homology to CRTAM, designated in the present application as PR0355 polypeptides.

11. PRQ357

Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be more easily manipulated to regulate the particular result of the protein-protein interaction. Thus, the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community. All proteins containing leucine-rich repeats are drought to be involved in protein-protein interactions. Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. The crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape. These two features have been indicated as responsible for the protein-binding functions of proteins containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends Biochem. Sci.. 19(10):415-421 (Oct. 1994).

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

Fr. Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in the leucine rich motif in a complex associated with the bleeding disorder Bernard-Soulier syndrome, Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1): 111- 116 (July 1995), reporting that platelets have leucine rich repeats and Ruoslahti, E. I., et al., WO9110727-A by La

Jolla Cancer Research Foundation reporting that decorin binding to transforming growth factorβ has involvement in a treatment for cancer, wound healing and scarring. Related by function to this group of proteins is the insulin like growth factor (IGF), in that it is useful in wound-healing and associated therapies concerned with re-growth of tissue, such as connective tissue, skin and bone; in promoting body growth in humans and animals; and in stimulating other growth-related processes. The acid labile subunit (ALS) of IGF is also of interest in that it increases the half-life of

IGF and is part of the IGF complex in vivo.

Another protein which has been reported to have leucine-rich repeats is die SLIT protein which has been reported to be useful in treating neuro-degenerative diseases such as Alzheimer's disease, nerve damage such as in

Parkinson's disease, and for diagnosis of cancer, see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1 by Yale University. Also of interest is LIG-1, a membrane glycoprotein that is expressed specifically in glial cells in the mouse brain, and has leucine rich repeats and immunoglobulin-like domains. Suzuki, et al., J. Biol. Chem.

(U.S.), 271(37):22522 (1996). Other studies reporting on the biological functions of proteins having leucine rich repeats include: Tayar, N., et al., Mol. Cell Endocrinol.. (Ireland), 125(l-2):65-70 (Dec. 1996) (gonadotropin receptor involvement); Miura, Y., et al., Nippon Rinsho (Japan), 54(7): 1784-1789 (July 1996) (apoptosis involvement); Harris, P. C, et al., J. Am. Soc. Nephrol.. 6(4): 1125-1133 (Oct. 1995) (kidney disease involvement).

Efforts are therefore being undertaken by both industry and academia to identify new proteins having leucine rich repeats to better understand protein-protein interactions. Of particular interest are those proteins having leucine rich repeats and homology to known proteins having leucine rich repeats such as the acid labile subunit of insulin-like growth factor. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane -bound proteins having leucine rich repeats. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Natl. Acad.

Sci.. £3:7108-7113 (1996); U.S. Patent No. 5,536,637)].

We describe herein the identification and characterization of novel polypeptides having homology to the acid labile subunit of insulin-like growth factor, designated in the present application as PR0357 polypeptides.

12. PRQ715

Control of cell numbers in mammals is believed to be determined, in part, by a balance between cell proliferation and cell death. One form of cell death, sometimes referred to as necrotic cell death, is typically characterized as a pathologic form of cell death resulting from some trauma or cellular injury. In contrast, there is another, "physiologic" form of cell death which usually proceeds in an orderly or controlled manner. This orderly or controlled form of cell death is often referred to as "apoptosis" [see, e.g. , Barr et al., Bio/Technology. 12:487-493 (1994); Steller et al., Science. 267: 1445-1449 (1995)]. Apoptotic cell death naturally occurs in many physiological processes, including embryonic development and clonal selection in die immune system [Itoh et al., Cell. 66:233-243 (1991)]. Decreased levels of apoptotic cell death have been associated with a variety of pathological conditions, including cancer, lupus, and herpes virus infection [Thompson, Science. 267: 1456-1462 (1995)]. Increased levels of apoptotic cell death may be associated with a variety of other pathological conditions, including AIDS, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, retinitis pigmentosa, cerebellar degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury, and toxin-induced liver disease [see, Thompson, supra] . Apoptotic cell death is typically accompanied by one or more characteristic morphological and biochemical changes in cells, such as condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function. A variety of extrinsic and intrinsic signals are believed to trigger or induce such morphological and biochemical cellular changes [Raff, Nature. 356:397- 400 (1992); Steller, supra: Sachs et al., Blood. 82:15 (1993)]. For instance, they can be triggered by hormonal stimuli, such as glucocorticoid hormones for immature thymocytes, as well as withdrawal of certain growth factors [Watanabe-Fukunaga et al., Nature. 356:314-317 (1992)]. Also, some identified oncogenes such as myc, rel, and E1A, and tumor suppressors, like p53, have been reported to have a role in inducing apoptosis. Certain chemotherapy drugs and some forms of radiation have likewise been observed to have apoptosis-inducing activity [Thompson, supra! . Various molecules, such as tumor necrosis factor- " ("TNF-α"), tumor necrosis factor-β ("TNF-β" or

"lymphotoxin-α"), lymphotoxin-β ("LT-β"), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (also referred to as Fas ligand or CD95 ligand), and Apo-2 ligand (also referred to as TRAIL) have been identified as members of the tumor necrosis factor ("TNF") family of cytokines [See, e.g., Gruss and Dower, Blood. 85:3378-3404 (1995); Pitti et al., J. Biol. Chem.. 271:12687-12690 (1996); Wiley et al., Immunity. 3:673-682 (1995); Browning et al., Cell, 72:847-856 (1993); Armitage et al. Nature. 357:80-82 (1992)]. Among these molecules, TNF- α, TNF-β, CD30 ligand, 4-1BB ligand, Apo-1 ligand, and Apo-2 ligand (TRAIL) have been reported to be involved in apoptotic cell death. Both TNF-α and TNF-β have been reported to induce apoptotic death in susceptible tumor cells [Schmid et al., Proc. Natl. Acad. Sci.. 83:1881 (1986); Dealtry et al., Eur. J. Immunol.. 17:689 (1987)]. Zheng et al. have reported that TNF-α is involved in post-stimulation apoptosis of CD8-positive T cells [Zheng et al., Nature. 377:348-351 (1995)]. Other investigators have reported that CD30 ligand may be involved in deletion of self- reactive T cells in the thymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium on Programmed Cell Death, Abstr. No. 10, (1995)].

Mutations in the mouse Fas/Apo-1 receptor or ligand genes (called Ipr and gld, respectively) have been associated with some autoimmune disorders, indicating that Apo-1 ligand may play a role in regulating the clonal deletion of self-reactive lymphocytes in the periphery [Krammer et al., Curr. Op. Immunol.. 6:279-289 (1994); Nagata et al., Science. 267:1449-1456 (1995)]. Apo-1 ligand is also reported to induce post-stimulation apoptosis in CD4-positive T lymphocytes and in B lymphocytes, and may be involved in the elimination of activated lymphocytes when their function is no longer needed [Krammer et al., supra: Nagata et al., supra] . Agonist mouse monoclonal antibodies specifically binding to the Apo-1 receptor have been reported to exhibit cell killing activity that is comparable to or similar to that of TNF-α [Yonehara et al., J. Exp. Med.. 169:1747-1756 (1989)].

Induction of various cellular responses mediated by such TNF family cytokines is believed to be initiated by their binding to specific cell receptors. Two distinct TNF receptors of approximately 55-kDa (TNFR1) and 75- kDa (TNFR2) have been identified [Hohman et al., J. Biol. Chem.. 264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci.. 87:3127-3131 (1990); EP 417,563, published March 20, 1991] and human and mouse cDNAs corresponding to both receptor types have been isolated and characterized [Loetscher et al., Cell. 61:351 (1990); Schall et al., CeU, 61:361 (1990); Smith et al., Science. 248:1019-1023 (1990); Lewis et al., Proc. Natl. Acad. Sci.. 88:2830-2834 (1991); Goodwin et al., Mol. Cell. Biol.. 11:3020-3026 (1991)]. The TNF family ligands identified to date, with the exception of lymphotoxin-α, are type II transmembrane proteins, whose C-terminus is extracellular. In contrast, most receptors in the TNF receptor (TNFR) family identified to date are type I transmembrane proteins. In both the TNF Ugand and receptor families, however, homology identified between family members has been found mainly in the extracellular domain ("ECD"). Several of the TNF family cytokines, including TNF-α, Apo-1 ligand and CD40 Ugand, are cleaved proteolytically at the cell surface; the resulting protein in each case typically forms a homotrimeric molecule that functions as a soluble cytokine. TNF receptor family proteins are also usually cleaved proteolytically to release soluble receptor ECDs that can function as inhibitors of the cognate cytokines. Recently, other members of the TNFR family have been identified. Such newly identified members of the

TNFR family include CAR1, HVEM and osteoprotegerin (OPG) [Brojatsch et al., Cell, 82:845-855 (1996); Montgomery et al., Cell, 22:427-436 (1996); Marsters et al., J. Biol. Chem.. 222:14029-14032 (1997); Simonet et al., CeU, 89:309-319 (1997)]. Unlike other known TNFR-like molecules, Simonet et al., supra, report that OPG contains no hydrophobic transmembrane-spanning sequence. For a review of the TNF family of cytokines and their receptors, see Grass and Dower, supra.

Applicants herein describe the identification and characterization of novel polypeptides having homology to members of the tumor necrosis factor family of polypeptides, designated herein as PR0715 polypeptides.

13. PRQ353 The complement proteins comprise a large group of serum proteins some of which act in an enzymatic cascade, producing effector molecules involved in inflammation. The complement proteins are of particular importance in regulating movement and function of cells involved in inflammation. Given the physiological importance of inflammation and related mechanisms in vivo, efforts are currently being under taken to identify new, native proteins which are involved in inflamation. We describe herein the identification and characterization of novel polypeptides which have homology to complement proteins, designated herein as PR0353 polypeptides.

14. PRQ361

The mucins comprise a family of glycoproteins which have been implicated in carcinogenesis. Mucin and mucin-like proteins are secreted by both normal and transformed cells. Both qualitative and quantitative changes in mucins have been implicated in various types of cancer. Given the medical importance of cancer, efforts are currently being under taken to identify new, native proteins which may be useful for the diagnosis or treatment of cancer. The chitinase proteins comprise a family of which have been implicated in pathogenesis responses in plants. Chitinase proteins are produced by plants and microorganisms and may play a role in the defense of plants to injury. Given the importance of plant defense mechanisms, efforts are currently being under taken to identify new, native proteins which may be useful for modulation of pathogenesis-related responses in plants. We describe herein the identification and characterization of novel polypeptides which have homology to mucin and chitinase, designated in the present application as PR0361 polypeptides.

15. PRQ365

Polypeptides such as human 2-19 protein may function as cytokines. Cytokines are low molecular weight proteins which function to stimulate or inhibit the differentiation, proliferation or function of immune cells. Cytokines often act as intercellular messengers and have multiple physiological effects. Given the physiological importance of immune mechanisms in vivo, efforts are currently being under taken to identify new, native proteins which are involved in effecting the immune system. We describe herein the identification and characterization of novel polypeptides which have homology to the human 2-19 protein, designated heein as PR0365 polypeptides.

SUMMARY OF THE INVENTION

1. PRQ241

Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to biglycan protein, wherein the polypeptide is designated in the present application as "PR0241 " .

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0241 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0241 polypeptide having amino acid residues 1 to 379 of Figure 2 (SEQ ID NO:2), 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 PR0241 polypeptide. In particular, the invention provides isolated native sequence PR0241 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 379 of Figure 2 (SEQ ID N0:2). Another embodiment of the present invention is directed to a PR0241 polypeptide lacking the N-terminal signal peptide, wherein the PR0241 polypeptide comprises about amino acids 16 to 379 of the full-length PR0241 amino acid sequence (SEQ ID N0:2).

2. PRQ243 AppUcants have identified a cDNA clone (DNA35917-1207) that encodes a novel polypeptide, designated in the present application as "PR0243" .

In one embodiment, the invention provides an isolated nucleic acid molecule having at least about 80% sequence identity to (a) a DNA molecule encoding a PR0243 polypeptide comprising the sequence of amino acids 24 to 954 of Fig. 4 (SEQ ID NO:7), or (b) the complement of the DNA molecule of (a). The sequence identity preferably is about 85 % , more preferably about 90 % , most preferably about 95 % . In one aspect, the isolated nucleic acid has at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity with a polypeptide having amino acid residues 1 to 954 of Fig. 4 (SEQ ID NO:7). Preferably, the highest degree of sequence identity occurs within the four (4) conserved cysteine clusters (amino acids 51 to 125; amino acids 705 to 761; amino acids 784 to 849; and amino acids 897 to 931) of Fig. 4 (SEQ ID NO:7). In a further embodiment, the isolated nucleic acid molecule comprises DNA encoding a PR0243 polypeptide having amino acid residues 24 to 954 of Fig. 4 (SEQ ID NO:7), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the invention provides a nucleic acid of the full length protein of clone DNA35917- 1207, deposited with the ATCC under accession number ATCC 209508, alternatively the coding sequence of clone DNA35917-1207, deposited under accession number ATCC 209508.

In yet another embodiment, the invention provides isolated PR0243 polypeptide. In particular, the invention provides isolated native sequence PR0243 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 24 to 954 of Figure 4 (SEQ ID NO:7). Native PR0243 polypeptides with or without the native signal sequence (amino acids 1 to 23 in Figure 4 (SEQ ID NO:7), and with or without the initiating methionine are specifically included. Alternatively, the invention provides a PR0243 polypeptide encoded by the nucleic acid deposited under accession number ATCC 209508.

3. PRQ299

Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0299" .

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0299 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0299 polypeptide having amino acid residues 1 to 737 of Figure 9 (SEQ ID NO: 15), 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 PR0299 polypeptide. In particular, the invention provides isolated native sequence PR0299 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 737 of Figure 9 (SEQ ID NO: 15). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0299 polypeptide.

4. PRQ323

AppUcants have identified a cDNA clone that encodes a novel polypeptide having homology to a microsomal dipeptidase protein, wherein the polypeptide is designated in the present application as "PR0323" .

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a

PR0323 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0323 polypeptide having amino acid residues 1 to 433 of Figure 13 (SEQ ID N0:24), 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 PR0323 polypeptide. In particular, the invention provides isolated native sequence PR0323 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 433 of Figure 13 (SEQ ID N0:24).

5. PRQ327

Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to prolactin receptor, wherein the polypeptide is designated in the present application as "PR0327" . In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0327 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0327 polypeptide having amino acid residues 1 to 422 of Figure 17 (SEQ ID NO:32), 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 PR0327 polypeptide. In particular, the invention provides isolated native sequence PR0327 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 422 of Figure 17 (SEQ ID NO:32).

6. PRQ233 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0233".

PR0233 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0233 polypeptide having amino acid residues 1 to 300 of Figure 19 (SEQ ID N0:37), 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 PR0233 polypeptide. In particular, the invention provides isolated native sequence PR0233 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 300 of Figure 19 (SEQ ID N0:37).

7. PRQ344

Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptides are designated in the present application as "PR0344".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0344 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0344 polypeptide having amino acid residues 1 to 243 of Figure 21 (SEQ ID N0:42), 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 PR0344 polypeptide. In particular, the invention provides isolated native sequence PR0344 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 243 of Figure 21 (SEQ ID N0:42).

8. PRQ347

Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to cysteine-rich secretory protein-3, wherein the polypeptide is designated in the present application as "PR0347" .

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0347 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0347 polypeptide having amino acid residues 1 to 455 of Figure 23 (SEQ ID NO: 50), 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 PR0347 polypeptide. In particular, the invention provides isolated native sequence PR0347 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 455 of Figure 23 (SEQ ID NO:50).

9. PRQ354

Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to the heavy chain of the inter-alpha-trypsin inhibitor (ITI), wherein the polypeptide is designated in the present application as "PR0354" .

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0354 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0354 polypeptide having amino acid residues 1 to 694 of Figure 25 (SEQ ID N0:55), 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 PR0354 polypeptide. In particular, the invention provides isolated native sequence PR0354 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 694 of Figure 25 (SEQ ID NO:55).

10. PRQ355 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0355" .

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0355 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0355 polypeptide having amino acid residues 1 to 440 of Figure 27 (SEQ ID N0:61), 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 PR0355 polypeptide. In particular, the invention provides isolated native sequence PR0355 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 440 of Figure 27 (SEQ ID NO:61). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0355 polypeptide.

11. PRQ357

AppUcants have identified a cDNA clone that encodes a novel polypeptide having homology to insulin-like growth factor (IGF) acid labile subunit (ALS), wherein the polypeptide is designated in the present application as "PR0357" .

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0357 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0357 polypeptide having amino acid residues 1 through 598 of Figure 29 (SEQ ID N0:69), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PR0357 polypeptide. In particular, the invention provides isolated native sequence PR0357 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 through 598 of Figure 29 (SEQ ID NO:69). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0357 polypeptide.

12. PRQ715

AppUcants have identified cDNA clones tiiat encode novel polypeptides having homology to tumor necrosis factor family polypeptides, wherein the polypeptides are designated in the present application as "PR0715" .

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0715 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0715 polypeptide having amino acid residues 1 to 250 of Figure 31 (SEQ ID N0:76), 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 PR0715 polypeptide. In particular, the invention provides isolated native sequence PR0715 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 250 of Figure 31 (SEQ ID N0:76). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0715 polypeptide.

13. PRQ353 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptides are designated in the present application as "PR0353" .

PR0353 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0353 polypeptide having amino acid residues 1 to 281 of Figure 35 (SEQ ID N0:86), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides an isolated PR0353 polypeptide. In particular, the invention provides isolated native sequence PR0353 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 281 of Figure 35 (SEQ ID NO:86).

14. PRQ361

Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0361 ".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0361 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0361 polypeptide having amino acid residues 1 to 431 of Figure 37 (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. The isolated nucleic acid sequence may comprise the cDNA insert of the vector deposited on February 5, 1998 as ATCC 209621 which includes the nucleotide sequence encoding PR0361. In another embodiment, the invention provides isolated PR0361 polypeptide. In particular, the invention provides isolated native sequence PR0361 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 431 of Figure 37 (SEQ ID NO:91). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0361 polypeptide having amino acids 1-379 of the amino acids sequence shown in Figure 37 (SEQ ID N0:91). Optionally, the PR0361 polypeptide is obtained or is obtainable by expressing the polypeptide encoded by the cDNA insert of the vector deposited on February 5, 1998 as ATCC 209621.

15. PRQ365

Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0365" .

PR0365 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PR0365 polypeptide having amino acid residues 1 to 235 of Figure 39 (SEQ ID N0:99), 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 isolated nucleic acid comprises DNA encoding the PR0365 polypeptide having amino acid residues 21 to 235 of Figure 39 (SEQ ID N0:99), 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 PR0365 polypeptide. In particular, the invention provides isolated native sequence PR0365 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 235 of Figure 39 (SEQ ID N0:99). An additional embodiment of the present invention is directed to an amino acid sequence comprising residues 21 to 235 of Figure 39 (SEQ ID N0:99).

16. Additional Embodiments

In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the above or below described polypeptides. A host cell comprising any such vector is also provided. By way of example, the host cells may be CHO cells, E. coli, or 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 from the cell culture.

In other embodiments, the invention provides chimeric molecules comprising 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 immunoglobulin.

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 genomic and cDNA nucleotide sequences, wherein those probes may be derived from any of the above or below described nucleotide sequences. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a nucleotide sequence (SEQ ID NO:l) of a native sequence PR0241 cDNA, wherein SEQ ID NO:l is a clone designated herein as "UNQ215" and/or "DNA34392-1170".

Figure 2 shows the amino acid sequence (SEQ ID NO:2) derived from the coding sequence of SEQ ID NO:l shown in Figure 1. Also presented in Figure 2 are the locations of a putative signal peptide, a potential leucine zipper region and a potential N-glycosylation site.

Figure 3 shows a nucleotide sequence (SEQ ID NO:6) of a native sequence PR0243 cDNA, wherein SEQ ID NO:6 is a clone designated herein as "UNQ217" and/or "DNA35917-1207" .

Figure 4 shows the amino acid sequence (SEQ ID NO:7) derived from the coding sequence of SEQ ID NO:6 shown in Figure 3. Figure 5 shows the organization of the genomic clones in the THPO region of human chromosome 3q27-q28.

Figures 6A-B show the expression of PR0243 in human adult and fetal tissues. Fig. 6A is a northern blot of human adult and fetal tissues hybridized to a human chordin cDNA (PR0243) probe. The lower panel shows an actin control. Fig. 6B is a diagram of the human chordin (PR0243) cDNA with the positions of the codons encoding the conserved cysteine blocks shown. The extent of the probe used is showed by the solid line. Figure 7 shows PR0243 in situ hybridization of adult human tissues giving a positive signal in the cleavage line of the developing synovial joint forming between the femoral head and acetabulum.

Figure 8 shows a nucleotide sequence (SEQ ID NO: 14) of a native sequence PR0299 cDNA, wherein SEQ ID NO: 14 is a clone designated herein as "UNQ262" and/or "DNA39976-1215" .

Figure 9 shows the amino acid sequence (SEQ ID NO: 15) derived from the coding sequence of SEQ ID NO: 14 shown in Figure 8.

Figure 10 shows a nucleotide sequence designated herein as DNA28847 (SEQ ID NO:18). Figure 11 shows a nucleotide sequence designated herein as DNA35877 (SEQ ID NO: 19). Figure 12 shows a nucleotide sequence (SEQ ID NO:23) of a native sequence PR0323 cDNA, wherein SEQ ID NO:23 is a clone designated herein as "UNQ284" and/or "DNA35595-1228". Figure 13 shows the amino acid sequence (SEQ ID NO:24) derived from the coding sequence of SEQ ID

NO:23 shown in Figure 12.

Figure 14 shows a single-stranded nucleotide sequence (SEQ ID NO:29) containing the nucleotide sequence

(nucleotides 79-1416) of a chimeric fusion protein between a PR0323-derived polypeptide and a portion of an IgG constant domain, wherein the chimeric fusion protein is designated herein as "PR0454" . The single-stranded nucleotide sequence (SEQ ID NO:29) encoding the PR0323/IgG fusion protein (PR0454) is designated herein as

"DNA35872".

Figure 15 shows the amino acid sequence (SEQ ID NO:30) derived from nucleotides 79-1416 of the nucleotide sequence shown in Figure 14. The junction in the PR0454 amino acid sequence between the PR0323- derived sequences and the IgG-derived sequences appears between amino acids 415-416 in the figure. Figure 16 shows a nucleotide sequence (SEQ ID N0:31) of a native sequence PR0327 cDNA, wherein SEQ

ID NO:31 is a clone designated herein as "UNQ327" and/or "DNA38113-1230" .

Figure 17 shows the amino acid sequence (SEQ ID N0:32) derived from the coding sequence of SEQ ID NO:31 shown in Figure 16. Figure 18 shows a nucleotide sequence (SEQ ID NO:36) of a native sequence PR0233 cDNA, wherein SEQ ID N0:36 is a clone designated herein as "UNQ207" and/or "DNA34436-1238" .

Figure 19 shows the amino acid sequence (SEQ ID N0:37) derived from the coding sequence of SEQ ID N0:36 shown in Figure 18.

Figure 20 shows a nucleotide sequence (SEQ ID N0:41) of a native sequence PR0344 cDNA, wherein SEQ ID N0:41 is a clone designated herein as "UNQ303 " and/or "DNA40592-1242" .

Figure 21 shows the amino acid sequence (SEQ ID NO:42) derived from the coding sequence of SEQ ID NO:41 shown in Figure 20.

Figure 22 shows a nucleotide sequence (SEQ ID NO:49) of a native sequence PR0347 cDNA, wherein SEQ ID N0:49 is a clone designated herein as "UNQ306" and/or "DNA44176-1244" . Figure 23 shows the amino acid sequence (SEQ ID NO:50) derived from the coding sequence of SEQ ID

N0:49 shown in Figure 22.

Figure 24 shows a nucleotide sequence (SEQ ID NO:54) of a native sequence PR0354 cDNA, wherein SEQ ID N0:54 is a clone designated herein as "UNQ311 " and/or "DNA44192-1246" .

Figure 25 shows the amino acid sequence (SEQ ID N0:55) derived from the coding sequence of SEQ ID NO: 54 shown in Figure 24.

Figure 26 shows a nucleotide sequence (SEQ ID NO:60) of a native sequence PR0355 cDNA, wherein SEQ ID NO:60 is a clone designated herein as "UNQ312" and/or "DNA39518-1247".

Figure 27 shows the amino acid sequence (SEQ ID N0:61) derived from the coding sequence of SEQ ID NO:60 shown in Figure 26. Figure 28 shows a nucleotide sequence (SEQ ID NO:68) of a native sequence PR0357 cDNA, wherein SEQ

ID NO:68 is a clone designated herein as "UNQ314" and/or "DNA44804-1248" .

Figure 29 shows the amino acid sequence (SEQ ID NO:69) derived from the coding sequence of SEQ ID NO: 68 shown in Figure 28.

Figure 30 shows a nucleotide sequence (SEQ ID NO:75) of a native sequence PR0715 cDNA, wherein SEQ ID N0:75 is a clone designated herein as "UNQ383" and/or "DNA52722-1229" .

Figure 31 shows the amino acid sequence (SEQ ID N0:76) derived from the coding sequence of SEQ ID N0:75 shown in Figure 30.

Figure 32 shows a comparison of the amino acid sequences of human tumor necrosis factor-α (TNFA_HUMAN) (SEQ ID NO:77) with the amino acid sequence (SEQ ID N0:76) derived from nucleotides 114- 863 of DNA52722-1229. Identical amino acids are boxed.

Figure 33 shows a comparison of the amino acid sequence (SEQ ID NO:76) derived from nucleotides 114- 863 of DNA52722-1229 with the amino acid sequences of a variety of members of the tumor necrosis family of proteins (SEQ ID NOS:78-84). Identical amino acids are boxed.

Figure 34 shows a nucleotide sequence (SEQ ID NO:85) of a native sequence PR0353 cDNA, wherein SEQ ID NO:85 is a clone designated herein as "UNQ310" and/or "DNA41234- 1242" .

Figure 35 shows the amino acid sequence (SEQ ID NO: 86) derived from the coding sequence of SEQ ID NO:85 shown in Figure 34.

Figure 36 shows a nucleotide sequence (SEQ ID NO:90) of a native sequence PR0361 cDNA, wherein SEQ ID NO:90 is a clone designated herein as "UNQ316" and/or "DNA45410-1250". Figure 37 shows the amino acid sequence (SEQ ID NO:91) derived from the coding sequence of SEQ ID NO:90 shown in Figure 36.

Figure 38 shows a nucleotide sequence (SEQ ID NO:98) of a native sequence PR0365 cDNA, wherein SEQ ID N0:98 is a clone designated herein as "UNQ320" and/or "DNA46777-1253".

Figure 39 shows the amino acid sequence (SEQ ID NO:99) derived from the coding sequence of SEQ ID NO:98 shown in Figure 38.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (i.e., PRO/number) refers to specific polypeptide sequences as described herein. The terms "PRO/number polypeptide" and "PRO/number" as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein). The PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence PRO polypeptide" specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e.g. , an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally- occmring alleUc variants of the polypeptide. In various embodiments of the invention, the native sequence PR0241 polypeptide is a mature or fuU-length native sequence PR0241 polypeptide comprising amino acids 1 to 379 of Figure 2 (SEQ ID NO:2), the native sequence PR0243 is a mature or full-length native sequence polypeptide comprising amino acids 24 to 954 of Fig. 4 (SEQ ID NO:7), with or without the N-terminal signal sequence (residues 1 to about 23), and witii or without die initiating methionine at position 1, the native sequence PR0299 polypeptide is a mature or full-length native sequence PR0299 polypeptide comprising amino acids 1 to 737 of Figure 9 (SEQ ID NO: 15) or the native sequence PR0299 polypeptide is an extracellular domain of the full-length PR0299 protein, wherein the putative transmembrane domain of the full-length PR0299 protein is encoded by nucleotides beginning at nucleotide 2022 as shown in Figure 8, the native sequence PR0323 polypeptide is a mature or full-length native sequence PR0323 polypeptide comprising amino acids 1 to 433 of Figure 13 (SEQ ID N0:24), die native sequence PR0327 polypeptide is a mature or full-length native sequence PR0327 polypeptide comprising amino acids 1 to 422 of Figure 17 (SEQ ID N0:32), the native sequence PR0233 polypeptide is a mature or full-length native sequence PR0233 polypeptide comprising amino acids 1 to 300 of Figure 19 (SEQ ID N0:37), the native sequence PR0344 polypeptide is a mature or fuU-length native sequence PR0344 polypeptide comprising amino acids 1 to 243 of Figure 21 (SEQ ID N0:42), the native sequence PR0347 polypeptide is a mature or full-length native sequence PR0347 polypeptide comprising amino acids 1 to 455 of Figure 23 (SEQ ID NO:50), the native sequence PR0354 polypeptide is a mature or full-length native sequence PR0354 polypeptide comprising amino acids 1 to 694 of Figure 25 (SEQ ID N0:55), the native sequence PR0355 polypeptide is a mature or full-length native sequence PR0355 polypeptide comprising amino acids 1 to 440 of Figure 27 (SEQ ID NO:61) or die native sequence PR0355 polypeptide is an extracellular domain of the fuU-lengdi PR0355 protein, wherein the putative transmembrane domain of the full-length PR0355 protein is encoded by nucleotides beginning at nucleotide 1138 as shown in Figure 26, tiie native sequence

PR0357 polypeptide is a mature or full-length native sequence PR0357 polypeptide comprising amino acids 1 through 598 of Figure 29 (SEQ ID NO:69) or the native sequence PR0357 polypeptide is an extracellular domain of die full-length PR0357 protein, wherein the putative transmembrane domain of the full-length PR0357 protein is encoded by nucleotides 1518-1572 of SEQ ID N0:68, or alternatively, 1491-1572 of SEQ ID N0:68, the native sequence PR0715 polypeptide is a mature or full-length native sequence PR0715 polypeptide comprising amino acids

1 to 250 of Figure 31 (SEQ ID NO:76), the native sequence PR0353 polypeptide is a mature or full-length native sequence PR0353 polypeptide comprising amino acids 1 to 281 of Figure 35 (SEQ ID NO:86) or the native sequence

PR0353 polypeptide is an extracellular domain of the full-length PR0353 protein, the native sequence PR0361 polypeptide is a mature or full-length native sequence PR0361 polypeptide comprising amino acids 1 to 431 of Figure 37 (SEQ ID NO:91) or the native sequence PR0361 polypeptide is an extracellular domain of the full-length PR0361 protein, wherein the putative transmembrane domain of the full-length PR0361 protein is encoded by nucleotides beginning at nucleotide 1363 as shown in Figure 36 and the native sequence PR0365 polypeptide is a mature or full-length native sequence PR0365 polypeptide comprising amino acids 1 to 235 of Figure 39 (SEQ ID NO:99).

The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have less than 1 % of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified.

"PRO polypeptide variant" means an active PRO polypeptide as defined above or below having at least about

80% amino acid sequence identity with the full-length native sequence PRO polypeptide sequence as disclosed herein.

Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, and even more preferably at least about 90% amino acid sequence identity, yet more preferably at least about 95% amino acid sequence identity and most preferably at least about 99% amino acid sequence identity with the amino acid sequence of the full-length native amino acid sequence as disclosed herein.

With regard to PR0243 variants, the phrase "PR0243 variant" means an active PR0243 as defined below having at least about 80% amino acid sequence identity to (a) a DNA molecule encoding a PR0243 polypeptide, with or widiout its native signal sequence, or (b) the complement of the DNA molecule of (a). In a particular embodiment, the PR0243 variant has at least about 80% amino acid sequence homology with the PR0243 having the deduced amino acid sequence shown in Fig. 4 (SEQ ID NO:7) for a full-length native sequence PR0243. Such PR0243 variants include, for instance, PR0243 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the sequence of Fig. 4 (SEQ ID NO:7). Preferably, the nucleic acid or amino acid sequence identity is at least about 85%, more preferably at least about 90% , and even more preferably at least about

95% .

"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific PRO polypeptide sequence, after aligning die 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 such as BLAST, BLAST-2, 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 a candidate sequence that are identical with the nucleotides in the PRO nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. "Isolated," when used to describe die 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 outer proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non- reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant ceUs, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step. An "isolated" PRO polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PRO polypeptide nucleic acid. An isolated PRO polypeptide nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated PRO polypeptide nucleic acid molecules therefore are distinguished from the specific PRO polypeptide nucleic acid molecule as it exists in namral cells. However, an isolated PRO polypeptide nucleic acid molecule includes PRO polypeptide nucleic acid molecules contained in cells that ordinarily express the PRO polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of namral cells.

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

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects die transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The term "antibody" is used in the broadest sense and specifically covers single anti-PRO polypeptide monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) and anti-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, i.e. , the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. "Active" or "activity" for the purposes herein refers to form(s) of PRO polypeptide which retain the biologic and/or immunologic activities of the specific native or naturally-occurring PRO polypeptide. As per PR0243, a preferred activity is the ability to bind to and affect, e.g. , block or otherwise modulate, an activity of chordin, wherein the activity preferably involves the regulation of notochord and muscle formation.

"Treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those akeady witi 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) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term "agonist" is used to refer to peptide and non-peptide analogs of the native PRO polypeptides (where native PRO polypeptide refers to pro-PRO polypeptide, pre-PRO polypeptide, prepro-PRO polypeptide, or mature PRO polypeptide) of the present invention and to antibodies specifically binding such native PRO polypeptides, provided that they retain at least one biological activity of a native PRO polypeptide. Preferably, the agonists of die present invention retain the quaUtative binding recognition properties and receptor activation properties of die native PRO polypeptide. The term "antagonist" is used to refer to a molecule inhibiting a biological activity of a native PRO polypeptide of the present invention wherein native PRO polypeptide refers to pro-PRO polypeptide, pre-PRO polypeptide, prepro-PRO polypeptide, or mature PRO polypeptide. Preferably, the antagonists herein inhibit the binding of a native PRO polypeptide of the present invention to a binding partner. A PRO polypeptide "antagonist" is a molecule which prevents, or interferes with, a PRO antagonist effector function (e.g. 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 monitoring binding of native PRO polypeptide in the presence and absence of the test antagonist molecule, for example. An antagonist of the invention also encompasses an antisense polynucleotide against the PRO polypeptide gene, which antisense polynucleotide blocks transcription or translation of the PRO polypeptide gene, tiiereby inhibiting its expression and biological activity.

"Stringent conditions" means (1) employing low ionic strength and high temperature for washing, for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C, or (2) employing during hybridization a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42^CC. Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6/8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 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% dextran sulfate, 2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.

"Moderately stringent conditions" are described in Sambrook et al., supra, and include the use of a washing solution and hybridization conditions (e.g., temperature, ionic strength, and %SDS) less stringent than described above. An example of moderately stringent conditions is a condition such as overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 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 die like.

"Southern analysis" or "Southern blotting" is a method by which the presence of DNA sequences in a restriction endonuclease digest of DNA or a DNA-containing composition is confirmed by hybridization to a known, labeled oligonucleotide or DNA fragment. Southern analysis typically involves electrophoretic separation of DNA digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nitrocellulose, nylon, or anotiier suitable membrane support for analysis widi a radiolabeled, biotinylated, or enzyme- labeled probe as described in sections 9.37-9.52 of Sambrook et al. , Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). "Northern analysis" or "Northern blotting" is a method used to identify RNA sequences that hybridize to a known probe such as an oUgonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment. The probe is labeled with a radioisotope such as ³²P, or by biotinylation, or with an enzyme. The RNA to be analyzed is usually electrophoretically 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., supra. II. Compositions and Methods of the Invention

1. Full-length PRQ241 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0241. In particular, Applicants have identified and isolated cDNA encoding a PR0241 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence ahgnment computer programs, Applicants found that portions of the PR0241 polypeptide have significant homology with the various biglycan proteins. Accordingly, it is presently believed that PR0241 polypeptide disclosed in the present application is a newly identified biglycan homolog polypeptide and may possess activity typical of biglycan proteins.

2. Full-length PRQ243 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0243. In particular, Applicants have identified and isolated cDNA encoding a PR0243 polypeptide, as disclosed in further detail in die Examples below. Using BLAST, BLAST-2 and FastA sequence aUgnment computer programs, Applicants found that a full-length native sequence PR0243 (shown in Figure 4 and SEQ ID N0:7) has 50% amino acid sequence identity with African clawed frog and Xenopus chordin and 77% homology with rat chordin. Accordingly, it is presently believed that PR0243 disclosed in the present application is a newly identified member of the chordin protein family and may possess ability to influence notochord and muscle formation by the dorsalization of the mesoderm.

3. Full-length PRQ299

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0299. In particular, Applicants have identified and isolated cDNA encoding a PR0299 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 PR0299 polypeptide have significant homology wiui the notch protein. Accordingly, it is presently believed that PR0299 polypeptide disclosed in die present appUcation is a newly identified member of the notch protein family and possesses signaling properties typical of the notch protein family.

4. Full-length PRQ323 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0323. In particular, Applicants have identified and isolated cDNA encoding a PR0323 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 PR0323 polypeptide have significant homology with various dipeptidase proteins. Accordingly, it is presently believed that PR0323 polypeptide disclosed in the present appUcation is a newly identified dipeptidase homolog that has dipeptidase activity 5. Full-length PRQ327 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0327. In particular, Applicants have identified and isolated cDNA encoding a PR0327 polypeptide, as disclosed in further detail in die Examples below. Using BLAST and FastA sequence aUgnment computer programs, Applicants found that portions of the PR0327 polypeptide have significant homology witii various prolactin receptor proteins. Accordingly, it is presently believed that PR0327 polypeptide disclosed in the present application is a newly identified prolactin receptor homolog and has activity typical of a prolactin receptor protein.

6. Full-length PRQ233 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0233. In particular, Applicants have identified and isolated cDNA encoding a PR0233 polypeptide, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0233 polypeptide have significant homology with various reductase proteins. AppUcants have also found tiiat the DNA encoding the PR0233 polypeptide has significant homology with proteins from Caenorhabditis elegans. Accordingly, it is presently beUeved that PR0233 polypeptide disclosed in die present application is a newly identified member of the reductase family and possesses the ability to effect the redox state of a cell typical of the reductase family.

7. Full-length PRQ344 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0344. In particular, Applicants have identified and isolated cDNA encoding PR0344 polypeptides, as disclosed in further detail in the Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0344 polypeptide have significant homology with the human and mouse complement proteins. Accordingly, it is presently believed that the PR0344 polypeptide disclosed in the present application is a newly identified member of the complement family and possesses tire ability to affect the inflammation process as is typical of the complement family of proteins.

8. Full-length PRQ347 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0347. In particular, Applicants have identified and isolated cDNA encoding a PR0347 polypeptide, as disclosed in further detail in die Examples below. Using BLAST and FastA sequence aUgnment computer programs, Applicants found that portions of the PR0347 polypeptide have sigmficant homology wiui various cysteine-rich secretory proteins. Accordingly, it is presently believed that PR0347 polypeptide disclosed in die present application is a newly identified cysteine-rich secretory protein and may possess activity typical of the cysteine-rich secretory protein family. 9. Full-length PRQ354 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0354. In particular, Applicants have identified and isolated cDNA encoding a PR0354 polypeptide, as disclosed in further detail in die Examples below. Using BLAST and FastA sequence aUgnment computer programs, Applicants found that portions of the PR0354 polypeptide have significant homology with the inter-alpha-trypsin inhibitor heavy chain protein. Accordingly, it is presently believed that PR0354 polypeptide disclosed in the present appUcation is a newly identified inter-alpha-trypsin inhibitor heavy chain homolog.

10. Full-length PRQ355 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0355. In particular, Applicants have identified and isolated cDNA encoding a PR0355 polypeptide, as disclosed in further detail in die Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0355 polypeptide have significant homology with the CRTAM protein. Applicants have also found that the DNA encoding the PR0355 polypeptide also has homology to the thymocyte activation and developmental protein, the H20A receptor, the H20B receptor, the poliovirus receptor and the Cercopithecus aethiops AGM delta 1 protein. Accordingly, it is presently beUeved that PR0355 polypeptide disclosed in the present application is a newly identified member of the CRTAM protein family.

11. Full-length PRQ357 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0357. In particular, Applicants have identified and isolated cDNA encoding a PR0357 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 PR0357 polypeptide have significant homology with the acid labile subunit of insulin-like growth factor. Applicants have also found that non- coding regions of die DNA44804-1248 aUgn with a human gene signature as described in WO 95/14772. Applicants have further found tiiat non-coding regions of the DNA44804-1248 align with the adenovirus type 12/human recombinant viral DNA as described in Deuring and Doerfler, Gene. 26:283-289 (1983). Based on die coding region homology, it is presendy beUeved that PR0357 polypeptide disclosed in the present application is a newly identified member of the leucine rich repeat family of proteins, and particularly, is related to the acid labile subunit of insulinlike growtii factor. As such, PR0357 is likely to be involved in binding mechanisms, and may be part of a complex.

12. Full-length PRQ715 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0715. In particular, Applicants have identified and isolated cDNA molecules encoding PR0715 polypeptides, as disclosed in further detail in die Examples below. Using BLAST and FastA sequence aUgnment computer programs, Applicants found that various portions of the PR0715 polypeptides have sigmficant homology with the various members of die tumor necrosis family of proteins. Accordingly, it is presently believed that the PR0715 polypeptides disclosed in die present application are newly identified members of the tumor necrosis factor family of proteins.

13. Full-length PRQ353 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in die present application as PR0353. In particular, Applicants have identified and isolated cDNA encoding PR0353 polypeptides, as disclosed in further detail in the Examples below. Using BLAST and, FastA sequence alignment computer programs, Applicants found that various portions of the PR0353 polypeptides have significant homology with the human and mouse complement proteins. Accordingly, it is presently believed that the PR0353 polypeptides disclosed in die present application are newly identified members of the complement protein family and possesses the abiUty to effect the inflammation process as is typical of the complement family of proteins.

14. Full-length PRQ361 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0361. In particular, Applicants have identified and isolated cDNA encoding a PR0361 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 PR0361 polypeptide have significant homology with the mucin and chitinase proteins. Accordingly, it is presently believed that PR0361 polypeptide disclosed in the present application is a newly identified member of the mucin and/or chitinase protein families and may be associated witii cancer, plant pathogenesis or receptor functions typical of the mucin and chitinase protein families, respectively.

15. Full-length PRQ365 Polypeptides

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0365. In particular, Applicants have identified and isolated cDNA encoding a PR0365 polypeptide, as disclosed in further detail in die Examples below. Using BLAST and FastA sequence alignment computer programs, Applicants found mat various portions of the PR0365 polypeptide have significant homology witii the human 2-19 protein. Accordingly, it is presently believed that PR0365 polypeptide disclosed in the present application is a newly identified member of the human 2-19 protein family.

16. PRO Polypeptide Variants In addition to die fuU-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 DNA, or by syntiiesis of the desired PRO polypeptide. Those skilled in die art wiU appreciate that amino acid changes may alter post-translational processes of the PRO polypeptides, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics. Variations in the native full-length sequence PRO polypeptides or in various domains of the PRO polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO polypeptide tiiat results in a change in the amino acid sequence of the PRO polypeptide as compared with the native sequence PRO polypeptide. Optionally the variation is by substitution of at least one amino acid witii any other amino acid in one or more of the domains of me 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 comparing the sequence of the PRO polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substimtions of amino acids in die sequence and testing die resulting variants for activity in the in vitro assay described in the Examples below.

In particular embodiments, conservative substitutions of interest 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, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and die products screened.

Table 1

Original Exemplary Preferred Residue Substimtions Substitutions

Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gin; lys; arg arg He (I) leu; val; met; ala; phe; norleucine leu

Leu (L) norleucine; ile; val; met; ala; phe ile

Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

Substantial modifications in function or immunological identity of the PRO polypeptide are accomplished by selecting substimtions that differ significantly in their effect on maintaining (a) e structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues tiiat influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

The variations can be made using methods known in die art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res.. 13:4331 (1986); Zoller et al., Nucl. Acids Res.. 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene. 34:315 (1985)], restriction selection mutagenesis [WeUs et al., Philos. Trans. R. Soc. London SerA. 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the desired PRO polypeptide variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among diis group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins. (W.H. Freeman & Co., N.Y.); Chothia, L Mol. Biol.. 150:1 (1976)]. If alanine substimtion does not yield adequate amounts of variant, an isoteric amino acid can be used.

17. Modifications of PRO Polypeptides

Covalent modifications of PRO polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of the PRO polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO polypeptide. Derivatization witii 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 purifying anti-PRO polypeptide antibodies, and vice-versa. Commonly used crosslinking agents include, e.g. , l,l-bis(diazoacetyl)-2-phenyledιane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N- maleimido-l,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or fhreonyl residues, metirylation of the α-amino groups of lysine, arginine, and histidine side chains [T.E.

Creighton, Proteins: Structure and Molecular Properties. W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of die PRO polypeptides included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in a native sequence PRO polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence PRO polypeptide, and/or alteration of the ratio and/or composition of the sugar residues attached to the glycosylation site(s).

Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO polypeptide (for O-linked glycosylation sites). The PRO polypeptide amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids. Anotiier means of increasing the number of carbohydrate moieties on the PRO polypeptide polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem.. pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophvs.. 259:52 (1987) and by Edge et al., Anal. Biochem.. 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by die use of a variety of endo- and exo- glycosidases as described by Thotakura et al., Meth. Enzvmol.. 138:350 (1987). Another type of covalent modification of PRO polypeptides of the invention comprises linking die PRO polypeptide to one of a variety of nonproteinaceous polymers, e.g. , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

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

Various tag polypeptides and their respective antibodies are well known in die art. Examples include poly- histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.. 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies diereto [Evan et al., Molecular and Cellular Biology. 5:3610-3616 (1985)]; and die Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering. 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnologv. 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science. 255:192-194 (1992)]; an α-tubuUn epitope peptide [Skinner et al., J. Biol. Chem.. 266:15163- 15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA. 82:6393- 6397 (1990)].

18. Preparation of PRO Polypeptides The description below relates primarily to production of PRO polypeptides by culturing cells transformed or transfected with a vector containing the desired PRO polypeptide nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare the PRO polypeptide. For instance, die PRO polypeptide sequence, or portions thereof, may be produced by direct peptide synthesis using solid- phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis. W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated syntiiesis may be accomplished, for instance, using an Applied Biosystems Peptide Syntiiesizer (Foster City, CA) using manufacturer's instructions. Various portions of the desired PRO polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce die full-length PRO polypeptide.

A. Isolation of DNA Encoding PRO Polypeptides

DNA encoding PRO polypeptides may be obtained from a cDNA library prepared from tissue believed to possess the desired PRO polypeptide mRNA and to express it at a detectable level. Accordingly, human PRO polypeptide DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. The PRO polypeptide-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis.

Libraries can be screened witii probes (such as antibodies to die desired PRO polypeptide or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding the desired PRO polypeptide is to use PCR methodology [Sambrook et al., supra: Dieffenbach et al., PCR PrimeπA Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such tiiat it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include die use of radiolabels like ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra. Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as BLAST, ALIGN, 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 necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.

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 nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach. M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

Mediods of transfection are known to die ordinarily skiUed artisan, for example, CaP0₄ and electroporation.

Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generaUy used for prokaryotes or other ceUs that contain substantial cell-wall barriers. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene. 23:315 (1983) and

WO 89/05859 published 29 June 1989. For mammalian cells widiout such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology. 52:456-457 (1978) can be employed. General aspects of mammaUan ceU host system transformations have been described in U.S. Patent No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact.. 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA1. 26:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g. , polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzvmologv. 185:527-537 (1990) and Mansour et al., Nature. 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31 ,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. , Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g. , B. licheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31 ,537); E. coli strain W3110 (ATCC 27,325); and K5 772 (ATCC 53,635). These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1 A2, which has the complete genotype tonA ; E. coli W3110 strain 9E4, which has the complete genotype tortA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompTkan^r; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)I69 degP ompT rbs7 ilvG karf; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an£. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vitro methods of cloning, e.g. , PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature. 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al , Bio/Technologv. 9: 968-975 (1991)) such as, e.g. , K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al, Bacteriol.. 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al. , Bio/Technologv. 8: 135 (1990)), K . thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al. , Basic Microbiol.. 28: 265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al. , Proc. Natl. Acad. Sci. USA. 16.: 5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 pubUshed 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al , Biochem. Biophvs. Res. Commun.. 112: 284-289 [1983]; Tilburn et al , Gene. 26: 205-221 [1983]; Yelton et al , Proc. Natl. Acad. Sci. USA. 8L 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J.. 4: 475-479 [1985]). Methylo tropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs. 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 Sf9, as well as plant ceUs. 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., J. Gen Virol .. 26:59 (1977)); Chinese hamster ovary ceUs/-DHFR (CHO, Uriaub and Chasin, Proc. Natl. Acad. Sci. USA. 22:4216 (1980)); mouse sertoU ceUs (TM4, Mather, Biol. Reprod.. 22:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriate host cell is deemed to be within die skill in the art.

C. Selection and Use of a Replicable Vector

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

The PRO polypeptide of interest may be produced recombinantly not only directly, but also as a fusion polypeptide witii a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at die 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 die 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, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces α-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 mammaUan ceU expression, mammaUan signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders. Both expression and cloning vectors contain a nucleic acid sequence tiiat enables d e 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 repUcation from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker.

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

An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO polypeptide nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is die CHO cell line deficient in DHFR activity, prepared and propagated as described by Uriaub et al., Proc. Natl. Acad. Sci. USA. 22:4216 (1980). A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature. 282:39 (1979); Kingsman et al., Gene. 2:141 (1979); Tschemper et al., Gene. 10:157 (1980)]. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4- 1 [Jones, Genetics. 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operably linked to die PRO polypeptide nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature. 225:615 (1978); Goeddel et al., Nature. 2£i:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res.. 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA. 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding d e desired PRO polypeptide. Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem.. 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzvme Reg.. 2:149 (1968); Holland, Biochemistry. 17:4900 (1978)], such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosρhate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. PRO polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g. , the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding die desired PRO polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into die vector at a position 5' or 3' to the PRO polypeptide coding sequence, but is preferably located at a site 5' from the promoter.

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

StiU other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO polypeptides in recombinant vertebrate cell culture are described in Gething et al., Nature. 223:620-625 (1981); Mantei et al., Nature. 281 :40-46 (1979); EP 117,060; and EP 117,058.

D. Detecting Gene Amplification/Expression

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

Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of ceUs or tissue sections and assay of ceU culture or body fluids, to quantitate directly the expression of gene product. Antibodies usefiil for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to a PRO polypeptide DNA and encoding a specific antibody epitope.

E. Purification of Polypeptide

Forms of PRO polypeptides may be recovered from culture medium or from host cell ly sates. If membrane- bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of 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 purify PRO polypeptides from recombinant ceU proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope- tagged forms of the PRO polypeptide. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzvmologv. 182 (1990); Scopes, Protein Purification: Principles and Practice. Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO polypeptide produced.

19. Uses for PRO Polypeptides

Nucleotide sequences (or their complement) encoding the PRO polypeptides of the present invention have various appUcations in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA. PRO polypeptide-encoding nucleic acid will also be useful for the preparation of PRO polypeptides by the 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 full-length 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 die PRO polypeptide nucleic acid sequences. Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from the nucleotide sequence of any of die DNA molecules disclosed herein or from genomic sequences including promoters, enhancer elements and introns of native sequence PRO polypeptide encoding DNA. By way of example, a screening method will comprise isolating the coding region of the PRO polypeptide gene using the known DNA sequence to syndiesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as ³²P or³⁵ S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the specific PRO polypeptide gene of die present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.

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

The 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 . The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.

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

Nucleic acids which encode a PRO polypeptide or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in me development and screening of therapeutically useful reagents. A transgenic animal (e.g., 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, e.g., an embryonic stage. A transgene 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 die PRO polypeptide in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding die PRO polypeptide. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for PRO polypeptide transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding a PRO polypeptide introduced into die germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding die PRO polypeptide. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated witii its overexpression. In accordance witii this facet of the invention, an animal is treated witii the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential tiierapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO polypeptides can be used to construct a PRO polypeptide "knock out" animal which has a defective or altered gene encoding the PRO polypeptide of interest as a result of homologous recombination between the endogenous gene encoding the PRO polypeptide and altered genomic DNA encoding the PRO polypeptide introduced into an embryonic ceU of the animal. For example, cDNA encoding a PRO polypeptide can be used to clone genomic DNA encoding die 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 e.g., Thomas and

Capecchi, CeU, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with die endogenous DNA are selected [see e.g., Li et al., CeU, 62:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem CeUs: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can tiien be implanted into a suitable pseudopregnant female foster animal and die embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in tiieir 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.

When in vivo administration of a PRO polypeptide is employed, normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 μg/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 Uterature; 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 treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from mat to another organ or tissue.

Where sustained-release administration of a PRO polypeptide is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder requiring administration of the PRO polypeptide, microencapsulation of the PRO polypeptide is contemplated. Microencapsulation of recombinant proteins for sustained release has been successfully performed witii human growm hormone (rhGH), interferon- (rhlFN- ), interleukin-2, and MN rgpl20. Johnson et al , Nat. Med.. 2: 795-799 (1996); Yasuda. Biomed. Ther.. 22: 1221- 1223 (1993); Hora et al , Bio/Technology. 8: 755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems, " in Vaccine Design: The Subunit and Adjuvant Approach. PoweU and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S Pat. No. 5,654,010.

The sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body. Moreover, the degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. For example, for a formulation that can provide a dosing of approximately 80 g/kg/day in mammals with a maximum body weight of 85 kg, die largest dosing would be approximately 6.8 mg of the PRO polypeptide per day. In order to achieve this dosing level, a sustained- release formulation which contains a maximum possible protein loading (15-20% w/w PRO polypeptide) with the lowest possible initial burst (<20%) is necessary. A continuous (zero-order) release of the PRO polypeptide from microparticles for 1-2 weeks is also desirable. In addition, the encapsulated protein to be released should maintain its integrity and stability over the desired release period.

PR0241 polypeptides of the present invention which possess biological activity related to tiiat of the endogenous biglycan protein may be employed both in vivo for therapeutic purposes and in vitro. Those of ordinary skiU in die art will well know how to employ the PR0241 polypeptides of the present invention for such purposes.

Chordin is a candidate gene for a dysmorphia syndrome known as Cornelia de Lange Syndrome (CDL) which is characterized by distinctive facial features (low anterior hairline, synophrys, antenerted nares, maxillary prognathism, long philtram, 'carp' mouth), prenatal and postnatal growth retardation, mental retardation and, often but not always, upper limb abnormalities. There are also rare cases where CDL is present in association with thrombocytopenia. The gene for CDL has been mapped by linkage to 3q26.3 (OMIM #122470). Xchd involvement in early Xenopus patterning and nervous system development makes CHD in intriguing candidate gene. CHD maps to the appropriate region on chromosome 3. It is very close to THPO, and deletions encompassing both THPO and

CHD could result in rare cases of thrombocytopenia and developmental abnormalities. In situ analysis of CD revealed tiiat almost all adult tissues are negative for CHD expression, the only positive signal was observed in the cleavage line of the developing synovial joint forming between the femoral head and acetabulum (hip joint) implicating

CHD in the development and presumably growth of long bones . Such a function, if disrupted, could result in growth retardation.

The human CHD amino acid sequence predicted from the cDNA is 50% identical (and 66% conserved) to Xchd. All 40 cysteines in the 4 cysteine-rich domains are conserved. These cysteine rich domains are similar to those observed in thrombospondin, procollagen and von Willebrand factor. Bornstein, P. FASEB J 6: 3290-3299 (1992); Hunt, L. & Barker, W. Biochem. Biophys. Res. Commun. 144: 876-882 (1987). The human CHD locus (genomic PR0243) comprises 23 exons in 9.6 kb of genomic DNA. The initiating methionine is in exon 1 and die stop codon in exon 23. A CpG island is located at the 5' and of the gene, beginning approximately 100 bp 5' of exon 1 and extends through the first exon and ends witiiin the first intron. The THPO and CHD loci are organized in a head-to-head fashion with approximately 2.2 kb separating their transcription start sites. At the protein level, PR0243 is 51 % identical to Xenopus chordin (Xchd). All forty cysteines in the one amino terminal and tiiree carboxy terminal cysteine-rich clusters are conserved.

PR0243 is a 954 amino acid polypeptide having a signal sequence at residues 1 to about 23. There are 4 cysteine clusters: (1) residues about 51 to about 125; (2) residues about 705 to about 761; (3) residues about 784 to about 849; and (4) residues about 897 to about 931. There are potential leucine zippers at residues about 315 to about 396, and N-glycosylation sites at residues 217, 351 , 365 and 434. PR0299 polypeptides and portions thereof which have homology to the notch protein may be useful for in vivo therapeutic purposes, as well as for various other applications. The identification of novel notch proteins and related molecules may be relevant to a number of human disorders such as those effecting development. Thus, the identification of new notch proteins and notch-like molecules is of special importance in that such proteins may serve as potential tiierapeutics 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 PR0299.

PR0323 polypeptides of the present invention which possess biological activity related to that of one or more endogenous dipeptidase proteins may be employed both in vivo for therapeutic purposes and in vitro. Those of ordinary skill in the art will well know how to employ the PR0323 polypeptides of the present invention for such purposes.

PR0327 polypeptides of the present invention which possess biological activity related to tiiat of the endogenous prolactin receptor protein may be employed both in vivo for therapeutic purposes and in vitro. Those of ordinary skiU in the art will well know how to employ the PR0327 polypeptides of the present invention for such purposes. PR0327 polypeptides which possess die ability to bind to prolactin may function both in vitro and in vivo as prolactin antagonists.

PR0233 polypeptides and portions thereof which have homology to reductase may also be useful for in vivo therapeutic purposes, as well as for various other applications. The identification of novel reductase proteins and related molecules may be relevant to a number of human disorders such as inflammatory disease, organ failure, adierosclerosis, cardiac injury, infertility, birth defects, premature aging, AIDS, cancer, diabetic complications and mutations in general. Given that oxygen free radicals and antioxidants appear to play important roles in a number of disease processes, the identification of new reductase proteins and reductase-like molecules is of special importance in tiiat 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. PR0344 polypeptides and portions thereof which have homology to complement proteins may also be useful for in vivo therapeutic purposes, as well as for various other applications. The identification of novel complement proteins and related molecules may be relevant to a number of human disorders such as effecting the inflammatory response of cells of the immune system. Thus, die identification of new complement proteins and complement-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 PR0344.

PR0347 polypeptides of the present invention which possess biological activity related to that of cysteine- rich secretory proteins may be employed both in vivo for therapeutic purposes and in vitro. Those of ordinary skill in the art will well know how to employ the PR0347 polypeptides of the present invention for such purposes.

PR0354 polypeptides of the present invention which possess biological activity related to that of the heavy chain of die inter-alpha-trypsin inhibitor protein may be employed both in vivo for therapeutic purposes and in vitro. Those of ordinary skill in the art will well know how to employ the PR0354 polypeptides of the present invention for such purposes. PR0355 polypeptides and portions thereof which have homology to CRTAM may also be useful for in vivo therapeutic purposes, as well as for various other applications. The identification of novel molecules associated with T cells may be relevant to a number of human disorders such as conditions involving the immune system in general. Given that the CRTAM protein binds antibodies which play important roles in a number of disease processes, the identification of new CRTAM proteins and CRTAM -like molecules is of special importance in tiiat 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 PR0355.

PR0357 can be used in competitive binding assays witii ALS to determine its activity with respect to ALS. Moreover, PR0357 can be used in assays to determine if it prolongs polypeptides which it may complex with to have longer half-lives in vivo. PR0357 can be used similarly in assays with carboxypeptidase, to which it also has homology. The results can be applied accordingly.

PR0715 polypeptides of die present invention which possess biological activity related to that of the tumor necrosis factor family of proteins may be employed botii in vivo for therapeutic purposes and in vitro. Those of ordinary skill in die art will well know how to employ the PR0715 polypeptides of the present invention for such purposes. PR0715 polypeptides wUl be expected to bind to tiieir specific receptors, thereby activating such receptors. Variants of the PR0715 polypeptides of the present invention may function as agonists or antagonists of their specific receptor activity.

PR0353 polypeptides and portions thereof which have homology to the complement protein may also be useful for in vivo therapeutic purposes, as well as for various other applications. The identification of novel complement proteins and related molecules may be relevant to a number of human disorders such as effecting the inflammatory response of cells of the immune system. Thus, the identification of new complement proteins complement-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 PR0353.

PR0361 polypeptides and portions thereof which have homology to mucin and/or chitinase proteins may also be useful for in vivo therapeutic purposes, as well as for various other applications. The identification of novel mucin and/or chitinase proteins and related molecules may be relevant to a number of human disorders such as cancer or those involving cell surface molecules or receptors. Thus, die identification of new mucin and/or chitinase proteins 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 appUcations. As a result, there is particular scientific and medical interest in new molecules, such as PR0361.

PR0365 polypeptides and portions thereof which have homology to the human 2-19 protein may also be useftil for in vivo therapeutic purposes, as well as for various other applications. The identification of novel human 2-19 proteins and related molecules may be relevant to a number of human disorders such as modulating the binding or activity of cells of the immune system. Thus, the identification of new human 2-19 proteins and human 2-19 protein-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 PR0365. 20. Anti-PRO Polypeptide Antibodies

The present invention further provides anti-PRO polypeptide antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

A. Polyclonal Antibodies The anti-PRO polypeptide antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant wtil be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the PRO polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole Umpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, syntiietic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in die art without undue experimentation.

B. Monoclonal Antibodies

The anti-PRO polypeptide antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma metiiods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizmg agent to eUcit lymphocytes that produce or are capable of producing antibodies that will specifically bind to die immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the PRO polypeptide of interest or a fusion protein thereof. GeneraUy, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused witii an immortalized ceU line using a suitable fusing agent, such as polyetiiylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usuaUy transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma ceU lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and tiiymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol , 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, (1987) pp. 51-63]. The culture medium in which die hybridoma cells are cultured can tiien be assayed for the presence of monoclonal antibodies directed against the PRO polypeptide of interest. Preferably, the binding specificity of monoclonal antibodies produced by die hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in die art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. , 102:220 (1980).

After the desired hybridoma ceUs are identified, die clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in

U.S. Patent No. 4,816,567. DNA encoding die monoclonal antibodies of d e invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specificaUy to genes encoding die heavy and Ught chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host ceUs. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Patent No. 4,816,567;

Morrison et al., supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobuUn polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen- combining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Meuiods for preparing monovalent antibodies are well known in die art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in die Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with anotiier amino acid residue or are deleted so as to prevent crosslinking.

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

C. Humanized Antibodies The anti-PRO polypeptide antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neitiier in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to tiiose of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typicaUy that of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al.,

Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol , 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.

Humanization can be essentiaUy performed foUowing the method of Winter and co-workers [Jones et al., Nature, 321:

522-525 (1986); Riechmann et al, Nature, 332:323-327 (1988); Verhoeyen et al. Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Human antibodies can also be produced using various techniques known in die art, including phage display

Ubraries [Hoogenboom and Winter, /. Mol. Biol, 222:381 (1991); Marks et al, J. Mol. Biol , 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies

(Cole et al., Monoclonal Antibodies and Cancer Ηierapy, Alan R. Liss, p. 77 (1985) and Boerner et al, J. Immunol ,

147(11:86-95 (1991)].

D. Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.

Mediods for making bispecific antibodies are known in the art. Traditionally, die recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific stracmre. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, EMBO J. , 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have die first heavy- chain constant region (CHI) containing die site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

E. Heteroconjugate 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 immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and metiιyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

21. Uses for Anti-PRO Polypeptide Antibodies The anti-PRO polypeptide antibodies of the invention have various utilities. For example, anti-PRO polypeptide antibodies may be used in diagnostic assays for a PRO polypeptide, e.g. , detecting its expression in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques. 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, eidier directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as ³H, ^l4C, ³²P, ³⁵ S, or¹²⁵ I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in d e art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al. Nature, 144:945 (1962); David et al . Biochemistry, 13:1014 (1974); Pain et al, J. Immunol. Meth. , 40:219 (1981); and Nygren, J. Histochem. and Cytochem. , 30:407 (1982).

Anti-PRO polypeptide antibodies also are useful for the affinity purification of PRO polypeptide from recombinant cell culture or natural sources. In this process, the antibodies against the PRO polypeptide are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in die art. The immobilized antibody then is contacted witii a sample containing the PRO polypeptide to be purified, and thereafter the support is washed witii a suitable solvent that will remove substantially all the material in the sample except the PRO polypeptide, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the PRO polypeptide from the antibody.

Chordin (CHD) is a candidate gene for a dysmorphia syndrome known as Cornelia de Lange Syndrome (CDL) which is characterized by distinctive facial features (low anterior hairline, synophrys, antenerted nares, maxiUary prognathism, long philtram, 'carp' mouth), prenatal and postnatal growth retardation, mental retardation and, often but not always, upper limb abnormalities. There are also rare cases where CDL is present in association with thrombocytopenia. The gene for CDL has been mapped by linkage to 3q26.3 (OMIM #122470). Xchd (Xenopus chordin) involvement in early Xenopus patterning and nervous system development makes CHD in intriguing candidate gene. CHD maps to the appropriate region on chromosome 3. It is very close to THPO, and deletions encompassing both THPO and CHD could result in rare cases of thrombocytopenia and developmental abnormaUties. In situ analysis of CD revealed that almost all adult tissues are negative for CHD expression, the only positive signal was observed in the cleavage line of the developing synovial joint forming between d e femoral head and acetabulum (hip joint) implicating CHD in the development and presumably growth of long bones. Such a function, if disrupted, could result in growth retardation. The human CHD amino acid sequence predicted from the cDNA is 50% identical (and 66% conserved) to

Xchd. All 40 cysteines in the 4 cysteine-rich domains are conserved. These cysteine rich domains are similar to those observed in thrombospondin, procollagen and von Willebrand factor. Bornstein, P. FASEB J 6: 3290-3299 (1992); Hunt, L. & Barker, W. Biochem. Biophys. Res. Commun. 144: 876-882 (1987).

Antibodies to PR0243 chordin can be made which bind die polypeptide in conditions characterized by overexpression of PR0243.

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

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

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

EXAMPLE 1 : Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNA Encoding Therefor

The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot pubUc database were used to search EST databases. The EST databases included public databases (e.g., Dayhoff, GenBank), and proprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altschul and Gish, Methods in Enzymologv 266: 460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons with a Blast score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, WA;

(http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).

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

Based upon the consensus sequences obtained as described above, oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide. Forward (.f) and reverse (.r) PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe (.p) sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are syndiesized when the consensus sequence is greater than about l-1.5kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs. The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commerciaUy available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Not! site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science. 253: 1278-1280 (1991)) in the unique Xhol and Notl sites.

EXAMPLE 2: Isolation of cDNA clones bv Amylase Screening

1. Preparation of oligo dT primed cDNA library mRNA was isolated from a human tissue of interest using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2). This RNA was used to generate an oligo dT primed cDNA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System). In this procedure, d e double stranded cDNA was sized to greater than 1000 bp and the Sall/Notl tinkered cDNA was cloned into Xhol/Notl cleaved vector. pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restriction enzyme site preceding die Xhol/Notl cDNA cloning sites.

2. Preparation of random primed cDNA library

A secondary cDNA library was generated in order to preferentially represent the 5' ends of the primary cDNA clones. Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY.O using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above). In this procedure the double stranded cDNA was sized to 500-1000 bp, tinkered with blunt to Notl adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector. pSST-AMY.O is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding die cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites. Thus, cDNAs cloned into this vector that are fused in frame with amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies.

3. Transformation and Detection

DNA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was then electroporated as recommended by die manufacturer. Subsequendy, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37 °C for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates containing ampicilUn and incubated for 16 hours (37 °C). Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet using standard protocols, e.g. CsCl-gradient. The purified DNA was then carried on to die yeast protocols below. The yeast metiiods were divided into three categories: (1) Transformation of yeast with the plasmid/cDNA combined vector; (2) Detection and isolation of yeast clones secreting amylase; and (3) PCR amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-ll, his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employed that have deficient post-translational pathways. Such mutants may have translocation deficient alleles in secll, secll, secβl, witii truncated secll being most preferred. Alternatively, antagonists (including antisense nucleotides and/or ligands) which interfere with the normal operation of these genes, other proteins implicated in this post translation pathway (e.g., SECόlp, SEC72p, SEC62p, SEC63p, TDJlp or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with d e amylase-expressing yeast. Transformation was performed based on d e protocol outlined by Gietz et al., Nucl. Acid. Res.. 2Q:1425

(1992). Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and grown overnight at 30°C. The YEPD broth was prepared as described in Kaiser et al., Methods in Yeast Genetics. Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). The overnight culture was then diluted to about 2 x 10⁶ cells/ml (approx. 00^=0.1) into fresh YEPD broth (500 ml) and regrown to 1 x 1⁷0 cells/ml (approx. 00^=0.4-0.5).

The cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The supernatant was discarded and die cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

Transformation took place by mixing die prepared cells (100 μl) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 μg, vol. < 10 μl) in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes. The cells were then heat shocked at 42°C for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) Mowed by recentrifugation. The cells were then diluted into TE (1 ml) and aliquots (200 μl) were spread onto the selective media previously prepared in 150 mm growth plates (VWR).

Alternatively, instead of multiple small reactions, the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al., Metiiods in Yeast Genetics. Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 208- 210 (1994). Transformants were grown at 30°C for 2-3 days. The detection of colonies secreting amylase was performed by including red starch in the selective growth media. Starch was coupled to die red dye (Reactive Red-120, Sigma) as per the procedure described by Biely et al., Anal. Biochem.. 122:176-179 (1988). The coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0 (50-100 mM final concentration).

The positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies. Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar. Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly.

4. Isolation of DNA bv PCR Amplification

When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile water (30 μl) in a 96 well plate. At this time, the positive colonies were either frozen and stored for subsequent analysis or immediately amplified. An aliquot of cells (5 μl) was used as a template for the PCR reaction in a 25 μl volume containing: 0.5 μl Klentaq (Clontech, Palo Alto, CA); 4.0 μl 10 mM dNTP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μl forward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. The sequence of the forward oligonucleotide 1 was:

5 '-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3 ' (SEQ ID NO: 16) The sequence of reverse oligonucleotide 2 was:

5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3 ' (SEQ ID NO: 17)

PCR was then performed as follows: a. Denature 92°C, 5 minutes b. 3 cycles of: Denature 92°C, 30 seconds

Anneal 59° C, 30 seconds

Extend 72°C, 60 seconds c. 3 cycles of: Denature 92°C, 30 seconds

Anneal 57 °C, 30 seconds

Extend 72°C, 60 seconds

25 cycles of: Denature 92°C, 30 seconds

Anneal 55 °C, 30 seconds

Extend 72°C, 60 seconds e. Hold 4°C

The underlined regions of the oligonucleotides annealed to die ADH promoter region and the amylase region, respectively, and ampUfied a 307 bp region from vector pSST-AMY.O when no insert was present. Typically, the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers. Thus, the total product of the PCR reaction from an empty vector was 343 bp. However, signal sequence-fused cDNA resulted in considerably longer nucleotide sequences.

FoUowing die PCR, an aliquot of the reaction (5 μl) was examined by agarose gel electrophoresis in a 1 % agarose gel using a Tris-Borate-EDTA (TBE) buffering system as described by Sambrook et al., supra. Clones resulting in a single strong PCR product larger than 400 bp were further analyzed by DNA sequencing after purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc., Chats worth, CA).

EXAMPLE 3: Isolation of cDNA Clones Encoding Human PRQ241

A consensus DNA sequence was assembled relative to other EST sequences as described in Example 1 above. This consensus sequence is herein designated DNA30876. Based on die DNA30876 consensus sequence, oUgonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained die sequence of interest, and

2) for use as probes to isolate a clone of the full-length coding sequence for PR0241. PCR primers (forward and reverse) were synthesized: forward PCR primer 5'-GGAAATGAGTGCAAACCCTC-3' (SEQ ID NO:3) reverse PCR primer 5'-TCCCAAGCTGAACACTCATTCTGC-3' (SEQ ID NO:4) Additionally, a synthetic oligonucleotide hybridization probe was constracted from the consensus DNA30876 sequence which had the following nucleotide sequence hybridization probe

5'-GGGTGACGGTGTTCCATATCAGAATTGCAGAAGCAAAACTGACCTCAGTT-3' (SEQ ID NO:5)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication with the PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0241 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB29).

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0241

[herein designated as UNQ215 (DNA34392-1170)] (SEQ ID NO:l) and the derived protein sequence for PR0241. The entire nucleotide sequence of UNQ215 (DNA34392-1170) is shown in Figure 1 (SEQ ID NO:l). Clone

UNQ215 (DNA34392-1170) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 234-236 and ending at the stop codon at nucleotide positions 1371-1373 (Figure 1). The predicted polypeptide precursor is 379 amino acids long (Figure 2). The full-length PR0241 protein shown in Figure

2 has an estimated molecular weight of about 43,302 daltons and a pi of about 7.30. Clone UNQ215 (DNA34392- 1170) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209526.

Analysis of the amino acid sequence of the full-length PR0241 polypeptide suggests that it possess significant homology to the various biglycan proteoglycan proteins, thereby indicating that PR0241 is a novel biglycan homolog polypeptide.

EXAMPLE 4: Isolation of cDNA Clones Encoding Human PRQ243 by Genomic Walking

Introduction: Human dirombopoietin (THPO) is a glycosylated hormone of 352 amino acids consisting of two domains. The N-terminal domain, sharing 50% simUarity to erythropoietin, is responsible for the biological activity. The C-terminal region is required for secretion. The gene for thrombopoietin (THPO) maps to human chromosome 3q27-q28 where die six exons of this gene span 7 kdobase base pairs of genomic DNA (Gurney et al, Blood 85: 981- 988 (1995). In order to determine whether there were any genes encoding THPO homologues located in close proximity to THPO, genomic DNA fragments from this region were identified and sequenced. Three PI clones and one PAC clones (Genome Systems Inc., St. Louis, MO; cat. Nos. Pl-2535 and PAC-6539) encompassing the THPO locus were isolated and a 140 kb region was sequenced using the ordered shotgun strategy (Chen et al, Genomics 12: 651-656 (1993)), coupled with a PCR-based gap filling approach. Analysis reveals that the region is gene-rich with four additional genes located very close to THPO: tumor necrosis factor-receptor type 1 associated protein 2 (TRAP2) and elongation initiation factor gamma (elF4g), chloride channel 2 (CLCN2) and RNA polymerase II subunit hRPB17. While no THPO homolog was found in die region, four novel genes have been predicted by computer-assisted gene detection (GRAIL)(Xu et al, Gen. Engin. 16: 241-253 (1994), the presence of CpG islands (Cross, S. and Bird, A., Curr. Opin. Genet. & Devel. 5: 109-314 (1995), and homology to known genes (as detected by WU-BLAST2.0)(Altschul and Gish, Methods Enzymol. 266: 460-480 (1996) (http : //blast, wustl . edu/blast/README.html) .

PI and PAC clones: The initial human PI clone was isolated from a genomic PI tibrary (Genome Systems Inc.,

St. Louis, MO; cat. no.: Pl-2535) screened with PCR primers designed from the THPO genomic sequence (A.L. Gurney, et al. Blood 85: 981-88 (1995). PCR primers were designed from the end sequences derived from this PI clone were then used to screen PI and PAC Ubraries (Genome Systems, Cat. Nos.: Pl-2535 & PAC-6539) to identify overlapping clones.

Ordered Shotgun Strategy: The Ordered Shotgun Strategy (OSS) (Chen et al, Genomics 12: 651-656 (1993)) involves the mapping and sequencing of large genomic DNA clones witii a hierarchical approach. The PI or PAC clone was sonicated and die fragments subcloned into lambda vector (λBluestar) (Novagen, Inc., Madison, WI; cat. no. 69242-3). The lambda subclone inserts were isolated by long-range PCR (Barnes, W. Proc. Natl. Acad. Sci. USA 21: 2216-2220 (1994) and the ends sequenced. The lambda-end sequences were overlapped to create a partial map of the original clone. Those lambda clones witii overlapping end-sequences were identified, the insets subcloned into a plasmid vector (pUC9 or pUC18) and the ends of the plasmid subclones were sequenced and assembled to generate a contiguous sequence. This directed sequencing strategy minimizes the redundancy required while allowing one to scan for and concentrate on interesting regions.

In order to define better the THPO locus and to search for other genes related to the hematopoietin family, four genomic clones were isolated from this region by PCR screening of human PI and PAC libraries (Genome System, Inc., Cat. Nos.: Pl-2535 and PAC-6539). The sizes of the genomic fragments are as follows: Pl.t is 40 kb; Pl .g is 70 kb; Pl .u is 70 kb; and PAC.z is 200 kb. The relationships between these four genomic clones are illustrated in Figure 5. Approximately 80% of the 200 kb genomic DNA region was sequenced by the Ordered Shotgun Strategy (OSS) (Chen et al, Genomics 17: 651-56 (1993), and assembled into contigs using AutoAssembler™ (Applied Biosystems, Perkin Elmer, Foster City, CA, cat. no. 903227). The preliminary order of these contigs was determined by manual analysis. There were 46 contigs and filling in the gaps was employed. Table 2 summarized die number and sizes of the gaps. Table 2 Summary of the gaps in the 140 kb region

Size of gap number

<50 bp 13

50-150 bp 7 150-300 bp 7

300-1000 bp 10

1000-5000 bp 7

> 5000 bp 2 ( 15,000 bp)

DNA sequencing: ABI DYE-primer™ chemistry (PE Applied Biosystems, Foster City, CA; Cat. No.: 402112) was used to end-sequence d e lambda and plasmid subclones. ABI DYE-terminater™ chemistry (PE Applied Biosystems, Foster City, CA, Cat. No: 403044) was used to sequence the PCR products with their respective PCR primers. The sequences were collected witii an ABI377 instrument. For PCR products larger than lkb, walking primers were used. The sequences of contigs generated by die OSS strategy in Auto Assembler™ a (PE Applied Biosystems, Foster City, CA; Cat. No: 903227) and the gap-filling sequencing trace files were imported into Sequencher™ (Gene Codes Corp., Ann Arbor, MI) for overlapping and editing.

PCR-Based gap filling Strategy: Primers were designed based on die 5'- and 3 '-end sequenced of each contig, avoiding repetitive and low quatity sequence regions. All primers were designed to be 19-24-mers with 50-70% G/C content. Oligos were synthesized and gel-purified by standard methods.

Since die orientation and order of the contigs were unknown, permutations of the primers were used in the amplification reactions. Two PCR kits were used: first, XL PCR kit (Perkin Elmer, Norwalk, CT; Cat. No.: N8080205), with extension times of approximately 10 minutes; and second, die Taq polymerase PCR kit (Qiagen Inc., Valencia, CA; Cat. No.: 201223) was used under high stringency conditions if smeared or multiple products were observed with the XL PCR kit. The main PCR product from each successful reactions was extracted from a 0.9% low melting agarose gel and purified with the Geneclean DNA Purification kit prior to sequencing.

Analysis: The identification and characterization of coding regions was carried out as follows: First, repetitive sequences were masked using RepeatMasker (A.F.A. Smit & P. Green, http://ftp.genome.washington.edu/RM/RM_details.html) which screens DNA sequences in FastA format against a Ubrary of repetitive elements and returns a masked query sequence. Repeats not masked were identified by comparing the sequence to the GenBank database using WUBLAST (Altschul, S & Gish, W., Methods Enzymol. 266: 460-480 (1996) and were masked manually.

Next, known genes were revealed by comparing the genomic regions against Genentech's protein database using d e WUBLAST2.0 algorithm and then annotated by aligning die genomic and cDNA sequences for each gene, respectively, using a Needleman-Wunch (Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) algorithm to find regions of local identity between sequences which are otiierwise largely dissimilar. The strategy results in detection of all exons of the five known genes in the region, THPO, TRAP2, elF4g, CLCN2 and hRPB17 (Table 3). Table 3 Summary of known genes located in the 140 kb region analyzed

Known genes Map position eukaryotic translation initiation factor 4 gamma 3q27-qter thrombopoietin 3q26-q27 chloride channel 2 3q26-qter

TNF receptor associated protein 2 not previously mapped

RNA polymerase II subunit hRPB17 not previously mapped

Finatiy, novel transcription units were predicted using a number of approaches. CpG islands (S. Cross & Bird, A., Curr. Opin. Genet. Dev. 5.: 109-314 (1995) islands were used to define promoter regions and were identified as clusters of sites cleaved by enzymes recognizing GC-rich, 6 or 8-mer palidromic sequences. CpG islands are usuaUy associated with promoter regions of genes. WUBLAST2.0 analysis of short genomic regions (10- 20 kb) versus GenBank revealed matches to ESTs. The individual EST sequences (or where possible, their sequence chromatogram files) were retrieved and assembled with Sequencher to provide a theoretical cDNA sequence (designated herein as DNA34415). GRAIL2 (ApoCom Inc., Knoxville, TN, command line version for the DEC alpha) was used to predict a novel exon. The five known genes in the region served as internal controls for the success of the GRAIL algorithm.

Isolation: Chordin cDNA clones were isolated from an oligo-dT-primed human fetal lung library. Human fetal lung polyA⁺ RNA was purchased from Clontech (cat #6528-1, lot #43777) and 5 mg used to constract a cDNA l i b r a r y in pKR5B (Genentech, LIB26) . The 3 ' -primer

(pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT) (SEQ ID NO:8) and the 5'-linker (pCGGACGCGTGGGGCCTGCGCACCCAGCT) (SEQ ID NO:9) were designed to introduce Sail and Notl restriction sites. Clones were screened wiui oUgonucleotide probes designed from the putative human chordin cDNA sequence (DNA34415) deduced by manually "splicing" together the proposed genomic exons of the gene. PCR primers flanking the probes were used to confirm the identity of the cDNA clones prior to sequencing.

The screening oligonucleotides probes were the following: OLI5640 34415.pl 5'-GCCGCTCCCCGAACGGGCAGCGGCTCCTTCTCAGAA-3' (SEQ ID NO: 10) and OLI5642 34415.p2 5'-GGCGCACAGCACGCAGCGCATCACCCCGAATGGCTC-3' (SEQ ID NO: 11); and the flanking probes used were the following:

OLI5639 34415.fl 5'-GTGCTGCCCATCCGTTCTGAGAAGGA-3' (SEQ ID NO: 12) and OLI5643 34415.r 5'-GCAGGGTGCTCAAACAGGACAC-3' (SEQ ID NO:13).

EXAMPLE 5: Northern Blot and in situ RNA Hybridization Analysis of PRQ243 Expression of PR0243 mRNA in human tissues was examined by Northern blot analysis . Human polyA +

RNA blots derived from human fetal and adult tissues (Clontech, Palo Alto, CA; Cat. Nos. 7760-1 and 7756-1) were hybridized to a ³²P-labelled cDNA fragments probe based on die full length PR0243 cDNA. Blots were incubated witii the probes in hybridization buffer (5X SSPE; 2X Denhardt's solution; 100 mg/mL denatured sheared salmon sperm DNA; 50% formamide; 2% SDS) for 60 hours at 42"C. The blots were washed several times in 2X SSC; 0.05% SDS for 1 hour at room temperature, foUowed by a high stringency wash 30 minute wash in O.lX SSC; 0.1 % SDS at 50"C and autoradiographed. The blots were developed after overnight exposure by phosphorimager analysis (Fuji).

As shown in Fig. 6, PR0243 mRNA transcripts were detected. Analysis of the expression pattern showed die strongest signal of the expected 4.0 kb transcript in adult and fetal liver and a very faint signal in the adult kidney. Fetal brain, lung and kidney were negative, as were adult heart, brain, lung and pancreas. Smaller transcripts were observed in placenta (2.0 kb), adult skeletal muscle (1.8 kb) and fetal liver (2.0 kb).

In situ hybridization of adult human tissue of PR0243 gave a positive signal in the cleavage line of the developing synovial joint forming between the femoral head and acetabulum. All other tissues were negative. Additional sections of human fetal face, head, limbs and mouse embryos were examined. Expression in human fetal tissues was observed adjacent to developing limb and facial bones in die perosteal msenchyme. The expression was highly specific and was often adjacent to areas undergoing vascularization. Expression was also observed in the developing temporal and occipital lobes of the fetal brain, but was not observed elsewhere in die brain. In addition, expression was seen in die ganglia of the developing inner ear. No expression was seen in any of the mouse tissues with the human probes (see Figure 7). In situ hybridization was performed using an optimized protocol, using PCR-generating ³³P-labeled riboprobes. (Lu and Gillett, Cell Vision I : 169-176 (1994)). Formalin-fixed, paraffin-embedded human fetal and adult tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at -37°C, and further processed for in situ hybridization as described by Lu and Gillett (1994). A [³³P]-UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55 °C overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

EXAMPLE 6: Isolation of cDNA clones Encoding Human PRQ299

A cDNA sequence designated herein as DNA28847 (Figure 10; SEQ ID NO: 18) was isolated as described in Example 2 above. After further analysis, a 3' truncated version of DNA28847 was found and is herein designated DNA35877 (Figure 11; SEQ ID NO: 19). Based on the DNA35877 sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained die sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0299. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, with the PCR primer pair. A positive tibrary was then used to isolate clones encoding die gene of interest using the probe oligonucleotide and one of the primer pairs.

Forward and reverse PCR primers were synthesized: forward PCR primer (35877.fl) 5'-CTCTGGAAGGTCACGGCCACAGG-3' (SEQ ID NO:20) reverse PCR primer (35877.rl) 5'-CTCAGTTCGGTTGGCAAAGCTCTC-3' (SEQ ID NO:21) Additionally, a synthetic oligonucleotide hybridization probe was constracted from the DNA35877 sequence which had die following nucleotide sequence hybridization probe (35877.pl)

5'-CAGTGCTCCCTCATAGATGGACGAAAGTGTGACCCCCCTTTCAGGCGAGAGCTTTGCCAACCGAA CTGA-3' (SEQ ID NO:22) In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication with one of the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0299 sequence using the probe oligonucleotide.

RNA for construction of the cDNA Ubraries was isolated from human fetal brain tissue. The cDNA libraries used to isolate die cDNA clones were constracted by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Notl site, linked witii blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a denned orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain die Sfil site; see, Holmes et al., Science. 253:1278-1280 (1991)) in the unique Xhol and Notl sites.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0299 [herein designated as UNQ262 (DNA39976-1215)] (SEQ ID NO: 14) and the derived protein sequence for PR0299.

The entire nucleotide sequence of UNQ262 (DNA39976-1215) is shown in Figure 8 (SEQ ID NO: 14).

Clone UNQ262 (DNA39976-1215) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 111-113 and ending at the stop codon at nucleotide positions 2322-2324 (Figure 8). The predicted polypeptide precursor is 737 amino acids long (Figure 9). Important regions of the polypeptide sequence encoded by clone UNQ262 (DNA39976-1215) have been identified and include the following: a signal peptide corresponding to amino acids 1-28, a putative transmembrane region corresponding to amino acids 638-662, 10 EGF repeats, corresponding to amino acids 80-106, 121-203, 336-360, 378415, 416-441, 454-490, 491-528, 529-548, 567-

604, and 605-622, respectively, and 10 potential N-glycosylation sites, corresponding to amino acids 107-120, 204-

207, 208-222, 223-285, 286-304, 361-374, 375-377, 442-453, 549-563, and 564-566, respectively. Clone UNQ262 (DNA39976-1215) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209524.

Analysis of the amino acid sequence of the full-length PR0299 polypeptide suggests that portions of it possess significant homology to the notch protein, thereby indicating that PR0299 may be a novel notch protein homolog and have activity typical of the notch protein.

EXAMPLE 7: Isolation of cDNA Clones Encoding Human PRQ323

A consensus DNA sequence was assembled relative to other EST sequences as described in Example 1 above. This consensus sequence is herein designated DNA30875. Based on die DNA30875 consensus sequence, oUgonucleotides were synthesized; 1) to identify by PCR a cDNA library that contained die sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0323. PCR primers (two forward and one reverse) were synthesized: forward PCR primer 1 5'-AGTTCTGGTCAGCCTATGTGCC-3' (SEQ ID NO:25) forward PCR primer 2 5'-CGTGATGGTGTCTTTGTCCATGGG-3' (SEQ ID NO:26) reverse PCR primer 5'-CTCCACCAATCCCGATGAACTTGG-3' (SEQ ID NO:27) Additionally, a synthetic oligonucleotide hybridization probe was constracted from the consensus DNA30875 sequence which had the following nucleotide sequence hybridization probe 5'-GAGCAGATTGACCTCATACGCCGCATGTGTGCCTCCTATTCTGAGCTGGA-3' (SEQ ID NO:ll)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication witii the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0323 gene using the probe oligonucleotide and one of die PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal liver tissue (LIB6).

DNA sequencing of the clones isolated as described above gave die full-length DNA sequence for PR0323 [herein designated as UNQ284 (DNA35595-1228)] (SEQ ID NO:23) and die derived protein sequence for PR0323. The entire nucleotide sequence of UNQ284 (DNA35595-1228) is shown in Figure 12 (SEQ ID NO:23).

Clone UNQ284 (DNA35595-1228) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 110-112 and ending at die stop codon at nucleotide positions 1409-1411 (Figure 12). The predicted polypeptide precursor is 433 amino acids long (Figure 13). The full-lengtii PR0323 protein shown in Figure 13 has an estimated molecular weight of about 47,787 daltons and a pi of about 6.11. Clone UNQ284 (DNA35595-1228) has been deposited with ATCC and is assigned ATCC deposit no. 209528.

Analysis of the amino acid sequence of the full-length PR0323 polypeptide suggests that portions of it possess significant homology to various dipeptidase proteins, thereby indicating that PR0323 may be a novel dipeptidase protein.

EXAMPLE 8: Isolation of cDNA Clones Encoding Human PRQ327

An expressed sequence tag (EST) DNA database (LJFESEQ™, Incyte Pharmaceuticals, Palo Alto, CA) was searched and various EST sequences were identified which showed certain degrees of homology to human prolactin receptor protein. Those EST sequences were aligned using phrap and a consensus sequence was obtained. This consensus DNA sequence was then 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. The extended assembly sequence is herein designated DNA38110. The above searches were performed using die computer program BLAST or BLAST2 (Altshul et al., Methods in Enzvmologv 266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington; http: //bozeman.mbt. washington.edu/phrap .docs/phrap .html) .

Based upon die DNA38110 consensus sequence obtained as described above, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained die sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0327.

PCR primers (forward and reverse) were synthesized as follows: forward PCR primer 5'-CCCGCCCGACGTGCACGTGAGCC-3' (SEQ ID NO:33) reverse PCR primer 5'-TGAGCCAGCCCAGGAACTGCTTG-3' (SEQ ID NO:34)

Additionally, a synthetic oligonucleotide hybridization probe was constracted from the consensus DNA38110 consensus sequence which had the following nucleotide sequence hybridization probe 5'-CAAGTGCGCTGCAACCCCTTTGGCATCTATGGCTCCAAGAAAGCCGGGAT-3' (SEQ ID NO:35)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication witii the PCR primer pair identified above. A positive library was then used to isolate clones encoding die PR0327 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 (LIB26).

DNA sequencing of die clones isolated as described above gave die full-length DNA sequence for PR0327 [herein designated as UNQ288 (DNA38113-1230)] (SEQ ID NO: 16) and the derived protein sequence for PR0327.

The entire nucleotide sequence of UNQ288 (DNA38113-1230) is shown in Figure 16 (SEQ ID NO:31). Clone UNQ288 (DNA38113-1230) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 119-121 and ending at the stop codon at nucleotide positions 1385-1387 (Figure 16). The predicted polypeptide precursor is 422 amino acids long (Figure 17). The full-length PR0327 protein shown in Figure 17 has an estimated molecular weight of about 46,302 daltons and a pi of about 9.42. Clone UNQ288 (DNA38113-1230) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209530.

Analysis of the amino acid sequence of the full-length PR0327 polypeptide suggests that it possess significant homology to the human prolactin receptor protein, thereby indicating that PR0327 may be a novel prolactin binding protein.

EXAMPLE 9: Isolation of cDNA Clones Encoding Human PRQ233

A consensus DNA sequence was assembled relative to other EST sequences as described in Example 1 above. This consensus sequence is herein designated DNA30945. Based on the DNA30945 consensus sequence, oUgonucleotides were synuiesized: 1) to identify by PCR a cDNA library that contained die sequence of interest, and

2) for use as probes to isolate a clone of the full-length coding sequence for PR0233. PCR primers were synthesized as followed: forward PCR primer 5'-GGTGAAGGCAGAAATTGGAGATG-3' (SEQ ID NO:38) reverse PCR primer 5'-ATCCCATGCATCAGCCTGTTTACC-3' (SEQ ID NO:39)

Additionally, a syntiietic oligonucleotide hybridization probe was constructed from the consensus DNA30945 sequence which had die following nucleotide sequence hybridization probe

5 '-GCTGGTGTAGTCTATACATCAGATTTGTTTGCTACACAAGATCCTCAG-3 ' (SEQ ID NO:40)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication with the PCR primer pair identified above. A positive library was then used to isolate clones encoding die PR0233 gene using die probe oUgonucleoti.de. RNA for construction of the cDNA libraries was isolated from human fetal brain tissue. DNA sequencing of the clones isolated as described above gave die full-length DNA sequence for PR0233

[herein designated as UNQ207 (DNA34436-1238)] (SEQ ID NO:36) and the derived protein sequence for PR0233. The entire nucleotide sequence of UNQ207 (DNA34436-1238) is shown in Figure 18 (SEQ ID NO:36).

Clone UNQ207 (DNA34436-1238) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 101-103 and ending at the stop codon at nucleotide positions 1001-1003 (Figure 18). The predicted polypeptide precursor is 300 amino acids long (Figure 19). The full-length PR0233 protein shown in Figure 19 has an estimated molecular weight of about 32,964 daltons and a pi of about 9.52. In addition, regions of interest including the signal peptide and a putative oxidoreductase active site, are designated in Figure 19. Clone UNQ207 (DNA34436-1238) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209523

Analysis of the amino acid sequence of the full-length PR0233 polypeptide suggests that portions of it possess significant homology to various reductase proteins, thereby indicating at PR0233 may be a novel reductase.

EXAMPLE 10: Isolation of cDNA Clones Encoding Human PRQ344

A consensus DNA sequence was assembled relative to other EST sequences as described in Example 1 above. This consensus sequence is herein designated DNA34398. Based on the DNA34398 consensus sequencs, oUgonucleotides were syntiiesized: 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 PR0344.

Based on die DNA34398 consensus sequence, forward and reverse PCR primers were synthesized as follows: forward PCR primer (34398.fl) 5'-TACAGGCCCAGTCAGGACCAGGGG-3' (SEQIDNO:43) forward PCR primer (34398.f2) 5'-AGCCAGCCTCGCTCTCGG-3' (SEQIDNO:44) forward PCR primer (34398.f3) 5'-GTCTGCGATCAGGTCTGG-3' (SEQIDNO:45) reverse PCR primer (34398.rl) 5'-GAAAGAGGCAATGGATTCGC-3' (SEQ1DN0:46) reverse PCR primer (34398. r2) 5'-GACTTACACTTGCCAGCACAGCAC-3' (SEQK>NO:47)

Additionally, a synthetic oligonucleotide hybridization probe was constructed from the DNA34398 consensus sequence which had the following nucleotide sequence hybridization probe (34398.pl)

5'-GGAGCACCACCAACTGGAGGGTCCGGAGTAGCGAGCGCCCCGAAG-3' (SEQIDNO:48)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication with one of the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0344 genes using the probe oligonucleotide and one of die 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 PR0344

[herein designated as UNQ303 (DNA40592-1242)] (SEQ ID NO:41) and the derived protein sequence for PR0344.

The entire nucleotide sequence of UNQ303 (DNA40592-1242) is shown in Figure 20 (SEQ ID NO:41). Clone UNQ303 (DNA40592-1242) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 227-229 and ending at die stop codon at nucleotide positions 956-958 (Figure 20). The predicted polypeptide precursor is 243 amino acids long (Figure 21). Important regions of the amino acid sequence encoded by nucleotides 1 to 729 of PR0344 include the signal peptide, die start of the mature protein, and two potential N-myristoylation sites as shown in Figure 21. Clone UNQ303 (DNA40592-1242) has been deposited with the ATCC and is assigned ATCC deposit no. ATCC 209492

Analysis of the amino acid sequence of the full-length PR0344 polypeptides suggests that portions of them possess significant homology to various human and murine complement proteins, thereby indicating tiiat PR0344 may be a novel complement protein. EXAMPLE 11: Isolation of cDNA Clones Encoding Human PRQ347

A consensus DNA sequence was assembled relative to other EST sequences as described in Example 1 above. This consensus sequence is herein designated DNA39499. Based on die DNA39499 consensus sequence, oUgonucleotides 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 PR0347. PCR primers (forward and reverse) were synthesized as follows: forward PCR primer 5'-AGGAACTTCTGGATCGGGCTCACC-3' (SEQ ID NO:51) reverse PCR primer 5'-GGGTCTGGGCCAGGTGGAAGAGAG-3' (SEQ ID NO:52)

Additionally, a synthetic oligonucleotide hybridization probe was constracted from the consensus DNA39499 sequence which had die following nucleotide sequence hybridization probe

S'-GCCAAGGACTCCTTCCGCTGGGCCACAGGGGAGCACCAGGCCTTC-S' (SEQ ID NO:53)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication with the PCR primer pair identified above. A positive library was then used to isolate clones encoding die PR0347 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228).

DNA sequencing of the clones isolated as described above gave die full-length DNA sequence for PR0347 [herein designated as UNQ306 (DNA44176-1244)] (SEQ ID NO:49) and the derived protein sequence for PR0347. The entire nucleotide sequence of UNQ306 (DNA44176-1244) is shown in Figure 22 (SEQ ID NO:49). Clone UNQ306 (DNA44176-1244) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 123-125 and ending at the stop codon at nucleotide positions 1488-1490 (Figure 22). The predicted polypeptide precursor is 455 amino acids long (Figure 23). The full-length PR0347 protein shown in Figure 23 has an estimated molecular weight of about 50,478 daltons and a pi of about 8.44. Clone UNQ306 (DNA44176-1244) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209532

Analysis of the amino acid sequence of the full-length PR0347 polypeptide suggests that portions of it possess significant homology to various cysteine-rich secretory proteins, thereby indicating tiiat PR0347 may be a novel cysteine-rich secretory protein.

EXAMPLE 12: Isolation of cDNA Clones Encoding Human PRQ354

An expressed sequence tag (EST) DNA database (LEFESEQ™, Incyte Pharmaceuticals, Palo Alto, CA) was searched and various EST sequences were identified which possessed certain degress of homology with the inter- alpha-trypsin inhibitor heavy chain and with one another. Those homologous EST sequences were then aligned and a consensus sequence was obtained. The obtained consensus DNA sequence was then extended using repeated cycles of BLAST and phrap to extend die consensus sequence as far as possible using homologous EST sequences derived from both public EST databases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, CA). The extended assembly sequence is herein designated DNA39633. The above searches were performed using the computer program BLAST or BLAST2 (Altshul et al., Methods in Enzvmologv 266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" ( P h i l Green, University of Washington, Seattle , Washington; http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).

Based on the DNA39633 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained die sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0354. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about l-1.5kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, with the PCR primer pair. A positive Ubrary was then used to isolate clones encoding die gene of interest using the probe oligonucleotide and one of the primer pairs. PCR primers were synthesized as follows: forward PCR primer 1 (39633. fll 5'-GTGGGAACCAAACTCCGGCAGACC-3' (SEQ ID NO:56) forward PCR primer 2 (39633.f21 5'-CACATCGAGCGTCTCTGG-3' (SEQ ID NO:57) reverse PCR primer (39633. rll 5'-AGCCGCTCCTTCTCCGGTTCATCG-3' (SEQ ID NO:58) Additionally, a synthetic oligonucleotide hybridization probe was constracted from the consensus DNA39633 sequence which had die following nucleotide sequence hybridization probe 5'-TGGAAGGACCACTTGATATCAGTCACTCCAGACAGCATCAGGGATGGG-3' (SEQ ID NO:59)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication with the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0354 gene using the probe oligonucleotide and one of die PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB227). The cDNA Ubraries used to isolate the cDNA clones were constructed by standard metiiods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain die Sfil site; see, Holmes et al., Science. 253: 1278-1280 (1991)) in die unique Xhol and Notl sites.

DNA sequencing of die clones isolated as described above gave die full-length DNA sequence for PR0354 [herein designated as UNQ311 (DNA44192-1246)] (SEQ ID NO:54) and the derived protein sequence for PR0354. The entire nucleotide sequence of UNQ311 (DNA44192-1246) is shown in Figure 24 (SEQ ID NO:54).

Clone UNQ311 (DNA44192- 1246) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 72-74 and ending at die stop codon at nucleotide positions 2154-2156 (Figure 24). The predicted polypeptide precursor is 694 amino acids long (Figure 25). The full-length PR0354 protein shown in Figure 25 has an estimated molecular weight of about 77,400 daltons and a pi of about 9.54. Clone UNQ311 (DNA44192- 1246) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209531.

Analysis of the amino acid sequence of the full-length PR0354 polypeptide suggests that it possess significant homology to the inter-alpha-trypsin inhibitor heavy chain protein, thereby indicating that PR0354 may be a novel inter-alpha-trypsin inhibitor heavy chain protein homolog. EXAMPLE 13: Isolation of cDNA Clones Encoding Human PRQ355

A consensus DNA sequence was assembled relative to other EST sequences using BLAST and phrap as described in Example 1 above. This consensus sequence is herein designated DNA35702. Based on the DNA35702 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained die sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0355. Forward and reverse PCR primers were synthesized as follows: forward PCR primer (.fl 5'-GGCTTCTGCTGTTGCTCTTCTCCG-3' (SEQ ID NO: 62) forward PCR primer (.f21 5'-GTACACTGTGACCAGTCAGC-3' (SEQ ID NO:63) forward PCR primer (.f31 5'-ATCATCACAGATTCCCGAGC-3' (SEQ ID NO:64) reverse PCR primer (.rl) 5'-TTCAATCTCCTCACCTTCCACCGC-3' (SEQ ID NO:65) reverse PCR primer (.r2) 5'-ATAGCTGTGTCTGCGTCTGCTGCG-3' (SEQ ID NO:66)

Additionally, a synthetic oligonucleotide hybridization probe was constracted from the consensus DNA35702 sequence which had die following nucleotide sequence: hybridization probe 5'-CGCGGCACTGATCCCCACAGGTGATGGGCAGAATCTGTTTACGAAAGACG-3' (SEQ ID NO:67) In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication with one of the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0355 gene using die probe oligonucleotide. RNA for construction of the cDNA libraries was isolated from human fetal liver tissue.

DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0355 [herein designated as UNQ312 (DNA39518-1247)] (SEQ ID NO:60) and the derived protein sequence for PR0355.

The entire nucleotide sequence of UNQ312 (DNA39518-1247) is shown in Figure 26 (SEQ ID NO:60).

Clone UNQ312 (DNA39518-1247) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 22-24 and ending at the stop codon at nucleotide positions 1342-1344 (Figure 26). The predicted polypeptide precursor is 440 amino acids long (Figure 27). The full-length PR0355 protein shown in Figure 27 has an estimated molecular weight of about 48,240 daltons and a pi of about 4.93. In addition, regions of interest including die signal peptide, Ig repeats in the extracellular domain, potential N-glycosylation sites, and the potential transmembrane domain, are designated in Figure 27. Clone UNQ312 (DNA39518-1247) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209529.

Analysis of the amino acid sequence of the full-length PR0355 polypeptide suggests that portions of it possess significant homology to the CRTAM protein, thereby indicating mat PR0355 may be CRTAM protein.

EXAMPLE 14: Isolation of cDNA Clones Encoding Human PRQ357

The sequence expression tag clone no. "2452972" by Incyte Pharmaceuticals, Palo Alto, CA was used to begin a data base search. The extracellular 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 which overlapped with a portion of Incyte EST clone no. "2452972" . The EST databases included public EST databases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using die computer program BLAST or BLAST2 (Altshul et al., Methods in Enzvmolo v 266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequence. Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington; http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).

A consensus DNA sequence was then assembled relative to other EST sequences using phrap. This consensus sequence is herein designated DNA37162. In this case, the consensus DNA sequence was extended using repeated cycles of BLAST and phrap to extend die consensus sequence as far as possible using die sources of EST sequences discussed above.

Based on die DNA37162 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained die sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0357. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about l-1.5kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as ber Ausubel et al., Current Protocols in Molecular Biology, with the PCR primer pair. A positive Ubrary was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.

PCR primers were synthesized as follows: forward primer 1 : 5'-CCCTCCACTGCCCCACCGACTG-3' (SEQ ID NO:70); reverse primer 1 : 5'-CGGTTCTGGGGACGTTAGGGCTCG-3' (SEQ ID NO:71); and forward primer 2: 5'-CTGCCCACCGTCCACCTGCCTCAAT-3' (SEQ ID NO:72).

Additionally, two synthetic oligonucleotide hybridization probes were constructed from the consensus DNA37162 sequence which had die following nucleotide sequences: hybridization probe 1 : 5'-AGGACTGCCCACCGTCCACCTGCCTCAATGGGGGCACATGCCACC-3' (SEQ ID NO:73); and hybridization probe 2:

5'-ACGCAAAGCCCTACATCTAAGCCAGAGAGAGACAGGGCAGCTGGG-3' (SEQ ID NO:74).

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with a PCR primer pair identified above. A positive library was then used to isolate clones encoding the PR0357 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA Ubraries was isolated from human fetal liver tissue. The cDNA libraries used to isolate die cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science. 253:1278-1280 (1991)) in the unique Xhol and Notl sites.

DNA sequencing of die clones isolated as described above gave the full-length DNA sequence for PR0357

[herein designated as UNQ314 (DNA44804-1248)] (SEQ ID NO:68) and the derived protein sequence for PR0357.

The entire nucleotide sequence of UNQ314 (DNA44804-1248) is shown in Figure 28 (SEQ ID NO:68).

Clone UNQ314 (DNA44804-1248) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 137-139 and ending at the stop codon at nucleotide positions 1931-1933 (Figure 28). The predicted polypeptide precursor is 598 amino acids long (Figure 29). Clone UNQ314 (DNA44804-1248) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209527

Futher analysis shows a number of characteristics as shown in Figure 29. Figure 29 shows the amino acid sequence (SEQ ID NO:69) derived from nucleotides 137 through 1930 of SEQ ID NO:68. Molecular weight is 63,030 daltons; pi is 7.24; and NX(S/T) is 3. The putative transmembrane domain is shown in Figure 29 at amino acids 506 through 524. Alternatively, the transmembrane region begins with the "G" at amino acid 497. The potential N-glycosylation sites are underlined in Figure 29. The EGF-like domain cysteine pattern signature appeasr at amino acids 355 through 366. This region can also be found in milk fat globule protein from rat, notch or the hepatocyte growth factor converting protease. The signal peptide is also at amino acids 1-22 of Figure 29. The start of the homology to ALS and otiier leucine-repeat rich proteins in die extracellular domain begins at amino acid position 24.

Analysis of the amino acid sequence of the full-length PR0357 polypeptide therefore suggests that portions of it possess significant homology to ALS, tiiereby indicating that PR0357 may be a novel leucine rich repeat protein related to ALS.

EXAMPLE 15: Isolation of cDNA Clones Encoding Human PRQ715

A proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, CA) was searched for

EST sequences encoding polypeptides having homology to human TNF-α.. This search resulted in the identification of Incyte Expressed Sequence Tag No. 2099855. A consensus DNA sequence was then assembled relative to other EST sequences using seqext and "phrap"

( P h i l Green, University of Washington, Seattle , Washington; http://bozeman.mbt.washington.edu/phrap.docs/phrap.html). This consensus sequence is herein designated

DNA52092. Based upon die aUgnment of the various EST clones identified in this assembly, a single EST clone from the Merck/Washington University EST set (EST clone no. 725887, Accession No. AA292358) was obtained and its insert sequenced. The ftill-length DNA52722-1229 sequence was then obtained from sequencing the insert DNA from

EST clone no. 725887.

The entire nucleotide sequence of UNQ383 (DNA52722-1229) is shown in Figure 30 (SEQ ID NO:75).

Clone UNQ383 (DNA52722-1229) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 114-116 and ending at the stop codon at nucleotide positions 864-866 (Figure 30). The predicted polypeptide is 250 amino acids long (Figure 31). The full-length PR0715 protein shown in Figure 31 has an estimated molecular weight of about 27,433 daltons and a pi of about 9.85.

Analysis of the amino acid sequence of the full-length PR0715 polypeptide suggests that it possesses significant homology to members of the tumor necrosis factor family of proteins, thereby indicating tiiat PR0715 is a novel tumor necrosis factor protein.

EXAMPLE 16: Isolation of cDNA Clones Encoding Human PRQ353

A consensus DNA sequence was assembled relative to otiier EST sequences using phrap as described in Example 1 above. This consensus sequences is herein designated DNA36363. The consensus DNA sequence was 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 on the DNA36363 consensus sequence, oligonucleotides were syntiiesized: 1) to identify by PCR a cDNA library that contained d e sequence of interest, and 2) for use as probes to isolate a clone of die full-length coding sequence for PR0353.

Based on the DNA36363 consensus sequence, forward and reverse PCR primers were synthesized as follows: forward PCR primer (36363.fl) 5'-TACAGGCCCAGTCAGGACCAGGGG-3' (SEQIDNO:87) reverse PCR primer (36363.rl) 5'-CTGAAGAAGTAGAGGCCGGGCACG-3' (SEQIDNO:88).

Additionally, a synthetic oligonucleotide hybridization probe was constracted from the DNA36363 consensus sequence which had die following nucleotide sequence: hybridization probe 36363.pl 5'-CCCGGTGCTTGCGCTGCTGTGACCCCGGTACCTCCATGTACCCGG-3' (SEQIDNO:89)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication wiu one of the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0353 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue. DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0353

[herein designated as UNQ310 (DNA41234- 1242)] (SEQ ID NO:85) and the derived protein sequence for PR0353. The entire nucleotide sequence of UNQ310 (DNA41234- 1242) is shown in Figure 34 (SEQ ID NO:85).

Clone UNQ310 (DNA41234-1242) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 305-307 and ending at the stop codon at nucleotide positions 1148-1150 (Figure 34). The predicted polypeptide precursor is 281 amino acids long (Figure 35). Important regions of the amino acid sequence encoded by PR0353 include the signal peptide, corresponding to amino acids 1-26, the start of the mature protein at amino acid position 27, a potential N-glycosylation site, corresponding to amino acids 93-98 and a region which has homology to a 30 kd adipocyte complement-related protein precursor, corresponding to amino acids 99-281.

Clone UNQ310 (DNA41234-1242) has been deposited with the ATCC and is assigned ATCC deposit no. ATCC 209618

Analysis of the amino acid sequence of the full-length PR0353 polypeptides suggests that portions of them possess significant homology to portions of human and murine complement proteins, thereby indicating that PR0353 may be a novel complement protein.

EXAMPLE 17: Isolation of cDNA Clones Encoding Human PRQ361

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 DNA40654. Based on the DNA40654 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained die sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0361. Forward and reverse PCR primers were synthesized as follows: forward PCR primer .fll 5'-AGGGAGGATTATCCTTGACCTTTGAAGACC-3' (SEQ ID NO:92) forward PCR primer (.f21 5'-GAAGCAAGTGCCCAGCTC-3' (SEQ ID NO:93) forward PCR primer (,Q1 5'-CGGGTCCCTGCTCTTTGG-3' (SEQ ID NO:94) reverse PCR primer (.rl) 5'-CACCGTAGCTGGGAGCGCACTCAC-3' (SEQ ID NO:95) reverse PCR primer (.r2) 5'-AGTGTAAGTCAAGCTCCC-3' (SEQ ID NO:96)

Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA40654 sequence which had the following nucleotide sequence hybridization probe

5 '- GCTTCCTGACACTAAGGCTGTCTGCTAGTCAGAATTGCCTCAAAAAGAG-3 ' (SEQ ID NO:97)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication witii one of die PCR primer pairs identified above. A positive library was then used to isolate clones encoding d e PR0361 gene using die probe oligonucleotide. 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 PR0361

[herein designated as UNQ316 (DNA45410-1250)] (SEQ ID NO:90) and the derived protein sequence for PR0361. The entire nucleotide sequence of UNQ316 (DNA45410-1250) is shown in Figure 36 (SEQ ID NO:90).

Clone UNQ316 (DNA45410-1250) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 226-228 and ending at the stop codon at nucleotide positions 1519-1521 (Figure 36). The predicted polypeptide precursor is 431 amino acids long (Figure 37). The full-lengtii PR0361 protein shown in

Figure 37 has an estimated molecular weight of about 46,810 daltons and a pi of about 6.45. In addition, regions of interest including the transmembrane domain (amino acids 380-409) and sequences typical of the arginase family of proteins (amino acids 3-14 and 39-57) are designated in Figure 37. Clone UNQ316 (DNA45410-1250) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209621. Analysis of the amino acid sequence of the full-length PR0361 polypeptide suggests that portions of it possess significant homology to the mucin and/or chitinase proteins, thereby indicating that PR0361 may be a novel mucin and/or chitinase protein.

EXAMPLE 18: Isolation of cDNA Clones Encoding Human PRQ365 A consensus DNA sequence was assembled relative to otiier EST sequences using phrap as described in

Example 1 above. This consensus sequence is herein designated DNA35613. Based on die DNA35613 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 PR0365. Forward and reverse PCR primers were synthesized as follows: forward PCRprimer (.fl-35613 5'-AATGTGACCACTGGACTCCC-3' (SBQIDN .100) forward PCR primer (.f2-3561 1 5'-AGGCTTGGAACTCCCTTC-3' (SBQIDN 101) reverse PCR primer (.rl-356131 5'-AAGATTCTTGAGCGATTCCAGCTG-3' (SEQIDN01Q2)

Additionally, a synthetic oligonucleotide hybridization probe was constracted from the consensus DNA35613 sequence which had the following nucleotide sequence hybridization probe

5 '-AATCCCTGCTCTTCATGGTGACCTATGACGACGGAAGCACAAGACTG-3 ' (SBQIDNO.KB)

In order to screen several Ubraries for a source of a full-length clone, DNA from the libraries was screened by PCR ampUfication widi one of the PCR primer pairs identified above. A positive library was then used to isolate clones encoding the PR0365 gene using the probe oligonucleotide and one of the PCR primers. RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of die clones isolated as described above gave die full-length DNA sequence for PR0365 [herein designated as UNQ320 (DNA46777-1253)] (SEQ ID NO:98) and the derived protein sequence for PR0365.

The entire nucleotide sequence of UNQ320 (DNA46777-1253) is shown in Figure 38 (SEQ ID NO:98). Clone UNQ320 (DNA46777-1253) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 15-17 and ending at die stop codon at nucleotide positions 720-722 (Figure 38). The predicted polypeptide precursor is 235 amino acids long (Figure 39). Important regions of the polypeptide sequence encoded by Clone UNQ320 (DNA46777-1253) have been identified and include the following: a signal peptide corresponding to amino acids 1-20, the start of the mature protein corresponding to amino acid 21, and multiple potential N- glycosylation sites as shown in Figure 39. Clone UNQ320 (DNA46777-1253) has been deposited with ATCC and is assigned ATCC deposit no. ATCC 209619.

Analysis of the amino acid sequence of the full-length PR0365 polypeptide suggests that portions of it possess significant homology to die human 2-19 protein, thereby indicating that PR0365 may be a novel human 2-19 protein homolog.

EXAMPLE 19: Use of PRO Polvpeptide-Encoding Nucleic Acid as Hybridization Probes

The foUowing mediod describes use of a nucleotide sequence encoding a PRO polypeptide as a hybridization probe.

DNA comprising the coding sequence of of a PRO polypeptide of interest as disclosed herein may be employed as a probe or used as a basis from which to prepare probes to screen for homologous DNAs (such as those encoding naturally-occurring variants of the PRO polypeptide) in human tissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing eitiier library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled PRO polypeptide-encoding nucleic acid-derived probe to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1 % SDS, 0.1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42"C for 20 hours. Washing of the filters is performed in an aqueous solution of 0. lx SSC and 0.1 % SDS at 42"C.

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.

EXAMPLE 20: Expression of PRO Polypeptides in £. coli

This example illustrates preparation of an unglycosylated form of a desired PRO polypeptide by recombinant expression in E. coli.

The DNA sequence encoding die desired PRO polypeptide is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to die restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene. 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested witii restriction enzyme and dephosphorylated. The PCR amplified sequences are tiien ligated into die vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including die first six STII codons, polyhis sequence, and enterokinase cleavage site), the specific PRO polypeptide coding region, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strain using the methods described in

Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented witii 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 die ceUs for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in die art, and die solubilized PRO polypeptide can then be purified using a metal chelating column under conditions that allow tight binding of the protein.

PR0241 was successfully expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding PR0241 was initially amplified using selected PCR primers. The primers contained restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and otiier useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences were then ligated into an expression vector, which was used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(ladq). Transformants were first grown in LB containing 50 mg/ml carbenicillin at 30 °C with shaking until an O.D.600 of 3-5 was reached. Cultures were then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH₄)₂S0₄, 0.71 g sodium citrate-2H20, 1.07 g KC1, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as weU as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgS0₄) 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 1 L fermentations (6-10 g pellets) was resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02 M, respectively, and the solution was stirred overnight at 4°C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution was centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant was diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. Depending d e clarified extract was loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4°C. Protein concentration was estimated by its absorbance at 280 nm using die calculated extinction coefficient based on its amino acid sequence. The proteins were refolded by diluting sample slowly into freshly prepared refolding buffer consisting of:

20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes were chosen so that the final protein concentration was between 50 to 100 micrograms/ml. The refolding solution was stirred gently at 4°C for 12-36 hours. The refolding reaction was quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution was filtered dirough a 0.22 micron filter and acetonitrile was added to 2-10% final concentration. The refolded protein was chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0.1 % TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were pooled. Generally, the properly refolded species of most proteins are eluted at die lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usuaUy eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.

Fractions containing the desired folded PR0241 protein were pooled and die acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins were formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.

EXAMPLE 21 : Expression of PRO Polypeptides in Mammalian Cells

This example illustrates preparation of a glycosylated form of a desired PRO polypeptide by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector. Optionally, the PRO polypeptide-encoding DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO polypeptide DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRK5-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 optionaUy, nutrient components and/or antibiotics. About 10 μg pRK5-PRO polypeptide DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 21:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP0₄, and a precipitate is allowed to form for 10 minutes at 25"C. The precipitate is suspended and added to die 293 cells and allowed to settle for about four hours at 37"C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and die cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays . In an alternative technique, PRO polypeptide may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci.. 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PRO polypeptide DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed witii PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into die spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PRO polypeptide can tiien be concentrated and purified by any selected method, such as dialysis and/or column chromatography.

In another embodiment, PRO polypeptides can be expressed in CHO cells. The pRK5-PRO polypeptide can be transfected into CHO ceUs using known reagents such as CaP0₄ or DEAE-dextran. As described above, the cell cultures can be incubated, and die medium replaced with culture medium (alone) or medium containing a radiolabel such as ³⁵S-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 medium containing the expressed PRO polypeptide 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 subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged PRO polypeptide insert can tiien be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing die expressed poly-His tagged PRO polypeptide can tiien be concentrated and purified by any selected method, such as by Ni²⁺-chelate affinity chromatography.

PR0241 was successfully expressed in CHO cells by both a transient and a stable expression procedure. In addition, PR0243, PR0323 and PR0233 were successfully transiently expressed in CHO cells.

Stable expression in CHO ceUs was performed using the following procedure. The proteins were expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins were fused to an IgGl constant region sequence containing die hinge, CH2 and CH2 domains and/or is a poly-His tagged form. Following PCR amplification, the respective DNAs were subcloned in a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO 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 described in Lucas et al, Nucl. Acids Res. 24: 9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.

Twelve micrograms of die desired plasmid DNA were introduced into approximately 10 million CHO cells using commerciaUy available transfection reagents Superfect^* (Quiagen), Dosper^* or Fugene^* (Boehringer Mannheim). The cells were grown and described in Lucas et al , supra. Approximately 3 x 10^"7 cells are frozen in an ampule for further growth and production as described below.

The ampules containing the plasmid DNA were thawed by placement into water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells were then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells were transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37°C. After anotiier 2-3 days, a 250 mL, 500 mL and 2000 mL spinners were seeded with 3 x 10⁵ cells/mL. The cell media was exchanged witii fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in US Patent No. 5,122,469, issued June 16, 1992 was actually used. 3L production spinner is seeded at 1.2 x 10⁶ cells/mL. On day 0, the cell number pH were determined. On day 1, the spinner was sampled and sparging witii filtered air was commenced. On day 2, the spinner was sampled, die temperature shifted to 33 °C, and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion). Throughout the production, pH was adjusted as necessary to keep at around 7.2. After 10 days, or until viability dropped below 70%, the cell culture was harvested by centrifugtion and filtering through a 0.22 μm filter. The filtrate was either stored at 4°C or immediately loaded onto columns for purification.

For the poly-His tagged constructs, the proteins were purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni-NTA column equiUbrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, die column was washed witii additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80° C.

Immunoadhesin (Fc containing) constructs of were purified from the conditioned media as follows. The conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation. PR0241, PR0243, PR0299, PR0323, PR0327, PR0233, PR0344, PR0347, PR0354, PR0355, PR0357,

PR0353, PR0361 and PR0365 were also successfully transiently expressed in COS cells.

EXAMPLE 22: Expression of PRO Polypeptides in Yeast

The following method describes recombinant expression of a desired PRO polypeptide in yeast. First, yeast expression vectors are constructed for intracellular production or secretion of PRO polypeptides from the ADH2/GAPDH promoter. DNA encoding a desired PRO polypeptide, a selected signal peptide and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the PRO polypeptide. For secretion, DNA encoding the PRO polypeptide can be cloned into the selected plasmid, togedier with DNA encoding the ADH2/GAPDH promoter, the yeast alpha-factor secretory signal/leader sequence, and linker sequences (if needed) for expression of the PRO polypeptide.

Yeast cells, such as yeast strain ABI 10, can then be transformed witii the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain. Recombinant PRO polypeptide can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and tiien concentrating the medium using selected cartridge filters. The concentrate containing die PRO polypeptide may further be purified using selected column chromatography resins.

EXAMPLE 23: Expression of PRO Polypeptides in Baculovirus-Infected Insect Cells The foUowing method describes recombinant expression of PRO polypeptides in Baculoviras-infected insect cells.

The desired PRO polypeptide is fused upstream of an epitope tag contained witii a baculoviras expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the PRO polypeptide or the desired portion of the PRO polypeptide (such as the sequence encoding d e extraceUular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.

Recombinant baculoviras is generated by co-transfecting the above plasmid and BaculoGold™ virus DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28°C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression is performed as described by O'Reilley et al., Baculoviras expression vectors: A laboratory Manual, Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO polypeptide can then be purified, for example, by Ni²⁺-chelate affinity chromatography as foUows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al, Nature, 262: 175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1 % NP-40; 0.4 M KC1), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer

(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTA agarose column (commercially available from Qiagen) is prepared witii a bed volume of 5 mL, washed witii 25 mL of water and equUibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto die column at 0.5 mL per minute. The column is washed to baseline A₂₈₀ with loading buffer, at which point fraction collection is started.

Next, the column is washed witii a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH

6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀ baseline again, die column is developed witii a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by

SDS-PAGE and silver staining or western blot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).

Fractions containing the eluted His₁₀-tagged PRO polypeptide are pooled and dialyzed against loading buffer.

Alternatively, purification of die IgG tagged (or Fc tagged) PRO polypeptide can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography. PR0241, PR0327 and PR0344 were successfully expressed in baculoviras infected Sf9 insect cells. While the expression was actually performed in a 0.5-2 L scale, it can be readily scaled up for larger (e.g. 8 L) preparations. The proteins were expressed as an IgG construct (immunoadhesin), in which the protein extracellular region was fused to an IgGl constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly- His tagged forms. For expression in baculoviras infected Sf9 cells, following PCR amplification, the respective coding sequences were subcloned into a baculoviras expression vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly- His tagged proteins), and die vector and Baculogold^® baculoviras DNA (Pharmingen) were co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of d e commerciaUy available baculoviras expression vector pVL1393 (Pharmingen), witii modified polylinker regions to include the His or Fc tag sequences. The cells were grown in Hink's TNM-FH medium supplemented witii 10% FBS (Hyclone). Cells were incubated for 5 days at 28°C. The supernatant was harvested and subsequendy used for the first viral ampUfication by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells were incubated for 3 days at 28°C. The supernatant was harvested and the expression of the constructs in the baculoviras expression vector was determined by batch binding of 1 ml of supernatant to 25 mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.

The first viral ampUfication supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells were incubated for 3 days at 28°C. The supernatant was harvested and filtered. Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed.

The conditioned medium from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein construct were purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media were pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, d e column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% annitol, pH 6.8, witii a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.

Immunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows. The conditioned media were pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, die column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into rubes containing 275 mL of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the proteins was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.

PR0243, PR0323, PR0344 and PR0355 were successfully expressed in baculoviras infected Hi5 insect ceUs. While die expression was actually performed in a 0.5-2 L scale, it can be readily scaled up for larger (e.g. 8 L) preparations.

For expression in baculoviras-infected Hi5 insect cells, the PRO polypeptide-encoding DNA may be amplified witii suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculoviras expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the PRO polypeptide or the desired portion of the PRO polypeptide (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR witii 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 subcloned into the expression vector. For example, derivatives of pVL1393 can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3 '-of) the NAME sequence. Preferably, the vector construct is sequenced for confirmation.

Hi5 ceUs are grown to a confluency of 50% under die conditions of, 27°C, no C02, NO pen/strep. For each 150 mm plate, 30 ug of pIE based vector containing PRO polypeptide is mixed with 1 ml Ex-Cell medium (Media: Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401-78P (note: this media is light sensitive)), and in a separate tube, 100 ul of CellFectin (CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) is mixed with 1 ml of Ex-Cell medium. The two solutions are combined and allowed to incubate at room temperature for 15 minutes. 8 ml of Ex- CeU media is added to the 2ml of DNA/CeUFECTIN mix and this is layered on Hi5 cells that have been washed once witii Ex-CeU media. The plate is tiien incubated in darkness for 1 hour at room temperature. The DNA/CellFECTIN mix is then aspirated, and die cells are washed once with Ex-Cell to remove excess CellFECTIN . 30 ml of fresh Ex-Cell media is added and the cells are incubated for 3 days at 28°C. The supernatant is harvested and the expression of the PRO polypeptide in die baculoviras expression vector can be determined by batch binding of 1 ml of supernatent to 25 mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.

The conditioned media from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein comprising the PRO polypeptide is purified using a Ni-NTA column (Qiagen). Before purification, imidazole is added to d e conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4"C. After loading, the column is washed with additional equilibration buffer and the protein eluted witii equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently deslated into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.

Immunoadhesin (Fc containing) constracts of proteins are purified from the conditioned media as follows. The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, die column is washed extensively witii equilibration buffer before elution witii 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 mL of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of PRO polypeptide can be assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary.

EXAMPLE 24: Preparation of Antibodies that Bind to PRO Polypeptides

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, supra. Immunogens that may be employed include purified PRO polypeptide, fusion proteins containing the PRO polypeptide, and cells expressing recombinant PRO polypeptide on die cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized witii the PRO polypeptide immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted witii additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO polypeptide antibodies.

After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO polypeptide. Three to four days later, die mice are sacrificed and die spleen cells are harvested. The spleen cells are then fused (using 35 % polyethylene glycol) to a selected murine myeloma cell tine such as P3X63AgU.l, avaUable from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can men be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity against the PRO polypeptide. Determination of "positive" hybridoma ceUs secreting the desired monoclonal antibodies against die PRO polypeptide is within the skill in the art. The positive hybridoma ceUs can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO polypeptide monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roUer botdes. Purification of die monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.

EXAMPLE 25: Chimeric PRO Polypeptides

PRO polypeptides may be expressed as chimeric proteins with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in die FLAGS™ extension/affinity purification system (Immunex Corp., Seattle Wash.). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego Calif.) between the purification domain and the PRO polypeptide sequence may be useful to facilitate expression of DNA encoding the PRO polypeptide.

EXAMPLE 26: Purification of PRO Polypeptides Using Specific Antibodies

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, mature PRO polypeptide, or pre-PRO polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PRO polypeptide of interest. In general, an immunoaffinity column is constracted by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. PartiaUy purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and die derivative resin is washed according to die manufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from ceUs containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods 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 are grown.

A soluble PRO polypeptide-containing preparation is passed over the immunoaffinity column, and die column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e.g. , high ionic strength buffers in die presence of detergent). Then, the column is eluted under conditions that disrupt antibody /PRO polypeptide binding (e.g., 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 27: Drag Screening

This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drag screening techniques. The PRO polypeptide or fragment employed in such a test may eitiier be free in solution, affixed to a soUd support, borne on a cell surface, or located intracellularly. One method of drag screening utilizes eukaryotic or prokaryotic host cells which are stably transformed witii recombinant nucleic acids expressing the PRO polypeptide or fragment. Drags are screened against such transformed cells in competitive binding assays. Such cells, eitiier in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PRO polypeptide and its target cell or target receptors caused by die agent being tested.

Thus, the present invention provides methods of screening for drags or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the PRO polypeptide or fragment, or (ϋ) for the presence of a complex between the PRO polypeptide or fragment and die cell, by methods well known in die art. In such competitive binding assays, d e PRO polypeptide or fragment is typically labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell complex.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984. Briefly 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 polypeptide, die peptide test compounds are reacted with PRO polypeptide and washed. Bound PRO polypeptide is detected by metiiods well known in the art. Purified PRO polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drag screening assays in which neutralizing antibodies capable of binding PRO polypeptide specifically compete witii a test compound for binding to PRO polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PRO polypeptide.

EXAMPLE 28: Rational Drag Design

The goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest (i.e. , a PRO polypeptide) or of small molecules with which they interact, e.g. , agonists, antagonists, or inhibitors.

Any of these examples can be used to fashion drugs which are more active or stable forms of the PRO polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo (c.f., Hodgson, Bio/Technologv. 9:

19-21 (1991)).

In one approach, the three-dimensional 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 polypeptide must be ascertained to elucidate die structure and to determine active site(s) of the molecule. Less often, useful information regarding the stracmre of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural 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, Biochemistry. 31:7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Atiiauda et al , J. Biochem.. 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drag design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore. By virtue of the present invention, sufficient amounts of the PRO polypeptide may be made available to perform such analytical studies as X-ray crystaUography. In addition, knowledge of the PRO polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.

EXAMPLE 29: Ability of PRQ241 to Stimulate the Release of Proteoglycans from Cartilage

The abihty of PR0241 to stimulate the release of proteoglycans from cartilage tissue was tested as follows.

The metacarphophalangeal joint of 4-6 month old pigs was aseptically dissected, and articular cartilage was removed by free hand slicing being careful to avoid die underlying bone. The cartilage was minced and cultured in bulk for 24 hours in a humidified atmosphere of 95% air, 5% C0₂ in serum free (SF) media (DME/F12 1 : 1) woth 0.1 % BSA and lOOU/ml penicillin and lOOμg/ml streptomycin. After washing three times, approximately 100 mg of articular cartilage was aliquoted into micronics tubes and incubated for an additional 24 hours in the above SF media. PR0241 polypeptides were then added at 1 % eitiier alone or in combination with 18 ng/ml interleukin-lα, a known stimulator of proteoglycan release from cartilage tissue. The supernatant was then harvested and assayed for the amount of proteoglycans using the 1 ,9-dimethyl-methylene blue (DMB) colorimetric assay (Farndale and Buttle, Biochem. Biophvs. Acta 883:173-177 (1985)). A positive result in this assay indicates tiiat die test polypeptide wtil find use, for example, in the treatment of sports-related joint problems, articular cartilage defects, osteoarthritis or rheumatoid arthritis.

When PR0241 polypeptides were tested in the above assay, the polypeptides demonstrated a marked ability to stimulate release of proteoglycans from cartilage tissue both basally and after stimulation with interleukin-lα and at 24 and 72 hours after treatment, thereby indicating tiiat PR0241 polypeptides are useful for stimulating proteoglycan release from cartilage tissue.

EXAMPLE 30: In situ Hybridization

In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze die tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping.

In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision 1:169-176 (1994), using PCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37°C, and further processed for in situ hybridization as described by Lu and Gillett, supra. A [³³-P] UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55 °C overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks. ³³P-Riboprobe synthesis 6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA <2000 Ci/mmol) were speed vac dried. To each tube containing dried ³³P-UTP, the following ingredients were added: 2.0 μl 5x transcription buffer 1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM : 10 μ; each of 10 mM GTP, CTP & ATP + 10 μl H₂0) 1.0 μl UTP (50 μM)

1.0 μl Rnasin

1.0 μl DNA template (lμg) 1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually) The tubes were incubated at 37°C for one hour. 1.0 μl RQ1 DNase were added, followed by incubation at 37°C for 15 minutes. 90 μl TE (10 mM Tris pH 7.6/lmM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Microcon-50 ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 μl TE were added. 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II.

The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNA Mrk III were added to 3 μl of loading buffer. After heating on a 95° C heat block for three minutes, the gel was immediately placed on ice. The weUs of gel were flushed, die 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. ³³P-Hvbridization

A. Pretreatment of frozen sections

The sUdes were removed from the freezer, placed on aluminium trays and thawed at room temperamre for 5 minutes. The trays were placed in 55°C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in die fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperamre (25 ml 20 x SSC + 975 ml SQ H₂0). After deproteination in 0.5 μg/ml proteinase K for 10 minutes at 37°C (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in 0.5 x SSC for 10 minutes at room temperamre. The sections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each.

B. Pretreatment of paraffin-embedded sections The slides were deparaffinized, placed in SQ H₂0, and rinsed twice in 2 x SSC at room temperature, for

5 minutes each time. The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNase-free RNase buffer; 37°C, 15 minutes) - human embryo, or 8 x proteinase K (100 μl in 250 ml Rnase buffer, 37°C, 30 minutes) - formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above. C. Prehvbridization

The sUdes were laid out in a plastic box lined widi Box buffer (4 x SSC, 50% formamide) - saturated filter paper. The tissue was covered with 50 μl of hybridization buffer (3.75g Dextran Sulfate + 6 ml SQ H₂0), vortexed and heated in the microwave for 2 minutes with the cap loosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20 x SSC and 9 ml SQ H₂0 were added, d e tissue was vortexed well, and incubated at 42°C for 1-4 hours. D. Hybridization

1.0 x 10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heated at 95 °C for 3 minutes. The sUdes were cooled on ice, and 48 μl hybridization buffer were added per slide. After vortexing, 50 μl ³³P mix were added to 50 μl prehybridization on slide. The slides were incubated overnight at 55 °C. E. Washes Washing was done 2 x 10 minutes with 2xSSC, EDTA at room temperamre (400 ml 20 x SSC + 16 ml

0.25M EDTA, V_f=4L), foUowed by RNaseA treatment at 37°C for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer = 20 μg/ml), The slides were washed 2 x 10 minutes with 2 x SSC, EDTA at room temperamre. The stringency wash conditions were as follows: 2 hours at 55°C, 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA, V_f=4L). F. Oligonucleotides

In situ analysis was performed on a variety of DNA sequences disclosed herein. The oligonucleotides employed for these analyses are as follows. (1) DNA44804-1248 (PR03571 pi 5'-GGATTCTAATACGACTCACTATAGGGCTGCCCGCAACCCCTTCAACTG-3' (SEQ ID NO:104) p2 5'-CTATGAAATTAACCCTCACTAAAGGGACCGCAGCTGGGTGACCGTGTA-3' (SEQ ID NO: 105)

(2) DNA52722-1229 (PR07151 pi 5'-GGATTCTAATACGACTCACTATAGGGCCGCCCCGCCACCTCCT-3' (SEQ ID NO: 106) p2 5'-CTATGAAATTAACCCTCACTAAAGGGACTCGAGACACCACCTGACCCA-3' (SEQ ID NO:107) p3 5'-GGATTCTAATACGACTCACTATAGGGCCCAAGGAAGGCAGGAGACTCT-3' (SEQ ID NO:108) p4 5'-CTATGAAATTAACCCTCACTAAAGGGACTAGGGGGTGGGAATGAAAAG-3' (SEQ ID NO:109)

(3) DNA38113-1230 (PR03271 pi 5'-GGATTCTAATACGACTCACTATAGGGCCCCCCTGAGCTCTCCCGTGTA-3 ' (SEQ ID NO: 110) p2 5'-CTATGAAATTAACCCTCACTAAAGGGAAGGCTCGCCACTGGTCGTAGA-3 ' (SEQ ID NO: 111)

(4) DNA35917-1207 (PRQ2431 pi 5'-GGATTCTAATACGACTCACTATAGGGCAAGGAGCCGGGACCCAGGAGA-3' (SEQ ID NO: 112) p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGGGGGCCCTTGGTGCTGAGT-3' (SEQ ID NO:113)

G. Results

In situ analysis was performed on a variety of DNA sequences disclosed herein. The results from these analyses are as follows.

(1) DNA44804-1248 (PR03571

Low to moderate level expression at sites of bone formation in fetal tissues and in the malignant cells of an osteosarcoma. Possible signal in placenta and cord. All other tissues negative.

Fetal tissues examined (E12-E16 weeksl include: liver, kidney, adrenals, lungs, heart, great vessels, oesophagus, stomach, spleen, gonad, brain, spinal cord and body wall.

Adult human tissues examined: Uver, kidney, stomach, spleen, adrenal, pancreas, lung, colonic carcinoma, renal cell carcinoma and osteosarcoma. Acetominophen induced liver injury and hepatic cirrhosis.

Chimp Tissues examined: thyroid, parathyroid, lymph node, nerve, tongue, thymus, adrenal, gastric mucosa and salivary gland. Rhesus Monkey: cerebrum and cerebellum.

(2) DNA52722-1229 (PRQ7151

Generalized high signal seen over many tissues - highest signal seen over placenta, osteoblasts, injured renal tubules, injured liver, colorectal liver metastasis and gall bladder. Fetal tissues examined (E12-E16 weeksl include: placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb.

Adult human tissues examined: liver, kidney, adrenal, myocardium, aorta, spleen, lung, skin, chondrosarcoma, eye, stomach, colon, colonic carcinoma, prostate, bladder mucosa and gall bladder. Acetominophen induced liver injury and hepatic cirrhosis .

Rhesus Tissues examined: cerebral cortex (rm), hippocampus (rm)

Chimp Tissues examined: thyroid, parathyroid, lymph node, nerve, tongue, thymus, adrenal, gastric mucosa and salivary gland.

(3) DNA38113-1230 (PRQ3271

High level of expression observed in developing mouse and human fetal lung. Normal human adult lung, including bronchial epithelium, was negative. Expression in submucosa of human fetal trachea, possibly in smooth muscle ceUs. Expression also observed in non-trophoblastic cells of uncertain histogenesis in die human placenta. In the mouse expression was observed in die developing snout and in the developing tongue. All otiier tissues were negative. Speculated function: Probable role in bronchial development.

Fetal tissues examined (E12-E16 weeksl include: placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb. Adult tissues examined: Uver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm), hippocampus (rm), cerebellum (rm), penis, eye, bladder, stomach, gastric carcinoma, colon, colonic carcinoma, thyroid (chimp), parathyroid (chimp) ovary (chimp) and chondrosarcoma.

(4) DNA35917-1207 (PRQ2431

CorneUa de Lange syndrome (CdLS) is a congenital syndrome. That means it is present from birth. CdLS is a disorder that causes a delay in physical, intellectual, and langauge development. The vast majority of children with CdLS are mentally retarded, with the degree of mental retardation ranging from mild to severe. Reported IQ's from 30 to 85. The average IQ is 53. The head and facial features include small head size, thin eyebrows which often meet at the midline, long eyelashes, short upturned nose, thin downturned lips, lowset ears and high arched palate or cleft palate. Other characteristics may include language delay, even in die most mildly affected, delayed growth and smaU stature, low pitched cry, small hands and feet, incurved fifth fingers, simian creases, and excessive body hair. Diagnosis depends on die presence of a combination of these characteristics. Many of these characteristics appear in varying degrees. In some cases these characteristics may not be present or be so mild tiiat they will be recognized only when observed by a trained geneticist or other person familar with the syndrome. Although much is known about CdLS, recent reports suggest that there is much more to be learned. In this study additional sections of human fetal face, head, limbs and mouse embryos were examined. No expression was seen in any of the mouse tissues. Expression was only seen with the antisense probe.

Expression was observed adjacent to developing limb and facial bones in the perosteal mesenchyme. The expression was highly specific and was often adjacent to areas undergoing vascularization. The distribution is consistent with die observed skeletal abnormaUties in the Cornelia de Lange syndrome. Expression was also observed in the developing temporal and occipital lobes of the fetal brain, but was not observed elsewhere. In addition, expression was seen in the ganglia of die developing inner ear; the significance of this finding is unclear.

Though these data do not provide functional information, the distribution is consistent with the sites that are known to be affected most severely in this syndrome. Additionally, faint expression was observed at die cleavage line in the developing synovial joint forming between the femoral head and acetabulum (hip joint). If this pattern of expression were observed at sites of joint formation elsewhere, it might explain die facial and limb abnormalities observed in die Cornelia de Lange syndrome.

EXAMPLE 31 : Activity of PRQ243 mRNA in Xenopus Oocvtes In order to demonstrate that the human chordin clone (DNA35917-1207) encoding PR0243 is functional and acts in a manner predicted by the Xenopus chordin and Drosophila sog genes, supercoiled plasmid DNA from DNA35917-1207 was prepared by Qiagen and used for injection into Xenopus laevis embryos. Micro-injection of Xenopus chordin mRNA into ventrovegetal blastomeres induces secondary (twinned) axes (Sasai et al., Cell 79:779- 790 (1994)) and Drosophila sog also induces a secondary axis when ectopically expresed on the ventral side of the Xenopus embryo (HoUey et al., Nature 376:249-253 (1995) and Schmidt et al., Development 121:4319-4328 (1995)). The abiUty of sog to function in Xenopus ooctyes suggests that the processes involved in dorsoventral patterning have been conserved during evolution. Methods Manipulation of Xenopus embryos: Adult female frogs were boosted with 200 LU. pregnant mare serum 3 days before use and with 800 U. of human chorionic gonadotropin the night before injection. Fresh oocytes were squeezed out from female frogs the next morning and in vitro fertilization of oocytes was performed by mixing oocytes witii minced testis from sacrificed male frogs. Developing embryos were maintained and staged according to Nieuwkoop and Faber, Normal Table of Xenopus laevis, N.-H. P. Co., ed. (Amsterdam, 1967). Fertilized eggs were dejellied with 2% cysteine (pH 7.8) for 10 minutes, washed once with distilled water and transferred to 0.1 x MBS with 5% Ficoll. Fertilized eggs were lined on injection trays in 0.1 X MBS with 5% FicoU. Two-ceU stage developing Xenopus embryos were injected with 200 pg of pRK5 containing wild type chordin (DNA35917-1207) or 200 pg of pRK5 without an insert as a control. Injected embryos were kept on trays for another 6 hours, after which they were transferred to 0.1 X MBS witii 50 mg/ml gentamycin until reaching Nieukwkoop stage 37-38. Results:

Injection of human chordin cDNA into single blastomeres resulted in die ventralization of the tadpole. The ventraUzation of the tadpole is visible in die shortening and kinking of the tail and the expansion of the cement gland. The ability of human chordin to function as a ventralizing agent in Xenopus shows that the protein encoded by DNA35917-1207 is functional and influences dorsal-ventral patterning in frogs and suggests that the processes involved in dorsoventral patterning have been conserved during evolution, with mechanisms in common between humans, flies and frogs.

Deposit of Material The fotiowing materials have been deposited with the American Type Culture Collection, 12301 Parklawn

Drive, Rockville, MD, USA (ATCC):

Material ATCC Pep. No. Deposit Date

DNA34392-1170 ATCC 209526 December 10, 1997

DNA35917-1207 ATCC 209508 December 3, 1997 DNA39976-1215 ATCC 209524 December 10, 1997 DNA35595-1228 ATCC 209528 December 10, 1997 DNA38113-1230 ATCC 209530 December 10, 1997 DNA34436-1238 ATCC 209523 December 10, 1997 DNA40592-1242 ATCC 209492 November 21, 1997 DNA44176-1244 ATCC 209532 December 10, 1997 DNA44192-1246 ATCC 209531 December 10, 1997 DNA39518-1247 ATCC 209529 December 10, 1997 DNA44804-1248 ATCC 209527 December 10, 1997 DNA52722-1229 ATCC 209570 January 7, 1998 DNA41234-1242 ATCC 209618 February 5, 1998 DNA45410-1250 ATCC 209621 February 5, 1998 DNA46777-1253 ATCC 209619 February 5, 1998

These deposit were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and die 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. and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the pubUc upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled tiiereto according to 35 USC § 122 and the Commissioner's rules pursuant thereto (including 37 CFR § 1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed tiiat if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be constraed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the constract deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionaUy equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that me written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be constraed as limiting he scope of the claims to the specific illustrations tiiat it represents. Indeed, various modifications of the invention in addition to those shown and described herein wdl become apparent to those skilled in die art from the foregoing description and fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. Isolated nucleic acid having at least 80% sequence identity to a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 9 (SEQ ID NO: 15), Figure 11 (SEQ ID NO:19), Figure 13 (SEQ ID NO:24), Figure 15 (SEQ ID NO:30), Figure 17 (SEQ ID NO:32), Figure 19 (SEQ ID NO:37), Figure 21 (SEQ ID NO:42), Figure 23 (SEQ ID NO:50), Figure 25 (SEQ ID NO:55), Figure 27 (SEQ ID NO:61), Figure 29 (SEQ ID NO:69), Figure 31 (SEQ ID NO:76), Figure 35 (SEQ ID NO:86), Figure 37 (SEQ ID NO:91), and Figure 39 (SEQ ID NO:99).

2. The nucleic acid of Claim 1, wherein said nucleotide sequence comprises a nucleotide sequence selected from the group consisting of the sequence shown in Figure 1 (SEQ ID NO:l), Figure 3 (SEQ ID NO:6),

Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO 18), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:29), Figure 16 (SEQ ID NO:31), Figure 18 (SEQ ID NO 36), Figure 20 (SEQ ID NO:41), Figure 22 (SEQ ID NO:49), Figure 24 (SEQ ID NO:54), Figure 26 (SEQ ID NO 60), Figure 28 (SEQ ID NO:68), Figure 30 (SEQ ID NO:75),

Figure 34 (SEQ ID NO:85), Figure 36 (SEQ ID NO:90), and Figure 38 (SEQ ID NO:98), or the complement thereof.

3. The nucleic acid of Claim 1, wherein said nucleotide sequence comprises a nucleotide sequence selected from the group consisting of the full-length coding sequence of the sequence shown in Figure 1 (SEQ ID NO: l), Figure 3 (SEQ ID NO:6), Figure 8 (SEQ ID NO: 14), Figure 10 (SEQ ID NO: 18), Figure 12 (SEQ ID NO:23), Figure 14 (SEQ ID NO:29), Figure 16 (SEQ ID NO:31), Figure 18 (SEQ ID NO:36), Figure 20 (SEQ ID NO:41), Figure 22 (SEQ ID NO:49), Figure 24 (SEQ ID NO:54), Figure 26 (SEQ ID NO:60), Figure 28 (SEQ ID NO:68), Figure 30 (SEQ ID NO:75), Figure 34 (SEQ ID NO:85), Figure 36 (SEQ ID NO:90), and Figure 38 (SEQ ID NO: 98), or the complement thereof.

4. Isolated nucleic acid which comprises the full-length coding sequence of the DNA deposited under accession number ATCC 209526, ATCC 209508, ATCC 209524, ATCC 209528, ATCC 209530, ATCC 209523,

ATCC 209492, ATCC 209532, ATCC 209531, ATCC 209529, ATCC 209527, ATCC 209570, ATCC 209618, ATCC 209621 or ATCC 209619.

5. A vector comprising the nucleic acid of Claim 1.

6. The vector of Claim 5 operably linked to control sequences recognized by a host cell transformed with the vector.

7. A host cell comprising the vector of Claim 5.

8. The host cell of Claim 7 wherein said cell is a CHO cell.

9. The host cell of Claim 7 wherein said cell is an E. coli.

10. The host cell of Claim 7 wherein said cell is a yeast cell.

11. A process for producing a PRO polypeptides comprising culturing the host cell of Claim 7 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 an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 9 (SEQ ID NO:15), Figure 11 (SEQ ID NO:19), Figure 13 (SEQ ID NO:24), Figure 15 (SEQ ID NO:30), Figure 17 (SEQ ID NO:32), Figure 19 (SEQ ID NO:37), Figure 21 (SEQ ID NO:42), Figure 23 (SEQ ID NO:50), Figure 25 (SEQ ID NO:55), Figure 27 (SEQ ID NO:61), Figure 29 (SEQ ID NO:69), Figure 31 (SEQ ID NO:76), Figure 35 (SEQ ID NO:86), Figure 37 (SEQ ID NO:91), and Figure 39 (SEQ ID NO:99).

13. Isolated PRO polypeptide having at least 80% sequence identity to die amino acid sequence encoded by the nucleotide deposited under accession number ATCC 209526, ATCC 209508, ATCC 209524, ATCC 209528, ATCC 209530, ATCC 209523, ATCC 209492, ATCC 209532, ATCC 209531, ATCC 209529, ATCC 209527, ATCC 209570, ATCC 209618, ATCC 209621 or ATCC 209619.

14. A chimeric molecule comprising a polypeptide according to Claim 12 fused to a heterologous amino acid sequence.

15. The chimeric molecule of Claim 14 wherein said heterologous amino acid sequence is an epitope tag sequence.

16. The chimeric molecule of Claim 14 wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.

17. An antibody which specifically binds to a PRO polypeptide according to Claim 12.

18. The antibody of Claim 17 wherein said antibody is a monoclonal antibody.