EP1112361A2

EP1112361A2 - Promotion or inhibition of angiogenesis and cardiovascularization

Info

Publication number: EP1112361A2
Application number: EP99946891A
Authority: EP
Inventors: Sherman Fong; Mary E. Gerritsen; Audrey Goddard; Austin L. Gurney; Kenneth J. Hillan; P. Mickey Williams; William I. Wood
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 1998-09-14
Filing date: 1999-09-13
Publication date: 2001-07-04
Also published as: JP2003524599A; CA2341767A1; ZA200101453B; MXPA01002546A; KR20010085792A; WO2000015792A3; AU5920099A; IL141537A0; WO2000015792A2

Abstract

Compositions and methods are disclosed for stimulating or inhibiting angiogenesis and/or cardiovascularization in mammals, including humans. Pharmaceutical compositions are based on polypeptides or antagonists thereto that have been identified for one or more of these uses. Disorders that can be diagnosed, prevented, or treated by the compositions herein included trauma such as wounds, various cancers, and disorders of the vessels including atherosclerosis and cardiac hypertrophy.

Description

PROMOTION OR INHIBITION OF ANGIOGENESIS AND CARDIOVASCULARIZATION

Background of the Invention Field of the Invention

The present invention relates to compositions and methods useful for promoting or inhibiting angiogenesis and/or cardiovasculaπzation in mammals in need of such biological effect This includes the diagnosis and treatment of cardiovascular disorders as well as oncological disorders

Description of Background

A Cardiac Disorders and Factors

Heart failure affects approximately five million Americans, and new cases of heart failure number about 400,000 each year It is the single most frequent cause of hospitaiization for people age 65 and older in the United States Recent advances in the management of acute cardiac diseases, including acute myocardial infarction, are resulting in an expanding patient population that will eventually develop chronic heart failure From 1979 to 1995 hospitalizations for congestive heart failure (CHF) rose from 377 000 to 872 000 (a 130 percent increase) and CHF deaths increased 1 16 percent

CHF is a syndrome characterized by left ventricular dysfunction, reduced exercise tolerance, impaired quality of life and markedly shortened life expectanc> The sine qua non of heart failure is an inability of the heart to pump blood at a rate sufficient to meet the metabolic needs of the bodv's tissues (in other words, there is insufficient cardiac output)

At least four major compensatory mechanisms are activated in the setting of heart failure to boost cardiac output, including peripheral vasoconstπction increased heart rate increased cardiac contractility and increased plasma volume These effects are mediated primarily by the sympathetic nervous system and the renm-angiotensm system See, Eichhorn, American Journal of Medicine. 104 163-169 ( 1998) Increased output from the sympathetic nervous system increases vascular tone, heart rate, and contractility Angiotensin II elevates blood pressure by 1 ) directly stimulating vascular smooth muscle contraction, 2) promoting plasma volume expansion by stimulating aldosterone and antidiuretic hormone secretion, 3) stimulating sympathetic-mediated vascular tone and 4) catalyzing the degradation of bradykinin which has vasodilatory and natπuretic activity See, review by Brown and Vaughan Circulation. 97 141 1 -1420 (1998) As noted below angiotensin II may also have directly deleterious effects on the heart by promoting myocyte necrosis (impairing systolic function) and intracar ac fibrosis (impairing diasto c and in some cases systolic function) See. Weber, Circulation, 96 4065-4082 ( 1998)

A consistent feature of congestive heart failure (CHF) is cardiac hypertrophy, an enlargement of the heart that is activated by both mechanical and hormonal stimuli and enables the heart to adapt to demands for increased cardiac output Morgan and Baker Circulation. 83 13-25 ( 1991) This hypertrophic response is frequently associated with a variety of distinct pathological conditions such as hypertension aortic stenosis myocardial infarction, cardiomyopathv valvular regurgitation. and intracardiac shunt all of which result in chronic hemodynamic overload

Hypertrophy is generally defined as an increase in size of an organ or structure independent of natural growth that does not involve tumor formation Hypertrophy of the heart is due either to an increase in the mass of __ the individual cells (myocytes). or to an increase in the number of cells making up the tissue (hyperplasia), or both While the enlargement of an embryonic heart is largely dependent on an increase in myocyte number (which continues until shortly after birth), post-natal cardiac myocytes lose their proliferative capacity Further growth occurs through hypertrophy of the individual cells

Adult myocyte hypertrophy is initially beneficial as a short term response to impaired cardiac function by permitting a decrease in the load on individual muscle fibers With severe long-standing overload, however, the hvpertrophied cells begin to deteriorate and die Katz, "Heart Failure" in Katz A M ed Physiology of the Heart (New York Raven Press, 1992) pp 638-668 Cardiac hypertrophy is a significant risk factor for both mortality and morbidity in the clinical course of heart failure Katz, Trends Cardiovasc Med . 5 37-44 (1995) For further details of the causes and pathology of cardiac hypertrophy see, e g , Heart Disease. A Textbook of Cardiovascular Medicine. Braunwald, E ed (W B Saunders Co , 1988), Chapter 14, "Pathophysiology of Heart Failure "

On a cellular level, the heart is composed of myocytes and surrounding support cells, geneπcally called non-myocytes While non-myocytes are primarily fibroblast/mesenchymal cells, they also include endothe al and smooth muscle cells Indeed, although myocytes make up most of the adult myocardial mass, they represent only about 30% of the total cell numbers present in heart In response to hormonal, physiological, hemodynamic, and pathological stimuli, adult ventricular muscle cells can adapt to increased workloads through the activation of a hypertrophic process This response is characterized by an increase in myocyte cell size and contractile protein content of individual cardiac muscle cells, without concomitant cell division and activation of embryonic genes, including the gene for atπal natπuretic peptide (ANP) Chien et al . FASEB J . 5 3037-3046 (1991). Chien et al , Annu Rev Physiol , 55 77-95 (1993) An increment in myocardial mass as a result of an increase in myocyte size that is associated with an accumulation of interstitial collagen within the extracellular matrix and around intramyocardial coronary arteries has been described in left ventricular hypertrophy secondary to pressure overload in humans Caspaπ et al , Cardiovasc Res ϋ 554-558 (1977), Schwarz et al . Am J Cardiol , 42 895-903

(1978), Hess et al , Circulation. 63 360-371 (1981), Pearlman et al , Lab Invest . 46 158-164 ( 1982)

It has also been suggested that paracπne factors produced by non-myocyte supporting cells may additionally be involved in the development of cardiac hypertrophy, and various non-myocyte derived hypertrophic factors, such as. leukocyte inhibitory factor (LIF) and endothe n, have been identified Metcalf, Growth Factors, 7 169- 173 (1992). Kurzrock e/ a/ . Endocrine Reviews. 12 208-217 (199H. Inoue et al . Proc Nati Acad Sci

USA, 86 2863-2867 (1989), Yanagisawa and Masaki, Trends Pharm Sci . K) 374-378 (1989), U S Patent No 5,573,762 (issued November 12, 1996) Further exemplary factors that have been identified as potential mediators ofcardiac hypertrophy include cardιotrophιn- 1 (CT-l ) (Pennιca e; α/ Proc Nat Acad Sci USA 92 1 142-1 146 (1995)), catecholamines, adrenocorticosteroids, angiotensin, and prostaglandins

At present, the treatment of cardiac hypertrophy vanes depending on the underlying cardiac disease Catecholamines, adrenocorticosteroids, angiotensin. prostaglandins, LIF, endothehn (including endothelιn-1. -2, and -3 and big endothehn), and CT-1 are among the factors identified as potential mediators of hypertrophy For example, beta-adrenergic receptor blocking drugs (beta-blockers, e g , propranolol. timolol. tertalolol, carteolol, nadolol, betaxolol, penbutolol. acetobutolol. atenolol. metoprolol. carvedilol, etc ) and verapamil have been used — extensively in the treatment of hypertrophic cardiomyopathy The beneficial effects of beta-blockers on symptoms (eg , chest pain) and exercise tolerance are largely due to a decrease in the heart rate with a consequent prolongation of diastole and increased passive ventricular filling Thompson et al , Br Heart J . 44 488-98 (1980),

Harrison et al , Circulation, 29 84-98 (1964) Verapamil has been described to improve ventricular filling and probably reducing myocardial ischemia Bonow et al , Circulation 72 853-64 (1985)

Nifedipine and diltiazem have also been used occasionally in the treatment of hypertrophic cardiomyopathy Lorell et al , Circulation 65 499-507 (1982). Betocchi et al . Am J Cardiol , 78 451-457 (1996) However, because of its potent vasodilating properties, nifedipine may be harmful, especially m patients with outflow obstruction Disopyramide has been used to relieve symptoms by virtue of its negative inotropic properties Pollick, N Engl J Med , 307 997-999 ( 1982) In many patients, however, the initial benefits decrease with time Wigle et al , Circulation, 92 1680- 1692 ( 1995) Antihypertensive drug therapy has been reported to have beneficial effects on cardiac hypertrophy associated with elevated blood pressure Examples of drugs used in antihypertensive therapy, alone or in combination, are calcium antagonists, e g , nitrendipine, adrenergic receptor blocking agents, e g , those listed above, angiotensin converting enzyme (ACE) inhibitors such as quinapπl, captopπl, enalapπl, ramipπl, benazepπl, fosmopπl, and lisinopπl, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamιde,and indapamide, and calcium channel blockers e , diltiazem, nifedipine, verapamil. and nicardipine For example, treatment of hypertension with diltiazem and captopπl showed a decrease in left ventricular muscle mass, but the Doppler indices of diastohc function did not normalize Szlachcic et al . Am J Cardiol , 63 198-201 (1989), Shahi et al , Lancet, 336 458-461 (1990) These findings were interpreted to indicate that excessive amounts of interstitial collagen may remain after regression of left ventricular hypertrophy Rossi et al , Am Heart J . 124 700-709 (1992) Rossi et al , supra, investigated the effect of captopπl on the prevention and regression of myocardial cell hypertrophy and interstitial fibrosis in pressure overload cardiac hypertrophy, in experimental rats

Agents that increase cardiac contractility directly (lontropic agents) were initially thought to benefit patients with heart failure because they improved cardiac output in the short term However, all positive inotropic agents except digoxigenin have been found to result in increased long-term mortality, in spite of short-term improvements in cardiac performance Massie, Curr Op in Cardiology. J_2 209-217 ( 1997), Reddy et al , Ourr

Opin Cardiol . \2 233-241 ( 1997) Beta-adrenergic receptor blockers have recently been advocated for use in heart failure Evidence from clinical trials suggests that improvements in cardiac function can be achieved without increased mortality, though documented improvements patient survival have not yet been demonstrated See also U S Pat Nos 5 935,924 5,624,806. 5,661.122. and 5.610.134 and WO 95/28173 regarding the use of cardiotropin- 1 or antagonists thereof or growth hormone and/or insulin-like growth factor-I in the treatment of CHF Another treatment modality is heart transplantation, but this is limited by the availability of donor hearts Endothehn is a vasoconstπcting peptide comprising 21 amino acids, isolated from swine arterial endothe al culture supernatant and structurally determined Yanagisawa et al , Nature, 332 41 1-415 ( 1988) Endothehn was later found to exhibit various actions, and endothehn antibodies as endothehn antagonists have- proven effective in the treatment of myocardial infarction, renal failure, and other diseases Since endothehn is present in live bodies and exhibits vasoconstπcting action, it is expected to be an endogenous factor involved m the regulation of the circulatory system, and may be associated with hypertension, cardiovascular diseases such as myocardial infarction, and renal diseases such as acute renal failure Endothehn antagonists are described, for example, in U S Pat No 5,773,414. JP Pat Publ 3130299/1991. EP 457.195, EP 460,679, and EP 552.489 A new endothehn B receptor for identifying endothehn receptor antagonists is described in U S Pat No 5,773,223

Current therapy for heart failure is primarily directed to using angiotensm-converting enzyme (ACE) inhibitors, such as captopπl, and diuretics These drugs improve hemodynamic profile and exercise tolerance and reduce the incidence of morbidity and mortality in patients with CHF Kramer et al , Circulation. 67(4)- 807-816 (1983), Captopπl Multicenter Research Group, J A C C , 2(4) 755-763 ( 1983), The CONSENSUS Trial Study Group. N Engl J Med . 316(23) 1429- 1435 ( 1987). The SOLVD Investigators. N Engl J Med .325(5) 293-302 (1991) Further, they are useful in treating hypertension, left ventricular dysfunction, atherosclerotic vascular disease, and diabetic nephropathy Brown and Vaughan, supra However, despite proven efficacy, response to

ACE inhibitors has been limited For example, while prolonging survival in the setting of heart failure. ACE inhibitors appear to slow the progression towards end-stage heart failure, and substantial numbers of patients on ACE inhibitors have functional class III heart failure

Moreover, improvement of functional capacity and exercise time is only small and mortality, although reduced, continues to be high The CONSENSUS Trial Study Group. N Engl J Med .316(23) 1429- 1453 (1987),

The SOLVD Investigators, N Engl J Med . 325(5) 293-302 ( 1991 ). Cohn et al , N Engl J Med . 325(5) 303-310 ( 1991), The Captopπl-Digoxin Multicenter Research Group, JAMA. 259(4) 539-544 ( 1988) Hence, ACE inhibitors consistently appear unable to relieve symptoms in more than 60% of heart failure patients and reduce mortality of heart failure only by approximately 15-20% For further adverse effects, see Brown and Vaughan, supra

An alternative to ACE inhibitors is represented by specific ATI receptor antagonists Clinical studies are planned to compare the efficacy of these two modalities in the treatment of cardiovascular and renal disease However, animal model data suggests that the ACE/Ang II pathway, while clearly involved in cardiac hypertrophy, is not the only, or even the primary pathway active in this role Mouse genetic "knockout" models have been made to test individual components of the pathway In one such model, the primary cardiac receptor for Ang II, AT-sub

IA, has been genetically deleted, these mice do not develop hypertrophy when Ang II is given experimentally (confirming the basic success of the model in eliminating hypertrophy secondary to Ang II) However, when the aorta is constricted in these animals (a model ot hypertensive cardiac stress) the hearts still become hypertrophic This suggests that alternative signaling pathways, not depending on this receptor (AT sub 1 A) are activated in hypertension ACE inhibitors would presumably not be able to inhibit these pathways See. Harada et al , Circulation. 97 1952- 1959 (1998) See also. Homcv Circulation. 97 1890-1892 (1998) regarding the enigma associated with the process and mechanism of cardiac hypertrophy

About 750 000 patients suffer from acute mvocardial infarction (AMI) annually and approximately one-fourth of all deaths in the United States are due to AMI In recent years, thrombolytic agents, e g , ^~~ streptokinase. urokinase, and in particular tissue plasminogen activator (t-PA) have significantly increased the survival of patients who suffered myocardial infarction When administered as a continuous intravenous infusion over 1 5 to 4 hours, t-PA produces coronary patency at 90 minutes in 69% to 90% of the treated patients Topol et al , Am J Cardiol 6J, 723-728 (1988), Neuhaus et al . J Am Coll Cardiol . V2 581-587 (1988), Neuhaus et al . J Am Coli Cardiol 14 1566- 1569 ( 1989) The highest patency rates have been reported with high dose or accelerated dosing regimens Topol. J Am Coll Cardiol , 15 922-924 (1990) t-PA may also be administered as a single bolus, although due to its relatively short half-life, it is better suited for infusion therap\ Tebbe et al , Am J Cardiol . 64 448-453 ( 1989) A t-PA variant, specifically designed to have longer half-life and very high fibrin specificity, TNK t-PA (a T103N I 17Q, KHRR(296-299)AAAAt-PA variant, Keyt etal Proc Nati Acad Sci USA, 9J_. 3670-3674 ( 1994)) is particularly suitable for bolus administration However, despite all these advances, the long-term prognosis of patient survival depends greatly on the post-infarction monitoring and treatment of the patients, which should include monitoring and treatment of cardiac hypertrophy

B Growth Factors

Various naturally occurring polypeptides reportedly induce the proliferation of endothelial cells Among those polypeptides are the basic and acidic fibroblast growth factors (FGF) (Burgess and Maciag Annual Rev Biochem 58 575 (1989)), platelet-derived endotheiialcell growth factor (PD-ECGF) (Ishιkawa e/α/ Nature.338 557 (1989)). and vascular endothelial growth factor (VEGF) Leimg et al Science 246 1306 ( 1989) Ferrara and

Henzel. Biochem Biophys Res Commun 161 851 (1989), Tischer et al , Biochem Biophvs Res Commun , 165 1 198 (1989), EP 471 754B granted July 31 , 1996

Media conditioned by cells transfected with the human VEGF (hVEGF) cDN A promoted the proliferation of capillary endothelial cells, whereas control cells did not Leung et al , Science, 246 1306 (1989) Several additional cDNAs were identified in human cDNA libraries that encode 121-, 189-, and 206-amιno acid isoforms of hVEGF (also collectively referred to as hVEGF-related proteins) The 121-amιno acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 1 16 and 159 in hVEGF The 189-amιno acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 1 16 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF) The 206-amιno acid protein differs from hVEGFby virtue of an insertion of 41 amino acids at residue 1 16 in hVEGF Houck et al . Mol Endocπn . 5 1806

(1991 ), Ferrara e/ α/ . J Cell Biochem . 47 21 1 ( 1991 ), Ferrara et al , Endocrine Reviews, 13 18 ( 1992). Keck et al , Science, 246 1309 ( 1989), Connolly et al , J Biol Chem , 264 20017 (1989), EP 370,989 published May 30. 1990

It is now well established that angiogenesis which involves the formation of new blood vessels from preexisting endothelium. is implicated in the pathogenesis of a variety of disorders These include solid tumors and metastasis, atherosclerosis, retrolental fibropiasia. hemangiomas. chronic inflammation, intraocular neovascular syndromes such as prohferative retinopathies, e , diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis Folkman etal J Biol Chem .267 10931-10934 (1992). Klagsbrun e/α/ Annu Rev Physiol . 53 217- - 239 (1991 ), and Garner A, "Vascular diseases" In Pathobiology of Ocular Disease A Dynamic Approach, Garner A. K ntworth GK, eds . 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710 In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasm, and for providing nourishment to the growing solid tumor Folkman et al . Nature. 339 58 (1989) The neovasculaπzation allows the tumor cells to acquire a growth advantage and prohferative autonomy compared to the normal cells Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors Weidner er α/ N Engl J Med. 324 1-6 (1991), Horak ef α/ Lancet, 340 1 120-1 124 (1992), Macchiaπni et al Lancet 340 145-146 (1992)

The search for positive regulators of angiogenesis has yielded many candidates, including aFGF, bFGF, TGF- α, TGF-β, HGF, TNF-cc. angiogenin, IL-8, etc Folkman et al J B C , supra and Klagsbrun et al , supra The negative regulators so far identified include thrombospondin (Good et αl , Proc Nati Acad Sci USA , 87 6624- 6628 (1990)), the 16-kilodalton N-terminal fragment of prolactin (Clapp et αl Endocrinology, 133 1292-1299 (1993)), angiostatin (O'Reilly er a/ , Cell, 79 315-328 (1994)), and endostatin O'Reilly et l Cell, 88 277-285

( 1996)

Work done over the last several years has established the key role of VEGF, not only in stimulating vascular endothelial cell proliferation, but also in inducing vascular permeability and angiogenesis Ferrara et αl Endocr Rev , 18 4-25 (1997) The finding that the loss of even a single VEGF allele results in embryonic lethality points to an irreplaceable role played bv this factor in the development and differentiation of the vascular system

Furthermore, VEGF has been shown to be a key mediator of neovasculaπzation associated with tumors and intraocular disorders Ferrara et αl , Endocr Rev , supr The VEGF mRNA is overexpressed by the majority of human tumors examined Berkman ef α/ J Clin Invest , 91 153-159 (1993). Brown et al . Human Pathol , 26 86-91 (1995), Brown et al Cancer Res . 53 4727-4735 (1993), Mattern e/ al Brit J Cancer, 73 931-934 (1996), Dvorak et al , Am J Pathol . 146 1029-1039 (1995)

Also, the concentration levels of VEGF in eye fluids are highly correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies Aiello et al , N_ Engl J Med , 331 1480-1487 (1994) Furthermore, recent studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by AMD Lopez et al Invest Ophthalmol Vis Sci . 37 855-868 (1996)

Anti- VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in nude mice (Kim e/ α/ Nature. 362 841-844 ( 1993), Warren et al J Clin Invest . 95 1789- 1797 (1995). Borgstrom et al Cancer Res , 56 4032-4039 ( 1996) Melnvk et al Cancer Res 56 921 -924 ( 1996)) and also inhibit intraocular angiogenesis in models of lschemic retinal disorders Adamis et al Arch Ophthalmol . 1 14 66-71 (1996) Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of solid tumors and various intraocular neovascular disorders Such antibodies are described, for example, in EP 817,648 published January 14 1998 and in PCT/US 98/06724 filed April 3. 1998

There exist several other growth factors and mitogens, including transforming oncogenes. that are capable of rapidly inducing a complex set of genes to be expressed by certain cells Lau and Nathans Molecular Aspects ot — Cellular Regulation, 6 165-202 ( 1991 ) These genes, which have been named immediate-early- or early-response genes, are transcriptionally activated within minutes after contact with a growth factor or mitogen. independent of de novo protein synthesis A group of these intermediate-early genes encodes secreted, extracellular proteins that are needed for coordination of complex biological processes such as differentiation and proliferation, regeneration, and wound healing Ryseck et al . Cell Growth Differ . 2 235-233 (1991)

Highly-related proteins that belong to this group include ceflO (Simmons et al , Proc Nati Acad Sci USA, 86 1 178-1 182 ( 1989)). cvr 61 which is rapidlv activated bv serum- or platelet-derived growth factor (PDGF) (O'Brien et al . Mol Cell Biol Jj) 3569-3577 ( 1990). human connective tissue growth factor (CTGF) (Bradham et al Cell Biol . 1 14 1285-1294 ( 1991 )) which is secreted by human vascular endothelial cells in high levels after activation with transforming growth factor beta (TGF-β), exhibits PDGF-hke biological and immunological activities, and competes with PDGF for a particular cell surface receptor, fisp-12 tRyseck et al , Cell Growth Differ , 2 235-233 (1991 )), human vascular IBP-like growth factor (VIGF) (WO 96/17931) and nov. normally arrested in adult kidney cells, which was found to be overexpressed in myeloblastosιs-assocιated-vιrus-type-1- mduced nephroblastomas Joloit et al , Mol Cell Biol . 12_. 10-21 (1992)

The expression of these immediate-early genes acts as "third messengers" in the cascade of events triggered by growth factors It is also thought that they are needed to integrate and coordinate complex biological processes, such as differentiation and wound healing in which cell proliferation is a common event As additional mitogens, insulin-like growth factor binding proteins (IGFBPs) have been shown, in complex with insulin-like growth factor (IGF), to stimulate increased binding of IGF to fibroblast and smooth muscle cell surface receptors Clemmons et al . J Clin Invest . 77 1548 (1986) Inhibitory effects of IGFBP on various IGF actions in vitro include stimulation of glucose transport by adipocytes, sulfate incorporation by chondrocytes, and thymidine incorporation in fibroblast Zapf et al , J Clin Invest . 63 1077 ( 1979) In addition, inhibitory effects of IGFBPs on growth factor-mediated mitogen activity in normal cells have been shown

C Need for Further Treatments

In view of the role of vascular endothelial cell growth and angiogenesis in manv diseases and disorders, it is desirable to have a means of reducing or inhibiting one or more of the biological effects causing these processes It is also desirable to have a means of assaying for the presence of pathogenic polypeptides in normal and diseased conditions, and especially cancer Further, in a specific aspect, as there is no generally applicable therapy for the treatment of cardiac hypertrophy, the identification of factors that can prevent or reduce cardiac myocyte hypertrophy is of primary importance in the development ot new therapeutic strategies to inhibit pathophysiological cardiac growth While there are several treatment modalities for various cardiovascular and oncologic disorders, there is still a need for additional therapeutic approaches

Summary ot the Invention

Accordingly the present invention concerns compositions and methods for promoting or inhibiting angiogenesis and/or cardiovascularization in mammals The present invention is based on the identification of __ proteins that test positive in various cardiovascular assays that test promotion or inhibition of certain biological activities Accordingly, the proteins are believed to be useful drugs for the diagnosis and/or treatment (including prevention) of disorders where such effects are desired, such as the promotion or inhibition of angiogenesis, inhibition or stimulation of vascular endothelial cell growth, stimulation of growth or proliferation of vascular endothelial cells, inhibition of tumor growth, inhibition of angiogenesis-dependent tissue growth, stimulation of angiogenesis-dependent tissue growth, inhibition of cardiac hypertrophy and stimulation of cardiac hypertrophy, e g , for the treatment of congestive heart failure In one embodiment, the present invention provides a composition comprising a PRO230 PR0216 or PRO302 polypeptide in admixture with a pharmaceutically acceptable carrier In one aspect, the composition comprises a therapeutically effective amount of the polypeptide In another aspect, the composition comprises a further active ingredient, namely, a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent Preferably, the composition is sterile The PRO230, PR0216 or PRO302 polypeptide may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability Preserved liquid pharmaceutical formulations might contain multiple doses of PRO230, PR0216 or PRO302 polypeptide, and might, therefore, be suitable for repeated use

In a further embodiment, the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of a PRO230, PR0216 or PRO302 polypeptide with a pharmaceutically acceptable carrier

In another embodiment, the present invention provides a composition comprising an agonist or antagonist of a PRO230. PR0216 or PRO302 polypeptide in admixture with a pharmaceutically acceptable carrier In one aspect, the composition comprises a therapeutically effective amount of the agonist or antagonist In another aspect, the composition comprises a further active ingredient, namely, a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent Preferably, the composition is sterile The

PRO230, PR0216 or PRO302 polypeptide agonist or antagonist may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability Preserved liquid pharmaceutical formulations might contain multiple doses of a PRO230 PR0216 or PRO302 polypeptide agonist or antagonist, and might, therefore, be suitable for repeated use In a further embodiment, the present invention provides a method for preparing such a composition usefuifor the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of a PRO230, PR0216 or PRO302 polypeptide agonist or antagonist with a pharmaceutically acceptable carrier

In yet another embodiment the present invention concerns a composition comprising an antι-PRO230. anti- PR0216 or antι-PRO302 antibody in admixture with a pharmaceutically acceptable carrier In one aspect, the composition comprises a therapeutically effective amount of the antιbod\ In another aspect, the composition comprises a further active ingredient, namelv. a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent Preferably, the composition is sterile The composition may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended ^~~ storage stability Preserved liquid pharmaceutical formulations might contain multiple doses of the antι-PRO230, antι-PR0216 or antι-PRO302 antibody, and might, therefore be suitable for repeated use In preferred embodiments, the antibody is a monoclonal antibody, an antibody fragment, a humanized antibody, or a single- chain antibody

In a further embodiment, the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of an antι-PRO230. antι-PR0216 or antι-PRO302 antibodv with a pharmaceuticallv acceptable carrier

In a still further aspect, the present invention provides an article of manufacture comprising

(a) a composition of matter comprising a therapeutically effective dosage of a PRO230, PR0216 or PRO302 polypeptide,

(b) a container containing said composition; and (c) a label affixed to said container, or a package insert included in said pharmaceutical product referring to the use of said PRO230, PR0216 or PRO302 polypeptide in the treatment of a cardiovascular, endothelial or angiogenic disorder

In another aspect, the present invention provides an article of manufacture comprising

(a) a composition of matter comprising a therapeutically effective dosage of a PRO230. PR0216 or PRO302 polypeptide agonist or antagonist

(b) a container containing said composition, and

(c) a label affixed to said container, or a package insert included in said pharmaceutical product referring to the use of said PRO230. PR0216 or PRO302 polypeptide agonist or antagonist in the treatment of a cardiovascular, endothelial or angiogenic disorder In yet another aspect, the present invention provides an article of maufacture comprising

(a) a composition of matter comprising a therapeutically effective dosage of an antι-PRO230. antι-PR0216 or antι-PR0302 antibody.

(b) a container containing said composition, and

(c) a label affixed to said container, or a package insert included in said pharmaceutical product referring to the use of said antι-PRO230. antι-PR0216 or antι-PRO302 antibody in the treatment of a cardiovascular, endothelial or angiogenic disorder

In another embodiment, the present invention provides a method for identifying an agonist of a PRO230, PR0216 or PRO302 polypeptide comprising

(a) contacting cells and a test compound to be screened under conditions suitable for the induction of a cellular response normally induced by a PRO230, PR0216 or PRO302 polypeptide, and

(b) determining the induction of said cellular response to determine if the test compound is an effective agonist, wherein the induction of said cellular response is indicative of said test compound being an effective agonist

In another embodiment, the present invention provides a method for identifying an agonist of a PRO230. — PR0216 or PRO302 polypeptide comprising

(a) contacting cells and a test compound to be screened under conditions suitable for the stimulation of cell proliferation by a PRO230, PR0216 or PRO302 polypeptide. and

(b) measuring the proliferation of said cells to determine if the test compound is an effective agonist, wherein the stimulation of cell proliferation is indicative of said test compound being an effective agonist

In another embodiment, the invention provides a method for identifying a compound that inhibits the activity of a PRO230 PR0216 or PRO302 polypeptide comprising contacting a test compound with a PRO230. PR0216 or PRO302 polypeptide under conditions and for a time sufficient to allow the test compound and polypeptide to interact and determining whether the activity of the PRO230, PR0216 or PRO302 polypeptide is inhibited In a specific preferred aspect, either the test compound or the PRO230, PR0216 or PRO302 polypeptide is immobilized on a solid support In another preferred aspect, the non-immobihzed component carries a detectable label In a preferred aspect, this method comprises the steps of (a) contacting cells and a test compound to be screened in the presence of PRO230, PR0216 or PRO302 polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO230, PR0216 or PRO302 polypeptide, and

(b) determining the induction of said cellular response to determine if the test compound is an effective antagonist In another preferred aspect, this process comprises the steps of

(a) contacting cells and a test compound to be screened in the presence of PRO230, PR0216 or PRO302 polypeptide under conditions suitable for the stimulation of cell proliferation by a PRO230, PR0216 or PRO302 polypeptide, and

(b) measuring the proliferation of the cells to determine if the test compound is an effective antagonist In another embodiment, the invention provides a method for identifying a compound that inhibits the expression of a PRO230, PR0216 or PRO302 polypeptide in cells that normally expresses the polypeptide. wherein the method comprises contacting the ceils with a test compound and determining whether the expression of the PRO230, PR0216 or PRO302 polypeptide is inhibited In a preferred aspect, this method comprises the steps of

(a) contacting cells and a test compound to be screened under conditions suitable for allowing expression of the PRO230. PR0216 or PRO302 polypeptide, and

(b) determining the inhibition of expression of said polypeptide

In a still further embodiment, the invention provides a compound that inhibits the expression of a PRO230, PR0216 or PRO302 polypeptide. such as a compound that is identified b\ the methods set forth above

Another aspect of the present invention is directed to an agonist or an antagonist of a PRO230. PR0216 or PRO302 polypeptide which may optionally be identified by the methods described above

One type of antagonist of a PRO230 PR0216 or PRO302 polypeptide that inhibits one or more of the functions or activities of the PRO230. PR0216 or PRO302 polypeptide is an antibody Hence, in another aspect, the invention provides an isolated antibody that binds a PRO230. PR0216 or PRO302 polypeptide In a preferred aspect, the antibody is a monoclonal antibody, which preferably has non-human complementarity-determining — region (CDR) residues and human framework-region (FR) residues The antibody may be labeled and may be immobilized on a solid support In a further aspect, the antibody is an antibody fragment a single-chain antibody, or a humanized antibody Preferably, the antibody specifically binds to the polypeptide

In a still further aspect, the present invention provides a method for diagnosing a disease or susceptibility to a disease which is related to a mutation in a PRO230. PR0216 or PRO302 polypeptide-encoding nucleic acid sequence compπsing

(a) isolating a nucleic acid sequence encoding a PRO230 PR0216 or PRO302 polypeptide from a sample derived from a host, and

(b) determining the presence or absence of said mutation in the PRO230, PR0216 or PRO302 polypeptide nucleic acid sequence, wherein the presence or absence of said mutation is indicative of the presence of said disease or susceptibility to said disease

In a still further aspect, the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises analyzing the level of expression of a gene encoding a PRO230,

PR0216 or PRO302 polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher oi lower expression level in the test sample as compared to the control sample is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in said mammal The expression of a gene encoding a PRO230. PR0216 or PRO302 polypeptide may optionally be accomplished bv measuring the level of mRNA or polypeptide in the test sample as compared to the control sample

In a still further aspect the present invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises detecting the presence or absence of a PRO230, PR0216 or PRO302 polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of said PRO230, PR0216 or PRO302 polypeptide in said test sample is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in said mammal

In a still further embodiment, the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal comprising (a) contacting an antι-PRO230. antι-PR0216 or antι-PRO302 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the PRO230. PR0216 or PRO302 polypeptide in the test sample, wherein the formation of said complex is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in the mammal The detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells ot the same cell tvpe A larger or smaller quantity of complexes formed in the test sample indicates the presence of a cardiovascular, endothelial or angiogenic dysfunction in the mammal from which the test tissue cells were obtained The antibody preferably carries a detectable label Complex formation can be monitored for example, by light microscopy flow cytometry, fluoπmetry, or other techniques known in the art The test sample is usually obtained from an individual suspected to have a cardiovascular, endothelial or angiogenic disorder

In another embodiment, the invention provides a method for determining the presence ot a PRO230, PR0216 — or PRO302 polypeptide in a sample comprising exposing a sample suspected of containing the PRO230, PR0216 or PRO302 polypeptide to an antι-PRO230, antι-PR0216 or antι-PRO302 antibody and determining binding of said antibody to a component of said sample In a specific aspect, the sample comprises a cell suspected of containing the PRO230, PR0216 or PRO302 polypeptide and the antibody binds to the cell The antibody is preferably detectably labeled and/or bound to a solid support

In further aspects, the invention provides a cardiovascular, endothelial or angiogenic disorder diagnostic kit compπsingan antι-PRO230. antι-PR0216 or antι-PRO302 antibody and a carrier in suitable packaging Preferably, such kit further comprises instructions for using said antibody to detect the presence of the PRO230. PR0216 or

PRO302 polypeptide Preferably, the carrier is a buffer, for example Preferably, the cardiovascular, endothelial or angiogenic disorder is cancer

In a further embodiment, the invention provides an article of manufacture, comprising a container, a label on the container, and a composition comprising a PRO230, PR0216 or PRO302 polypeptide contained within the container, wherein the label on the container indicates that the composition can be used for treating cardiovascular, endothelial or angiogenic disorders

In a further embodiment, the invention provides an article of manufacture, comprising a container, a label on the container, and a composition comprising a PRO230, PR0216 or PRO302 polypeptide agonist or antagonist contained within the container, wherein the label on the container indicates that the composition can be used for treating cardiovascular, endothelial or angiogenic disorders In a further embodiment, the invention provides an article of manufacture, comprising a container, a label on the container, and a composition comprising an antι-PRO230. antι-PR0216 or antι-PRO302 antibody contained within the container, wherein the label on the container indicates that the composition can be used for treating cardiovascular endothelial or angiogenic disorders

In yet another embodiment, the present invention provides a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of a PRO230, PR0216 or PRO302 polypeptide Preferably, the disorder is cardiac hypeπrophy trauma such as wounds or burns, or a type of cancer In a further aspect, the mammal is further exposed to angioplastv or a drug that treats cardiovascular, endothelial or angiogenic disorders such as ACE inhibitors or chemotherapeutic agents if the cardiovascular, endothelial or angiogenic disorder is a type of cancer Preferably, the mammal is human, preferably one who is at risk of developing cardiac hypertrophy and more preferably has suffered myocardial infarction

In another preferred aspect, the cardiac hypertrophy is characterized by the presence of an elevated level of PGF Alternatively, the cardiac hypertrophy may be induced by myocardial infarction, wherein preferably the __ administration of PRO230. PR0216 or PRO302 polypeptide is initiated within 48 hours, more preferably within 24 hours, following myocardial infarction In anotherpreferredembodiment.the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy and said PRO230, PR0216 or PRO302 polypeptide is administered together with a cardiovascular, endothelial or angiogenic agent The preferred cardiovascular, endothelial or angiogenic agent for this purpose is selected from the group consisting of an antihypertensive drug, an ACE inhibitor, an endothehn receptor antagonist and a thrombolytic agent If a thrombolvtic agent is administered, preferablv the PRO230, PR0216 or PRO302 polypeptide is administered following administration of such agent More preferably, the thrombolytic agent is recombinant human tissue plasmmogen activator

In another preferred aspect, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy and the PRO230, PR0216 or PRO302 polypeptide is administered following primary angioplasty for the treatment of acute myocardial infarction, preferably wherein the mammal is further exposed to angioplasty or a cardiovascular, endothelial, or angiogenic agent

In another preferred embodiment, the cardiovascular, endothelial or angiogenic disorder is a cancer and the PRO230, PR0216 or PRO302 polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent or a cytotoxic agent

In a further embodiment the invention concerns a method for treating a cardiovascular, endothelial or angiogenic disorder m a mammal comprising administering to the mammal an effective amount of an agonist of a PRO230, PR0216 or PRO302 polypeptide Preferably, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration Also preferred is where the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the agonist In a further embodiment, the invention concerns a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of an antagonist of a PRO230. PR0216 or PRO302 polypeptide Preferably, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration Also preferred is where the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the antagonist

In a further embodiment, the invention concerns a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of an antι-PRO230, antι-PR0216orantι-PRO302antιbody Preferably the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration Also preferred is where the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the antibody In still further embodiments, the invention provides a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal that suffers therefrom comprising administering to the mammal a nucleic acid molecule that codes for either (a) a PRO230. PR0216 or PRO302 polypeptide. (b) an agonist of a PRO230. - PR0216 or PRO302 polypeptide or (c) an antagonist of a PRO230, PR0216 or PRO302 polypeptide. wherein said agonist or antagonist may be an antι-PRO230. antι-PR0216 or antι-PRO302 antibody In a preferred embodiment, the mammal is human In another preferred embodiment, the gene is administered via ex vivo gene therapy In a further preferred embodiment, the gene is comprised within a vector, more preferably an adenoviral, adeno-associated viral, lentiviral. or retroviral vector

In yet another aspect, the invention provides a recombinant retroviral particle comprising a retroviral vector consisting essentially of a promoter nucleic acid encoding (a) a PRO230. PR0216 or PRO302 polypeptide. (b) an agonist polypeptide of a PRO230. PR0216 or PRO302 polypeptide. or (c) an antagonist polypeptide of a PRO230.

PR0216 or PRO302 polypeptide, and a signal sequence for cellular secretion of the polypeptide. wherein the retroviral vector is in association with retroviral structural proteins Preferably, the signal sequence is from a mammal, such as from a native PRO230, PR0216 or PRO302 polypeptide

In a still further embodiment, the invention supplies an ex vivo producer cell comprising a nucleic acid construct that expresses retroviral structural proteins and also comprises a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO230, PR0216 or PRO302 polypeptide, (b) an agonist polypeptide of a PRO230, PR0216 or PRO302 polypeptide or (c) an antagonist polypeptide of a PRO230. PR0216 or PRO302 polypeptide. and a signal sequence for cellular secretion of the polypeptide. wherein said producer cell packages the retroviral vector in association with the structural proteins to produce recombinant retroviral particles In yet another embodiment, the invention provides a method for inhibiting endothelial cell growth in a mammal comprising administering to the mammal (a) a PR0216 polypeptide. (b) an agonist of a PR0216 polypeptide, (c) an antagonist of a PR0216 polypeptide. or (d) an antι-PR0216 antibody, wherein endothelial cell growth m said mammal is inhibited Preferably, the mammal is human and the endothelial cell growth is associated with a tumor or a retinal disorder In yet another embodiment, the invention provides a method for stimulating endothelial cell growth in a mammal comprising administering to the mammal (a) a PRO230 polypeptide (b) an agonist of a PRO230 polypeptide (c) an antagonist of a PRO230 polypeptide. or (d) an antι-PRO230 antibody, wherein endothelial cell growth in said mammal is stimulated Preferably, the mammal is human in yet another embodiment, the invention provides a method for inhibiting angiogenesis induced by a PRO302 polypeptide in a mammal comprising administering a therapeutically effective amount of an antι-PRO302 antibody to the mammal Preferably, the mammal is a human, and more preferably the mammal has a tumor or a retinal disorder Brief Description of the Drawings Figures 1 A through I B show a nucleotide sequence (SEQ ID NO 1 ) ot a native sequence PRO230 cDNA. wherein SEQ ID NO 1 is a clone designated herein as DNA33223-1 136"

Figure 2 shows the amino acid sequence (SEQ ID NO 2) derived from the coding sequence of SEQ ID NO 1 shown in Figures I A through I B

Figure 3 shows a nucleotide sequence (SEQ ID NO 6) of a native sequence PR0216 cD A wherein SEQ ID NO 6 is a clone designated herein as DNA33087" __

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 a nucleotide sequence (SEQ ID NO 1 1 ) of a native sequence PRO302 cDNA wherein SEQ

ID NO 1 1 is a clone designated herein as "DNA40370- 1217"

Figure 6 shows the amino acid sequence (SEQ ID NO 12) derived from the coding sequence of SEQ ID NO 11 shown in Figure 5

Figures 7 A through 7D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Figures 7A-B) and % nucleic acid sequence identity (Figures 7C-D) using the

ALIGN-2 sequence comparison computer program, wherein 'PRO^"' represents the amino acid sequence of a hypothetιcalPRO230, PR0216 or PRO302 polypeptide of interest, "Comparison Protein^"" represents the ammo acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared '"PRO-DNA" represents a hypothetical PRO230-, PR0216- or PRO302-encodιng nucleic acid sequence of interest. "'Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA ' nucleic acid molecule of interest is being compared, "X", " Y", and "Z" each represent different hypothetical amino acid residues and "N", "L" and "V" each represent different hypothetical nucleotides

Figures 8A through 8Q provide the complete source code for the ALIGN-2 sequence comparison computer program This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN- 2 sequence comparison computer program

Detailed Description of the Invention I Definitions

The phrases "cardiovascular, endothelial and angiogenic disorder", "cardiovascular endothelial and angiogenic dysfunction", "'cardiovascular, endothelial or angiogenic disorder" and "cardiovascular, endothelial or angiogenic disfunction" are used interchangeably and refer in part to systemic disorders that affect vessels, such as diabetes mel tus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins, and/or lymphatics This would include indications that stimulate angiogenesis and/or cardiovascularization. and those that inhibit angiogenesis and/or cardiovascularization Such disorders include, for example, arterial disease, such as atherosclerosis, hypertension, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, and arterial restenosis. venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema, and other vascular disorders such as peripheral vascular disease, cancer such as vascular tumors, e g , hemangioma (capillary and cavernous) glomus tumors telangiectasia bacillary angiomatosis hemangioendothelioma angiosarcoma haemangiopeπcytoma Kaposi s sarcoma, lymphangioma and lymphangiosarcoma, tumor angiogenesis. trauma such as wounds burns, and other injured tissue, implant fixation scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis This would also include angina, myocardial infarctions such as acute myocardial infarctions, cardiac hypertrophy, and heart failure such as CHF

"Hypertrophy^"", as used herein, is defined as an increase in mass of an organ or structure independent of__ natural growth that does not involve tumor formation Hypertrophy of an organ or tissue is due either to an increase in the mass of the individual ceils (true hypertrophy), or to an increase in the number of cells making up the tissue (hyperplasia), or both Certain organs, such as the heart, lose the ability to divide shortly after birth Accordingly,

"'cardiac hypertrophy" is defined as an increase in mass of the heart, which, in adults, is characterized by an increase in myocyte cell size and contractile protein content without concomitant cell division The character of the stress responsible for inciting the hypertrophy, (e g , increased preload, increased afterload, loss of myocytes. as in myocardial infarction or primary depression of contractility) appears to plav a critical role in determining the nature of the response The early stage of cardiac hypertrophy is usually characterized morphologically by increases m the size ot myofibπls and mitochondria, as well as by enlargement of mitochondria and nuclei At this stage, while muscle cells are larger than normal, cellular organization is largely preserved At a more advanced stage ofcardiac hypertrophy, there are preferential increases in the size or number of specific organelles, such as mitochondria, and new contractile elements are added in localized areas of the cells, in an irregular manner Cells subjected to long-standing hypertrophy show more obvious disruptions in cellular organization, including markedly enlarged nuclei with highly lobulated membranes, which displace adjacent myofibπls and cause breakdown of normal Z-band registration The phrase "cardiac hypertrophy" is used to include all stages of the progression of this condition, characterized by various degrees of structural damage of the heart muscle, regardless of the underlying cardiac disorder. Hence, the term also includes physiological conditions instrumental in the development ofcardiac hypertrophy, such as elevated blood pressure, aortic stenosis, or mvocardial infarction

"Heart failure refers to an abnormality of cardiac function where the heart does not pump blood at the rate needed for the requirements of metabolizing tissues The heart failure can be caused by a number of factors, including lschemic. congenital, rheumatic, or ldiopathic forms

"Congestive heart failure" (CHF) is a progressive pathologic state where the heart is increasingly unable to supply adequate cardiac output (the volume of blood pumped by the heart over time) to deliver the oxygenated blood to peripheral tissues As CHF progresses, structural and hemodynamic damages occur While these damages have a variety of manifestations, one characteristic symptom is ventricular hypertrophy CHF is a common end result of a number of various cardiac disorders

"Myocardial infarction generally results from atherosclerosis of the coronary arteries, often with superimposed coronary thrombosis It may be divided into two major types transmural infarcts. in which myocardial necrosis involves the full thickness of the ventricular wall, and subendocardial (nontransmuraL) infarcts, in which the necrosis involves the subendocardium, the intramural myocardium, or both, without extending all the way through the ventricular wall to the epicardium Vlyocardial infarction is known to cause both a change in hemodynamic effects and an alteration in structure in the damaged and healthy zones of the heart Thus, for example, myocardial infarction reduces the maximum cardiac output and the stroke volume of the heart Also associated with myocardial infarction is a stimulation of the DNA synthesis occurring in the interstice as well as an increase in the formation of collagen in the areas of the heart not affected

As a result of the increased stress or strain placed on the heart in prolonged hypertension due. for example, to the increased total peripheral resistance, cardiac hypertrophy has long been associated with "h\ pertension^" A__ characteristic of the ventricle that becomes hypertrophic as a result of chronic pressure overload is an impaired diastohc performance Fouad et al J Am Coll Cardiol 4 1500- 1506 ( 1984). Smith et al J Am Coll Cardiol . 5 869-874 ( 1985) A prolonged left ventricular relaxation has been detected in early essential hypertension, in spite of normal or supranormal systolic function Hartford et al . Hypertension, 6 329-338 ( 1984) However, there is no close parallelism between blood pressure levels and cardiac hypertrophy Although improvement in left ventricular function in response to antihypertensive therapy has been reported in humans, patients variously treated with a diuretic (hvdrochlorothiazide), a β-blocker (propranolol). or a calcium channel blocker (diltiazem), have shown reversal of left ventricular hypertrophy, without improvement in diastohc function lnou\ e et al Am J

Cardiol . 53 1583-7 ( 1984)

Another complex cardiac disease associated with cardiac hypertrophy is ' hypertrophic cardiomyopathy^"' This condition is characterized by a great diversity of morphologic, functional, and clinical features (Maron et al N Engl J Med , 316 780-789 (1987), Spiπto et al N Engl J Med , 320 749-755 (1989), Louie and Edwards, Prog Cardiovasc Pis , 36 275-308 ( 1994), Wigle et al , Circulation, 92 1680- 1692 ( 1995)), the heterogeneity of which is accentuated by the fact that it afflicts patients of all ages Spiπto er α/ N Engl J Med . 336 775-785 ( 1997) The causative factors of hypertrophic cardiomyopathy are also diverse and little understood In general, mutations in genes encoding sarcomeπc proteins are associated with hypertrophic cardiomyopathv Recent data suggest that β-myosin heavv chain mutations may account for approximately 30 to 40 percent of cases of familial hypertrophic cardiomyopathy Watkins era/ N Engl J Med 326 1 108- 1 1 14 ( 1992). Schwartz etal Circulation

91_. 532-540 ( 1995), Marian and Roberts. Circulation. 92 1336- 1347 ( 1995), Thierfelder e/ al CeH. 77 701-712 (1994), Watkins et al Nat Gen , 1 1 434-437 ( 1995) Besides β-myosin heavv chain other locations of genetic mutations include cardiac troponin T, alpha topomyosin, cardiac myosin binding protein C. essential myosin light chain, and regulatory myosin light chain See. Malik and Watkins, Curr Opin Cardiol . 12. 295-302 (1997) Supravalvular "aortic stenosis^'' is an inherited vascular disorder characterized by narrowing of the ascending aorta, but other arteries, including the pulmonary arteries, may also be affected Untreated aortic stenosis may lead to increased intracardiac pressure resulting in myocardial hypertrophy and eventually heart failure and death The pathogenesis of this disorder is not fully understood, but hypertrophy and possibly hyperplasia of medial smooth muscle are prominent features of this disorder It has been reported that molecular variants of the elastm gene are involved in the development and pathogenesis of aortic stenosis U S Patent No 5.650,282 issued July 22, 1997

"Valvular regurgitation ^" occurs as a result of heart diseases resulting in disorders of the cardiac valves Various diseases, like rheumatic fever, can cause the shrinking or pulling apart of the valve orifice, while other diseases may result in endocarditis an inflammation of the endocardium or iin ing membrane of the atπoventπcular orifices and operation of the heart Defects such as the narrowing of the valve stenosis or the defective closing of the valve result in an accumulation ot blood in the heart cavity or regurgitation of blood past the valve If uncorrected, prolonged valvular stenosis or insufficiency may result in cardiac hypertrophy and associated damage to the heart muscle, which may eventually necessitate valve replacement

The treatment of all these, and other cardiovascular, endothelial and angiogenic disorders, which may or may not be accompanied by cardiac hypertrophy, is encompassed by the present invention __

The terms "cancer", "cancerous", and "malignant" refer to or describe the physiological condition in mammals that is typically characterized bv unregulated cell growth Examples of cancer include but are not limited to. carcinoma including adenocarcinoma. lymphoma, blastoma. melanoma, sarcoma, and leukemia More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin sandnon-Hodgkin s lymphoma, pancreatic cancer, g oblastoma. cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma. bladder cancer, breast cancer, colon cancer, colorectal cancer, endometπal carcinoma, salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer The preferred cancers for treatment herein are breast, colon, lung, melanoma, ovarian, and others involving vascular tumors as noted above

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells The term is intended to include radioactive isotopes ( g , ^|J'I, ^{l S}I, ⁹⁰Y, and ^l86Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer Examples of chemotherapeutic agents include alkylating agents, folic acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics pyπmidine analogs, 5-fluorouracιl. cisplatin, puπne nucleosides. amines, amino acids. tπazol nucleosides or corticosteroids Specific examples include Adπamycm. Doxorubicin. 5-Fluorouracιl,

Cytosine arabinoside ("Ara-C"), Cyclophosphamide. Thiotepa, Busulfan. Cytoxin, Taxol, Toxotere Methotrexate Cisplatin. Melphalan. Vinblastine. Bleomycin. Etoposide. Ifosfamide. Mitomycin C. Mitoxantrone, Vincreistine, Vmorelbine, Carboplat , Teniposide. Daunomycin, Carminomycin, Aminopteπn, Dactinomycin. Mitomycins. Esperamicms (see U S Pat No 4,675,187), Melphalan, and other related nitrogen mustards Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors, such as tamoxifen and onapπstone

A "growth-inhibitory agent" when used herein refers to a compound or composition that inhibits growth of a cell, such as an Wnt-overexpressing cancer cell, either in vitro or in vivo Thus, the growth-inhibitory agent is one which significantly reduces the percentage of malignant cells in S phase Examples of growth-inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G 1 arrest and M-phase arrest Classical M-phase blockers include the vincas (vincπstine and vinblastine). taxol, and topo II inhibitors such as doxorubicin, daunorubicin. etoposide, and bleomycin Those agents that arrest G 1 also spill over into S-phase arrest, for example. DNA alkylating agents such as tamoxifen, prednisone. dacarbazine, mechlorethamine. cisplatin. methotrexate. 5-fluorouracιl. and ara-C Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds , Chapter 1. entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al (WB Saunders Philadelphia, 1995), especially p 13 Additional examples include tumor necrosis factor (TNF). an antibody capable of inhibiting or neutralizing the angiogenic activity of acidic or basic FGF or hepatocvte growth factor (HGF), an antibody capable of inhibiting or neutralizing the coagulant activities of tissue factor, protein C. or protein S (see. WO 91/01753. published 21 February 1991 ), — or an antibody capable of binding to HER2 receptor (WO 89/06692), such as the 4D5 antibody (and functional equivalents thereof) (e g , WO 92/22653) "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a cardiovascular, endothelial. and angiogenic disorder The concept of treatment is used in the broadest sense, and specifically includes the prevention (prophylaxis), moderation, reduction, and curing of cardiovascular, endothelial. and angiogenic disorders of any stage Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a cardiovascular, endothelial. and angiogenic disorder such as hypertrophy Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented The disorder may result from any cause, including ldiopathic, cardiotrophic, or myotrophic causes, or ischemia or ischemic insults, such as myocardial infarction

"Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial effect, such as an anti-hypertrophic effect, for an extended period of time

"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 dogs, horses, cats, cows, sheep, pigs, etc Preferably, the mammal is human

Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order

The phrase "cardiovascular, endothelial or angiogenic agents" refers geneπcally to any drug that acts in treating cardiovascular, endothelial, and angiogenic disorders Examples of cardiovascular agents are those that promote vascular homeostasis by modulating blood pressure, heart rate, heart contractility, and endothelial and smooth muscle biology, all of which factors have a role in cardiovascular disease Specific examples of these include angiotensm-II receptor antagonists, endothehn receptor antagonists such as. for example, BOSENTAN™ and MOXONODIN™, interferon-gamma (IFN- γ), des-aspartate-angiotensin I. thrombolytic agents, e g , streptokinase, urokinase. t-PA, and a t-PA variant specifically designed to have longer half-life and very high fibrin specificity. TNK t-PA (a Tl 03N. N 1 17Q. KHRR(296-299)AAAA t-PA variant, Keyt et al , Proc Nati Acad Sci USA 91 , 3670-3674 (1994)), inotropic or hypertensive agents such as digoxigenin and β-adrenergic receptor blocking agents, e g , propranolol. timolol. tertalolol, carteolol, nadolol. betaxolol, penbutolol. acetobut&lol, atenolol, metoprolol, and carvedilol. angiotensin converting enzyme (ACE) inhibitors, e g , quinapπl, captopπl, enalapπl, ramipπl, benazepπl, fosinopπl. and lisinopπl, diuretics, e g , chlorothiazide, hydrochlorothiazide, hvdroflumethazide. methvlchlothiazide benzthiazide. dichloψhenamide acetazolamide. and indapamide. and calcium channel blockers, e g , diltiazem. nifedipine. verapamil. nicardipine One preferred category of this type is a therapeutic agent used for the treatment of cardiac hypertrophy or ot a physiological condition instrumental in the development ofcardiac hypertrophy such as elevated blood pressure, aortic stenosis, or myocardial infarction "Angiogenic agents" and "endothelial agents" are active agents that promote angiogenesis and/or endothelial cell growth, or. if applicable, vasculogenesis This would include factors that accelerate wound healing, such as growth hormone, insulin-like growth factor-I (IGF-I). VEGF, VIGF, PDGF, epidermal growth factor (EGF). CTGF _ and members of its family. FGF, and TGF-α and TGF-β

"Angiostatic agents" are active agents that inhibit angiogenesis or vasculogenesis or otherwise inhibit or prevent growth of cancer cells Examples include antibodies or other antagonists to angiogenic agents as defined above, such as antibodies to VEGF They additionally include cytotherapeutic agents such as cytotoxic agents, chemotherapeutic agents, growth-inhibitory agents, apoptotic agents, and other agents to treat cancer, such as anti-

HER-2, antι-CD20. and other bioactive and organic chemical agents

In a pharmacological sense, in the context ot the present invention, a "therapeutically effective amount" of an active agent such as a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto or an anti-

PRO230, antι-PR0216 or antι-PRO302 antibody, refers to an amount effective in the treatment of a cardiovascular, endothelial or angiogenic disorder in a mammal and can be determined empirically

As used herein, an "effective amount" of an active agent such as a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto or an antι-PRO230, antι-PR0216 or antι-PRO302 antibody, refers to an amount effective for carrying out a stated puφose, wherein such amounts may be determined empirically for the desired effect

Asusedherein.the terms "PRO230". " PR0216", "PRO302", "PRO230 polypeptide" "PR0216 polypeptide" or "PRO302 polypeptide" when used herein encompass native-sequence PRO230. PR0216 or PRO302 polypeptides and PRO230. PR0216 or PRO302 polypeptide variants (which are further defined herein) The PRO230. PR0216 or PRO302 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods

A "native sequence" PRO230, PR0216 or PRO302 polypeptide comprises a polypeptide having the same ammo acid sequence as a PRO230, PR0216 or PRO302 polypeptide derived from nature Such native sequence PRO230, PR0216 or PRO302 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means The term "native sequence" PRO230, PR0216 or PRO302 polypeptide specifically encompasses naturally-occurring truncated or secreted forms (e g , an extracellular domain sequence), naturally-occurπngvaπant forms (e g , alternatively spliced forms), and naturally-occurring allelic variants of the PRO230, PR0216 and PRO302 polypeptide In certain embodiments of the invention, the native-sequence PRO230, PR0216 or PRO302 polypeptide is a mature or full-length native sequence PRO230. PR0216 or PRO302 polypeptide comprising the amino acid sequence of Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7) or Figure 6 (SEQ ID NO 12). respectively Fragments of the respective native polypeptides herein include, but are not limited, to polypeptide variants from which the native N-termmal signal sequence has been fully or partially deleted or replaced by another sequence. and extracellulardomains of the respective native sequences, regardless whether such truncated (secreted) forms occur in nature Fragments are preterablv sufficient in length for the production ot an antibody specifically binding the corresponding native "PRO" polypeptide

PRO230 variant polypeptide means an active PRO230 polypeptide (other than a native sequence PRO230 polypeptide) as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2). (b) X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is anv amino acid residue from 17 to __ 26 of Figure 2 (SEQ ID NO 2) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2) "PR0216 variant polypeptide^" means an active PR0216 polypeptide (other than a native sequence PR0216 polypeptide) as defined below having at least about 80% amino acid sequence identity with the am o acid sequence of (a) residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO 7) or (b) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 7)

"PRO302 variant polypeptide^' means an active PRO302 polypeptide (other than a native sequence PRO302 polypeptide) as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12). (b) X to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12), wherein X is any amino acid residue from 21 to 30 of Figure 6 (SEQ ID NO 12) or (c) another specifically derived fragment of the amino acid sequence shown m Figure 6 (SEQ ID NO 12) Such PRO230, PR0216 or PRO302 variant polypeptides include, for instance, PRO230, PR0216 or PRO302 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C-termmus, as well as withm one or more internal domains, of the sequence of Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7) or Figure 6 (SEQ ID NO 12), respectively

Ordinarily, a PRO230 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% ammo acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% ammo acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any amino acid residue from 17 to 26 of Figure 2 (SEQ ID NO 2) or (c I another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2)

Ordinarily, a PR0216 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% ammo acid sequence identity, more preferably at least about

84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% am o acid __ sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% ammo acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% ammo acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 to 421 of the PR0216 polypeptide shown in Figure 4

(SEQ ID NO 7) or (b) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 7)

Ordinarily, a PRO302 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% ammo acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about

84% ammo acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% ammo acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preterablv at least about 92% ammo acid sequence identity, more preferably at least about 93% am o acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% ammo acid sequence identity with (a) residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12), (b) X to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12), wherein X is any amino acid residue from 21 to 30 of Figure 6 (SEQ ID NO 12) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO 12)

Variants do not encompass the native PRO230. PR0216 or PRO302 polypeptide sequence Ordinarily. PRO230. PR0216 and PRO302 variant polypeptides are at least about 10 amino acids in length, often at least about

20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 ammo acids in length, more often at least about 60 amino acids in

~)1 length, more often at least about 70 amino acids in length, more often at least about 80 ammo acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 300 amino acids in length, or more "Percent (%) amino acid sequence identity" with respect to the PRO230, PR0216 and PRO302 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the ammo acid residues in a PRO230. PR0216 or PRO302 sequence, after aligning the __ sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for puφoses 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, ALIGN-2 or Megahgn (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 For puφoses herein, however. % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2. wherein the complete source code for the ALIGN-

2 program is provided in Figures 8A-Q The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc , and the source code shown in Figures 8A-Q has been filed with user documentation in the U.S Copyright Office, Washington D C, 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech, Inc . South San Francisco, California or may be compiled from the source code provided in Figures 8A-Q The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary

For puφoses herein, the % amino acid sequence identity of a given amino acid sequence A to. with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to. with, or against a given amino acid sequence B) is calculated as follows

100 times the fraction X/Y

where X is the number of ammo acid residues scored as identical matches by the sequence alignment program

ALIGN-2 in that program s alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of ammo acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations. Figures 7A-B demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein^" to the am o acid sequence designated "PRO"

Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al . Nucleic Acids Res . 25 3389-3402 ( 1997)) The NCBI-BLAST2 sequence comparison program mav be downloaded from http //www ncbi nlm nih gov NCBI-BLAST2 uses several search parameters wherein all of those search parameters are set to default values including, for example, unmask = ves, strand = all, expected occurrences = 10. minimum low complexity length = 15/5, multi-pass e-value = 0 01 constant for multi-pass = 25 dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62 ~~

In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % ammo acid sequence identity of a given amino acid sequence A to. with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to. with, or against a given amino acid sequence B) is calculated as follows

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches bv the sequence alignment program

NCBI-BLAST2 in that program^'s alignment of A and B. and where Y is the total number of ammo acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A In addition % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzv ology, 266 460-480 (1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values, ; e . the adjustable parameters, are set with the following values overlap span = 1 , overlap fraction = 0 125, word threshold (T) = 1 1. and scoring matrix = BLOSUM62 For puφoses herein, a % ammo acid sequence identitv value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison ammo acid sequence of interest (/ e , the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of ammo acid residues of the PRO polypeptide of interest For example, in the statement "a polypeptide comprising an ammo acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B", the am o acid sequence

A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest

"PRO230 variant polynucleotide" or 'PRO230 variant nucleic acid sequence means a nucleic acid molecule which encodes an active PRO230 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 164 o the

PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes amino acids X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2). wherein X is any ammo acid residue from 17 to 26 of Figure 2 (SEQ ID NO 2) or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2) Ordinarily a PRO230 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity more preferably at least about 82% nucleic acid sequence identity more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about __ 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about

93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes amino acids X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any ammo acid residue from 17 to 26 of Figure 2 (SEQ ID NO 2) or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2) PRO230 polynucleotide variants do not encompass the native PRO230 nucleotide sequence

"PR0216 variant polynucleotide" or "PR0216 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0216 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO 7) or (b) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 7) Ordinarily a PR0216 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about

88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO 7) or (b) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 7) PR0216 polynucleotide variants do not encompass the native PR0216 nucleotide sequence "PRO302 variant polynucleotide" or "PRO302 variant nucleic acid sequence means a nucleic acid molecule which encodes an active PRO302 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 26 to 452 of the__ PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12), (b) a nucleic acid sequence which encodes amino acids X to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12), wherein X is any am o acid residue from 21 to 30 of Figure 6 (SEQ ID NO 12) or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO 12) Ordinarily, a PRO302 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12). (b) a nucleic acid sequence which encodes amino acids X to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12), wherein X is any amino acid residue from 21 to 30 of Figure 6 (SEQ ID NO 12) or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO 12) PRO302 polynucleotide variants do not encompass the native PRO302 nucleotide sequence Ordinarily, PRO230, PR0216 and PRO302 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more

"Percent (%) nucleic acid sequence identity" with respect to the PRO230. PR0216 and PRO302 polypeptide- encoding nucleic acid sequences identified herein is defined as the percentage ot nucleotides in a candidate sequence that are identical with the nucleotides in a PRO230. PR0216 or PRO302 polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity Alignment for puφoses 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, ALIGN-2 or Mega gn (DN ASTAR) 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 For puφoses herein, however. % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code forthe ALIGN-2 program is provided in Figures 8A-Q The ALIGN-2 sequence comparison computer program was authored by Genentech. Inc , and the source code shown in Figures 8A-Q has been filed with user documentation in the U S Copyright Office, Washington D C . 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech. Inc . South San Francisco, California or mav be compiled from the source code provided in Figures 8A- Q The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX

V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary

For puφoses herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches bv the sequence alignment program ALIGN-2 in that program s alignment of C and D. and where Z is the total number of nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D. the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C As examples of % nucleic acid sequence identity calculations. Figures 7C-D demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO- DNA"

Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al . Nucleic Acids Res . 25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih.gov NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5. multi-pass e- value = 0 01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62

In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with or against a given nucleic acid sequence D) is calculated as follows

100 times the fraction W/Z __

where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI- BLAST2 in that program s alignment of C and D, and where Z is the total number of nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C

In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST-2 computer program (Altschul et al Methods in Enzvmology. 266 460-480 (1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values. / e . the adjustable parameters, are set with the following values overlap span = 1 , overlap fraction = 0 125, word threshold (T) = 1 1. and scoring matrix = BLOSUM62 For puφoses herein, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide- encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide- encoding nucleic acid and the comparison nucleic acid molecule of interest (; e , the sequence against which the

PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide- encoding nucleic acid molecule of interest For example, in the statement "an isolated nucleic acid molecule compπsing a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest

In other embodiments, PRO230, PR0216 and PRO302 variant polynucleotides are nucleic acid molecules that encode an active PRO230, PR0216 or PRO302 polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO230, PR0216 or PRO302 polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7) or Figure 6 (SEQ

ID NO 12), respectively PRO230, PR0216 and PRO302 variant polypeptides may be those that are encoded by a PRO230, PR0216 or PRO302 variant polynucleotide

The term "positives", in the context of the ammo acid sequence identity comparisons performed as described above, includes ammo acid residues in the sequences compared that are not only identical, but also those that have similar properties Amino acid residues that score a positive value to an ammo acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 1 below) of the amino acid residue of interest For puφoses herein the % value of positives ot a given amino acid sequence A to with, or against a given am o acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to with or against a given ammo acid sequence B) is calculated as follows

100 times the fraction X/Y

where X is the number of amino acid residues scoring a positive value by the sequence alignment program ALIGN- — 2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B. the % positives of A to B will not equal the % positives of B to A

"Isolated", when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide. and mav include enzvmes. hormones, and other 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 cells, since at least one component of the PRO230, PR0216 or PRO302 natural environment will not be present Ordinarily, however, isolated polypeptide will be prepared by at least one purification step

An "isolated" nucleic acid molecule encoding a PRO230, PR0216 or PRO302 polypeptide or an "isolated" nucleic acid molecule encoding an antι-PRO230, antι-PR0216 or antι-PRO302 antibody 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 PRO230-. PR0216- or PRO302-encodmg nucleic acid or the natural source of the antι-PRO230-, antι-PR0216- or antι-PRO302-encodιng nucleic acid Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated An isolated PRO230-, PR0216- or PRO302-encodιng nucleic acid molecule or an isolated antι-PRO230-, antι-PR0216- or antι-PRO302-encodιng nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated nucleic acid molecules therefore are distinguished from the PRO230-. PR0216- or PRO302-encodιng nucleic acid molecule or from the antι-PRO230-, antι-PR0216- or antι-PRO302-encodιng nucleic acid molecule as it exists in natural cells However, an isolated nucleic acid molecule encoding a PRO230, PR0216 or PRO302 polypeptide or an isolated nucleic acid molecule encoding an antι-PRO230. antι-PR0216 or antι-PRO302 antibody includes PRO230-, PR0216- or PRO302-nucleιc acid molecules or antι-PRO230-. antι-PR0216- or antι-PRO302-nucleιc acid molecules contained in cells that ordinarily express PRO230, PR0216 or PRO302 polypeptides or arrti-

PRO230, antι-PR0216 or antι-PRO302 antibodies where, tor example, the nucleic acid molecule is in a chromosomal location different from that of natural cells The term 'control sequences" refers to DNA sequences necessary for the expression ot 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 πbosome 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 PRO230, PR0216 or PRO302 polypeptide if it is expressed as a preprotein that participates in the secretion of the — polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a πbosome 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 hgation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice

"Stπngencv" of hybridization reactions is readily determinable by one of ordinary skill in the art. and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hybπdizable sequence, the higher the relative temperature that can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so For additional details and explanation of stringency of hybridization reactions, see, Ausubel et al , Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995)

"Stringent conditions" or "high-stringency conditions", as defined herein, may be identified by those that ( 1 ) employ low ionic strength and high temperature for washing, for example, 0 015 M sodium chloπde/0 0015 M sodium cιtrate/0 1% sodium dodecyl sulfate at 50°C. (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0 1% bovine serum albumιn/0 1% Fιcoll/0 1% polyvιnylpyrrohdone/50mM sodium phosphate buffer at pH 6 5 with 750 niM sodium chloride 75 mM sodium citrate at 42°C, or (3) employ 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 (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" may be identified as described by Sambrook et al Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Press, 1989). and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength, and % SDS) less stringent than those described abαve

An example of moderately stringent conditions is overnight incubation at 37^CC in a solution comprising 20% formamide, 5 x SSC ( 150 mM NaCl, 15 mM tπsodium 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 aπisan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like

The modifier "epitope-tagged" w hen used herein refers to a chimeric polypeptide comprising a PRO230, PR0216 or PRO302 polypeptide fused to a "tag polypeptide" The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused The tag polypeptide preferably also is fairly unique so that the antibody does— not substantially cross-react with other epitopes Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)

"Active" or "activity" in the context of PRO230. PR0216 or PRO302 variants refers to form(s) of PRO230, PR0216 or PRO302 proteins that retain the biologic and/or lmmunologic activities of a native or naturally- occurring PRO230, PR0216 or PRO302 polypeptide

"Biological activity in the context of a molecule that antagonizes a PRO230 PR0216 or PRO302 polypeptide that can be identified by the screening assays disclosed herein ( , an organic or inorganic small molecule, peptide. etc ) is used to refer to the ability of such molecules to bind or complex with the PRO230, PR0216 or PRO302 polypeptide identified herein, or otherwise interfere with the interaction of the PRO230, PR0216 or PRO302 polypeptides with other cellular proteins or otherwise inhibits the transcription or translation of the PRO230, PR0216 or PRO302 polypeptide Particularly preferred biological activity includes cardiac hypertrophy, activity that acts on systemic disorders that affect vessels, such as diabetes mel tus. as well as diseases of the arteries, capillaries, veins, and/or lymphatics, and cancer

The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes one or more of the biological activities of a native PRO230, PR0216 or PRO302 polypeptide disclosed herein, for example, if applicable, its mitogenic or angiogenic activity Antagonists of a PRO230. PR0216 or PRO302 polypeptide may act bv interfering with the binding of a PRO230. PR0216 or PRO302 polypeptide to a cellular receptor, by incapacitating or killing cells that have been activated by a PRO230, PR0216 or PRO302 polypeptide, or by interfering with vascular endothelial cell activation after binding of a PRO230, PRO216 or PRO302 polypeptide to a cellular receptor All such points of intervention by a PRO230, PR0216 or PRO302 polypeptide antagonist shall be considered equivalent for puφoses of this invention The antagonists inhibit the mitogenic, angiogenic, or other biological activity of PRO230, PR0216 or PRO302 polypeptides, and thus are useful for the treatment of diseases or disorders characterized by undesirable excessive neovasculaπzation, including by way of example tumors and especially solid malignant tumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hypeφlasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation The antagonists also are useful for the treatment of diseases or disorders characterized by undesirable excessive vascular permeability, such as edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, lung inflammation, nephrotic syndrome, peπcardial effusion (such as that associated with pericarditis) and pleural effusion In a similar manner the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO230. PR0216 or PRO302 polypeptide disclosed herein Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibodv fragments, fragments, or amino acid sequence variants of native PRO230 PR0216 or PRO302 polypeptides. peptides. small organic molecules, etc

A "small molecule" is defined herein to have a molecular weight below about 500 daltons The term "PRO230, PR0216 or PRO302 polypeptide receptor" as used herein refers to a cellular receptor for __ a PRO230, PR0216 or PRO302 polypeptide. ordinarily a cell-surface receptor found on vascular endothelial cells, as well as variants thereof that retain the ability to bind a PRO230. PR0216 or PRO302 polypeptide "Antibodies" (Abs) and "immunoglobuhns" (Igs) are glycoproteins having the same structural characteristics

While antibodies exhibit binding specificity to a specific antigen, immunoglobuhns include both antibodies and other antibody-like molecules that lack antigen specificity Polypeptides of the latter kind are. for example, produced at low levels by the lymph system and at increased levels by myelomas The term "antibody" is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e g bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity

"Native antibodies" and "native immunoglobuhns" are usually heterotetrameπc glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced lntrachain disulfide bridges Each heavy chain has at one end a variable domain (V_H) followed by a number of constant domains Each light chain has a variable domain at one end (V_L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains

The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody to and for its particular antigen However, the variability is not evenly distributed throughout the variable domains of antibodies It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervaπable regions both in the light-chain and the heavy-chain variable domains The more highly conserved portions of variable domains are called the framework regions (FR) The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs which form loops connecting, and in some cases forming part of. the β-sheet structure The CDRs in each chain are held together in close proximity by the FR regions and. with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies See, Kabat et al , NIH Publ No 91 -3242 Vol I, pages 647-669 ( 1991 ) The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity "Antibody fragments" comprise a portion ot an intact antibody, preferably the antigen-binding or variable region of the intact antιbod\ Examples of antibody fragments include Fab. Fab', F(ab'),, and Fv fragments, diabodies. linear antibodies (Zapata et al . Protein Eng .8( 10) 1057-1062( 1995)), single-chain antibody molecules and multispecific antibodies formed from antibody fragments Papain digestion of antibodies producestwo identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, w hose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab')-. fragment that has two antigen-combining sites and is still capable of cross-linking __ antigen

"Fv" is the minimum antibodv fragment that contains a complete antigen-recognition and -binding site This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association

It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_L dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI ) of the heavy chain Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine resιdue(s) of the constant domains bear a free thiol group F(ab')_: antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them Other chemical couplings of antibody fragments are also known

The "light chains" of antibodies (immunoglobuhns) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of their constant domains

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobuhns can be assigned to different classes There are five major classes ot immunoglobuhns IgA. IgD, IgE IgG, and IgM and several of these may be further divided into subclasses (isotypes), e g , IgG 1 , IgG2, lgG3, IgG4. IgA, and IgA2 The heavy-chain constant domains that correspond to the different classes of immunoglobuhns are called α, δ, e γ, and μ, respectively The subunit structures and three-dimensional configurations of different classes of immunoglobuhns are well known The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies. / e . the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigenic site Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybπdoma culture, uncontaminated by other immunoglobuhns The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population ot antibodies, and is not to be construed as requiring production of the antibody by any particular method For example, the monoclonal antibodies to be used in accordance with the present invention mav be made by the hybπdoma method first described by Kohler et al Nature. 256 495 ( 1975), or may be made by recombinant DNA methods (see e g , U S PatentNo 4 816.567) The "monoclonal antibodies" may also be isolated from phage antibodv libraries using the techniques described in Clackson et al Nature. 352

624-628 (1991 ) and Marks et al J Mol Biol , 222 581-597 (1991), for example

The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobuhns) in which a— portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, w hile the remainder of the chaιn(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass as well as fragments of such antibodies, so long as they exhibit the desired biological activity U S Patent No 4 816.567, Morrison et al Proc Nati Acad Sci USA, 81 6851- 6855 (1984)

"Humanized" forms of non-human (e g , murine) antibodies are chimeric immunoglobuhns. immunoglobulin chains, or fragments thereof (such as Fv Fab. Fab' F(ab'), or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a 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 FR residues of the human immunoglobulin are replaced by corresponding non-human residues Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences These modifications are made to further refine and maximize antibody performance In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence The humanized antibody preferably also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin For further details, see Jones et al Nature 321 522-525 ( 1986). Reichmann et al Nature. 332 323-329 ( 1988) and Presta. Curr Op Struct Biol . 2 593-596 (1992) The humanized antibody includes a PRIMATIZED™ antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest

"Single-chain Fv" or "sFv" antibody fragments comprise the V_H and V_L domains of an antibody, wherein these domains are present in a single polypeptide chain Preferably, the Fv polypeptide further comprises a polypeptide linker between the V_H and V[ domains that enables the sFv to form the desired structure for antigen binding For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies Vol 1 13 Rosenburg and Moore, eds (Springer- Verlag New York, 1994), pp 269-315

The term "d bodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V„) connected to a light-chain variable domain (V_L) in the same polypeptide chain (V„ - V_L) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites Diabodies are described more fully in. for example, EP 404 097, WO 93'1 1 161. and Holhnger et al Proc Nati Acad Sci USA 90 6444-6448 ( 1993) An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or — nonproteinaceous solutes In preferred embodiments, the antibody will be purified ( 1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cup sequenator. or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain Isolated antibody includes the antibody m situ within recombinant cells, since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step The word "label" when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody The label may be detectable by itself (e g , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable

By "solid phase" is meant a non-aqueous matrix to which an antibody of the present invention can adhere Examples of solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysacchaπdes (e g , agarose), poiyacrylamides, polystyrene, polyvinyl alcohol and si cones In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275, 149 A "liposome" is a small vesicle composed of various types of lipids. phospholipids and/or surfactant that is useful for delivery of a drug (such as the PRO230. PR0216 or PRO302 poly peptide or antibodies thereto disclosed herein) to a mammal The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes

As used herein, the term "lmmunoadhesin" designates antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion ot an am o acid sequence with the desired binding specificity that is other than the antigen recognition and binding site of an antibody (; e . is "heterologous"), and an immunoglobulin constant domain sequence The adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence compπsing at least the binding site of a receptor or a ligand The immunoglobulin constant domain sequence in the lmmunoadhesin may be obtained from any immunoglobulin, such as IgG-1. IgG-

2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM II Compositions and Methods of the Invention

A PRO230 PRQ216 and PRO302 Variants

In addition to the full-length native sequence PRO230 PR0216 and PRO302 polypeptides described herein it is contemplated that PRO230. PR0216 and PRO302 variants can be prepared PRO230 PR0216 and PRO302 variants can be prepared by introducing appropriate nucleotide changes into the PRO230, PR0216 or PRO302 DNA, and/or by synthesis of the desired PRO230, PR0216 or PRO302 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO230 PR0216 or PRO302, such __ as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics

Variations in the native full-length sequence PRO230, PR0216 or PRO302 or in various domains of the PRO230, PR0216 or PRO302 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 PRO230. PR0216 or PRO302 that results in a change in the amino acid sequence of the PRO230 PR0216 or PRO302 as compared with the native sequence PRO230. PR0216 or PRO302 Optionally the variation is bv substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO230 PR0216 or PRO302 Guidance in determining which am o acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO230. PR0216 or PRO302 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 am o acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, ; e , conservative amino acid replacements Insertions or deletions may optionally be in the range of about 1 to 5 amino acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence

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 reference to amino acid classes are introduced and the products screened

Table 1

Original Exemplary Preferred

Residue Substitutions Substitutions

Ala (A) val. leu, lie val Arg (R) lys, gin, asn lys Asn (N) gin. his lys, arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro, ala ala Hιs (H) asn gin Ivs arg arg He (I) leu. val. met ala. phe norleucine leu

Leu (L) norleucine lie. val

Lys (K) arg gin, asn arg Met (M) leu, phe. lie leu Phe (F) leu. val. lie, ala tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Tφ (W) tyr, phe tyr Tyr (Y) tφ, phe. thr. ser phe Val (V) lie. leu. met. phe. ala, norleucine leu

Substantial modifications in function or immunological identity of the PRO230 PR0216 or PRO302 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hvdrophobicitv 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, lie,

(2) neutral hydrophilic cys, ser. thr,

(3) acidic asp, glu (4) basic asn, gin, his. lys, arg,

(5) residues that influence chain orientation gly, pro, and

(6) aromatic tφ, 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 the 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 . JO 6487 ( 1987)], cassette mutagenesis [Wells et al , Gene. 34 315 ( 1985)], restriction selection mutagenesis [Wells 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 PRO230, PR0216 or PRO302 variant

DNA

Scanning amino acid analysis can also be employed to identify one or more am o acids along a contiguous sequence Among the preferred scanning amino acids are relatively small, neutral ammo acids Such amino acids include alanine. glycine, serine. and cysteine Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells. Science. 244 1081 -1085 ( 1989)] Alanine is also typically preferred because it is the most common amino acid Further, it is frequently found in both buried and exposed

57 positions [Creighton, The Proteins ( W H Freeman & Co . N Y ) Chothia J Mol Biol 150 1 ( 1976)] If alanine substitution does not yield adequate amounts of variant, an lsoteπc amino acid can be used

B Modifications of PRO230 PRQ216 and PRO302 Covalent modifications of PRO230, PR0216 and PRO302 are included within the scope of this invention

One type of covalent modification includes reacting targeted amino acid residues of a PRO230. PR0216 or PRO302 polypeptide with an organic deπvatizing agent that is capable of reacting with selected side chains or the . N- or C- terminal residues of the PRO230, PR0216 or PRO302 Deπvatization with bifunctional agents is useful, for instance, for crosslinking PRO230, PR0216 or PRO302 to a water-insoluble support matrix or surface for use in the method for purifying antι-PRO230. antι-PR0216 or antι-PRO302 antibodies, and vice-versa Commonly used crosslinking agents include, e g , 1 , 1 -bιs(dιazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccmιmιde esters, forexample,esterswιth4-azιdosalιcyhcacιd. homobifunctional imidoesters, including disuccimmidyl esters such as 3,3'-dιthιobιs(succιnιmιdylpropιonate), bifunctional maleimides such as bιs-N-maleιmιdo- 1 8-octane and agents such as methyl-3-[(p-azιdophenyl)dιthιo]propιoιmιdate Other modifications include deamidation of glutaminyl and asparagmyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of prohne and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methy lation of the cc-amino groups of lysine, arginine, and histid e 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 the PRO230, PR0216 or PRO302 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended for puφoses herein to mean deleting one or more carbohydrate moieties found in native sequence PRO230, PR0216 or PRO302 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO230. PR0216 or PRO302 In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present

Addition of glycosylation sites to the PRO230. PR0216 or PRO302 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 PRO230. PR0216 or PRO302 (for O-linked glycosylation sites) The PRO230. PR0216 or PRO302 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO230, PR0216 or PRO302 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids

Another means of increasing the number of carbohydrate moieties on the PRO230. PR0216 or PRO302 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 1 1 September 1987 and in Ap n and Wπston CRC Cπt Rev Biochem , pp 259-306 ( 1981 ) Removal ot carbohydrate moieties present on the PRO230 PR0216 or PRO302 polypeptide mav be accomplished chemically or enzvmaticalK or by mutational substitution of codonsencoding for amino acid residues that serve as targets for glycosylation Chemical deglycosylation techniques are known in the art and described, for instance, bv Hakimudd et al Arch Biochem Biophvs . 259 52 ( 1987) and by Edge et al . Anal Biochem 1 18 131 (1981 ) Enzvmatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al , Meth Enzymol 138 350 ( 1987)

Another type of covalent modification of PRO230 PR0216 or PRO302 comprises linking the PRO230, — PR0216 or PRO302 polypeptide to one of a variety of nonprotemaceous polymers, e g , polyethylene glycol (PEG), 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 PRO230. PR0216 or PRO302 of the present invention may also be modified in a way to form a chimeric molecule comprising PRO230 PR0216 or PRO302 fused to another, heterologous polypeptide or amino acid sequence

In one embodiment, such a chimeric molecule comprises a fusion of the PRO230. PR0216 or PRO302 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 PRO230, PR0216 or PRO302 The presence of such epitope-tagged forms of the PRO230. PR0216 or PRO302 can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the PRO230, PR0216 or PRO302 to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag Various tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidine

(poly-His) or poly-histidine-glyαne (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, 6E 10. G4. B7 and 9E 10 antibodies thereto [Evan et al . Molecular and Cellular Biology. 5 3610-3616 ( 1985)], and the Heφes 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 , BioTechnology. 6 1204- 1210 ( 1988)], the KT3 epitope peptide

[Martin et al . Science. 255 192- 194 ( 1992)], an α-tubuhn 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 Nati Acad Sci USA, 87 6393-6397 ( 1990)1

In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO230, PR0216 or PRO302 with an immunoglobulin or a particular region of an immunoglobulin For a bivalent form of the chimeric molecule (also referred to as an 'lmmunoadhesin"), such a fusion could be to the Fc region of an IgG molecule The lg fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO230. PR0216 or PRO302 polypeptide in place of at least one variable region within an lg molecule In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge. CH2 and CH3 or the hinge, CHI, CH2 and CH3 regions of an IgG 1 molecule For the production of immunoglobulin fusions see also. -US

Patent No 5,428.130 issued June 27. 1995 C Preparation of the PRO230 PRQ216 and PRO302 polypeptides

The present invention provides newlv identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO230 PR0216 or PRO302 In particular. cDNAs encoding PRO230 PR0216 or PRO302 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed However, for sake of simplicity, in the present specification the protein encoded by DNA33223-1 136, DNA33087-1 158 or __ DNA40370-1217, as well as all further native homologues and variants included in the foregoing definition of PRO230, PR0216 or PRO302. will be referred to as "PRO230 PR0216 or PRO302 ', respectively, regardless of their origin or mode of preparation

The description below relates primarily to production of PRO230, PR0216 or PRO302 polypeptides by culturing cells transformed or transfected with a vector containing nucleic acid encoding PRO230, PR0216 or PRO302 polypeptides It is, of course, contemplated that alternative methods that are well known in the art may be employed to prepare PRO230. PR0216 or PRO302 For instance, the PRO230, PR0216 or PRO302 polypeptide sequence or portions thereof mav 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, C A, 1969), Merπfield, J Am Chem Soc , 85 2149-2154 (1963) In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, with an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions Various portions of PRO230, PR0216 or PRO302 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO230, PR0216 or PRO302 polypeptide

l Isolation of DNA Encoding PRO230, PRQ216 or PRO302 DNA encoding PRO230, PR0216 or PRO302 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA encoding PRO230, PR0216 or PRO302 and to express it at a detectable level Accordingly, DNAs encoding human PRO230, PR0216 or PRO302 can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in the Examples The gene encoding PRO230,

PR0216 or PRO302 polypeptide may also be obtained from a genomic library or by oligonucleotide synthesis

Libraries can be screened with probes (such as antibodies to the PRO230, PR0216 or PRO302 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 , supra An alternative means to isolate the gene encoding PRO230, PR0216 or PRO302 is to use PCR methodology Sambrook et al , supra. Dieffenbach et al . PCR Primer A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1995) The Examples below describe techniques for screening a cD A library The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened Methods ot labeling are well known in the art. and include the use ot radiolabels like ' P-labeled ATP biotinylation, or enzvme labeling Hybridization conditions, including moderate stringency and high stringency are provided in Sambrook el 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 __ ALIGN. DNAstar. and INHERIT, which employ various algorithms to measure homology

Nucleic acid having protein coding sequence may be obtained bv 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

ii Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described herein tor PRO230

PR0216 or PRO302 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

Methods of transfection are known to the ordinarily skilled artisan, for example CaP0₄ treatment 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 generally used for prokaryotes or other cells 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 without 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 mammalian cell 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 Nati Acad Sci (USA), 76 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 or polyornithine, may also be used For various techniques for transforming mammalian cells, see, Keown et al , Methods in Enzymology , 185 527-537 ( 1990) and Mansour et l . Nature, 336 348-352 (1988)

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokarvote, yeast, or higher eukaryote cells Suitable prokaryotes include, but are not limited to. eubacteπa. such as Gram-negative or Gram- positive organisms for example Enterobacteπaceae such as E colt Various E coli strains are pubhclv available, such as £ coli K12 strain MM294 (ATCC 31.446). E coli X I 776 (ATCC 3 1.537) E coli strain W31 10 (ATCC 27.325), and K5 772 (ATCC 53.635) Other suitable prokarvotic host cells include Enterobacteπaceae such as Escherichia. e g , E coli. Enterobacter. Erwima. Klebsiella. Proteus. Salmonella, e g , Salmonella tvphimurium, Serratia, e g , Serratia marcescans. and Shigella. as well as Bacilli such as B subtilis and B licheniformis (e g ,

B licheniformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P aeruginosa, and Streptomyces These examples are illustrative rather than limiting Strain W31 10 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 W31 10 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 W31 10 strain 1A2, which has the complete genotype tonA . E coli W31 10 strain 9E4, which has the complete genotype tonA ptri. E co// W31 10 strain 27C7 (ATCC 55.244) which has the complete genotype to 4 ptr3 phoA El 5 (argF-lac) 169 degP ompT karf, E coli W31 10 strain 37D6. which has the complete genotype tonA ptri phoA E15 (argF-lac)169 degP ompT rbs7 vG kari . E coli W31 10 stram 40B4. which is strain 37D6 with a non- kanamycin resistant degP deletion mutation, and an E coli strain having mutant periplasmic protease disclosed in

U S Patent No 4,946,783 issued 7 August 1990 Alternatively, m 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 vectors encoding PRO230, PR0216 or PRO302 Saccharomyces cerevisiae is a commonly used lowereukaryotichostmicroorganism Others include Schizosaccharomvces 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/Technology, 9 968-975 (1991)) such as, e g , K lactis (MW98-8C, CBS683. CBS4574, Louvencourt et al . J Bacteriol . 737 [19831). K frastlis (ATCC 12,424) K bitlgaricus (ATCC 16 045), K wickeramu (ATCC 24.178), A: waltu (ATCC 56.500). K drosophilarum (ATCC 36.906 Van den Berg et al . Bio/Technology. 8 135 ( 1990)), arrowia (EP 402.226), Pichia pastor is (EP 183.070. Sreekπshna et al . J Basic Microbiol . 28 265-278 [ 1988]), Candida Trichoderma reesta (EP 244,234), Neurospora crassa (Case et al , Proc Nati Acad Sci USA 76 5259-5263 [ 1979]), Schwanmomvces such as Schwanniomvces occidentals (EP394 538pubhshed31 October 1990), and filamentous fungi such as, e g Neurospora Penictlltum. Tolypocladtum (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A nidulans (Ballance et al , Biochem Biophvs Res Commun , 1 12 284-289 [19831, Tilburn et al , Gene. 26 205-221 [1983], Yelton et al . Proc Nati Acad Sci USA, 81 1470-1474 [1984]) and 4 niger (Kelly and Hynes, EMBOJ_. 4 475-479 [ 1985]) Methylotropic yeasts are suitable herein and include, but are not limited to yeast capable of growth on methanol selected from the genera consisting of Hansemda. 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 nucleic acid encoding glycosylated PRO230. PR0216 or PRO302 are derived from multicellular organisms Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera SP9 as well as plant cells Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkev kidney CV 1 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 36 59 ( 1977)), Chinese hamster ovarv cells/-DHFR (CHO. Urlaub and Chasm, Proc Nati Acad Sci USA, 77 4216 ( 1980)) mouse sertoli cells (TM4. Mather. Biol Reprod .23 243-

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 __ the skill m the art

in Selection and Use of a Rep cable Vector

The nucleic acid (e , cDN A or genomic DNA) encoding PRO230, PR0216 or PRO302 may be inserted into a rephcable vector for cloning (amplification of the DNA) or for expression Various vectors are publicly available The vector may, for example, be in the form of a plasmid. cosmid. viral particle or phage The appropriate nucleic acid sequence may be inserted into the vector a variety ot procedures in general, DNA is inserted into an appropriate restriction endonuclease sιte(s) using techniques known in the art Vector components generally include, but are not limited to. one or more of a signal sequence if the sequence is to be secreted, 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 hgation techniques that are known to the skilled artisan The PRO230. PR0216 or PRO302 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide In general, the signal sequence may be a component of the vector, or it may be a part of the DNA encoding PRO230, PR0216 or PRO302 that is inserted into the vector The signal sequence mav be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase. penicil nase. Ipp or heat-stable enterotoxin II leaders For veast secretion the signal sequence may be, e g , the yeast invertase leader, alpha factor leader(ιncludιng 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 mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Such sequences are well known for a vaπetv of bacteria, yeast, and viruses The origin of replication 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 clorung vectors in mammalian cells

Expression and cloning vectors will typicallv 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 . ampicilhn neomvcin. methotrexate. or tetracvchne. (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e . 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 nucleic acid encoding PRO230 PR0216 or PRO302. such as DHFR or thvmidine kinase

An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al Proc Nati Acad Sci USA. 77_. 4216 ( 1980) A suitable — selection gene for use in yeast is the trp 1 gene present in the yeast plasmid YRp7 Stinchcomb et al Nature. 282 39 (1979). Kmgsman et al . Gene. 7 141 ( 1979). Tschemper et al , Gene, 10 157 (1980) The trp\ 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 the nucleic acid sequence encoding PRO230, PR0216 or PRO302 to direct mR A 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 275 615 ( 1978), Goeddel et al . Nature. 281 544 ( 1979)), alkaline phosphatase. a tryptophan (tφ) promoter system (Goeddel, Nucleic Acids Res . 8 4057 (1980). EP 36,776), and hybrid promoters such as the tac promoter deBoer ef α/ . Proc Nati 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 PRO230, PR0216 or PRO302 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 Enzyme Reg . _ 1 149 ( 1968), Holland, Biochemistry, J_7 4900 ( 1978)), such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase. tπosephosphate isomerase, phosphoglucose isomerase and glucokinase

Other yeast promoters that 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-phosphate 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

PRO230, PR0216 or PRO302 nucleic acid 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 2.21 1 ,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) by heterologous mammalian promoters, e , the actin promoter or an immunoglobulin promoter, and by heat-shock promoters, provided such promoters are compatible with the host cell systems

Transcription of a DNA encoding the PRO230, PR0216 or PRO302 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-fetoprotem. 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 the vector at a position 5' or 3' to the sequence coding for PRO230, PR0216 or PRO302. 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 tor 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 PRO230, PR0216 or PRO302

Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO230, PR0216 or PRO302 in recombinant vertebrate cell culture are described in Gething et al Nature.293 620-625 ( 1981 ), Mantei et al . Nature. 281 40-46 ( 1979). EP 1 17.060. and EP 1 17,058

iv Detecting Gene Amplification/Expression Gene amplification 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 Nati Acad. Sci USA. 77 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 that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected Gene expression, alternatively, mav be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for 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 PRO230, PR0216 or PRO302 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA encoding PRO230, PR0216 or

PRO302 and encoding a specific antibody epitope

v Purification of Polypeptide Forms of PRO230, PR0216 or PRO302 polypeptides may be recovered from culture medium or from host cell lysates 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 nucleic acid encoding the PRO230, PR0216 or PRO302 polypeptide can be disrupted by various physical or chemical means, such as treeze-thaw cvchng. sonication. mechanical disruption, or cell-lysing agents

It may be desired to purify the PRO230, PR0216 or PRO302 polypeptide from recombinant cell proteins or polypeptides The following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC. chromatography on silica or on a cation- exchange resinsuch as DEAE.chromatofocusmg,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 PRO230, PR0216 or PRO302 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 Enzvmology, 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 PRO230, PR0216 or PRO302 produced

D Uses of the PRO230. PRQ216 or PRO302 polypeptides l Assays for Cardiovascular Endothelial and Angiogenic Activity Various assays can be used to test the polypeptide herein for cardiovascular endothelial and angiogenic activity Such assays include those provided in the Examples below

Assays for testing for endothehn antagonist activity, as disclosed in U S Pat No 5,773,414, include a rat heart ventricle binding assay where the polypeptide is tested for its ability to inhibit lodinized endothehn- 1 binding in a receptor assay, an endothehn receptor binding assay testing for intact cell binding of radiolabeled endothehn- 1 using rabbit renal artery vascular smooth muscle cells, an inositol phosphate accumulation assay where functional activity is determined in Rat-1 cells by measuring intra-cellular levels of second messengers, an arachidonic acid release assay that measures the ability of added compounds to reduce endothelin-stimulatedarachidonic acid release in cultured vascular smooth muscles, in vitro (isolated vessel) studies using endothe um from male New Zealand rabbits, and in vivo studies using male Sprague-Dawley rats Assays for tissue generation activity include without limitation those described in WO 95/16035 (bone, cartilage, tendon), WO 95/05846 (nerve, neuronal), and WO 91 /074 1 (skin, endothehum)

Assays for wound-healing activity include, for example, those described in Winter, Epidermal Wound Healing, Maibach. HI and Rovee, DT, eds (Year Book Medical Publishers, Inc , Chicago), pp 71 - 1 12, as modified by the article of Eaglstein and Mertz, J Invest Dermatol . 71_ 382-384 (1978) An assay to screen for a test molecule relating to a PRO230, PR0216 or PRO302 polypeptide that binds an endothehn B, (ETB , ) receptor polypeptide and modulates signal transduction activity involves providing a host cell transformed with a DNA encoding endothehn B, receptor polypeptide, exposing the cells to the test candidate, and measuring endothehn B, receptor signal transduction activity , as described, e . in U S Pat No 5,773.223

There are several cardiac hypertrophy assays In vitro assays include induction of spreading of adult rat cardiac myocytes In this assay, ventricular myocytes are isolated from a single (male Sprague-Dawley) rat, essentially following a modification of the procedure described in detail by Piper et al , "Adult ventricular rat heart muscle cells" in Cell Culture Techniques in Heart and Vessel Research H M Piper, ed (Berlin Spπnger-Verlag, 1 90), pp 36-60 This procedure permits the isolation ot adult ventricular mvocvtes and the long-term culture of these cells in the rod-shaped phenotype Phenvlephπne and Prostaglandin F_2π (PGF_^J have been shown to induce a spreading response in these adult cells The inhibition of myocyte spreading induced bv PGF_2α or PGF_2α analogs (e g fluprostenol) and phenylephrine by various potential inhibitors of cardiac hypertrophy is then tested One example of an in vivo assay is a test tor inhibiting cardiac h peπrophy induced by fluprostenol in vivo

This pharmacological model tests the ability of the PRO230, PR0216 or PRO302 polypeptide to inhibit cardiac hypertrophy induced in rats (e g , male Wistar or Sprague-Dawley) by subcutaneous injection of fluprostenol (an __ agonist analog of PGF_2α) It is known that rats with pathologic cardiac hypertrophy induced by myocardial infarction have chronically elevated levels of extractable PGF_2α in their myocardium Lai et al . Am J Phvsiol (Heart Circ Physiol ). 271 H2197-H2208 (1996) Accordingly, factors that can inhibit the effects of fluprostenol on myocardial growth in vivo are potentially useful for treating cardiac hypertrophy The effects of the PRO230, PR0216 or PRO302 polypeptide on cardiac hypertrophy are determined by measuring the weight of heart, ventricles, and left ventricle (normalized by body weight) relative to fluprostenol-treated rats not receiving the PRO230, PR0216 or PRO302 polypeptide Another example of an m vivo assay is the pressure-overload cardiac hypertrophy assay For in vivo testing it is common to induce pressure-overload cardiac hypertrophy by constriction of the abdominal aorta of test animals In a typical protocol, rats (e g , male Wistar or Sprague-Dawley) are treated under anesthesia, and the abdominal aorta of each rat is narrowed down just below the diaphragm Beznak M , Can J Biochem Physiol , 33 985-94 (1955) The aorta is exposed through a surgical incision, and a blunted needle is placed next to the vessel The aorta is constricted with a ligature of silk thread around the needle, which is immediately removed and which reduces the lumen of the aorta to the diameter of the needle This approach is described, for example, in Rossi et al , Am Heart J , 124 700-709 ( 1992) and O'Rourke and Reibel, P S E M B , 200 95-100 (1992)

In yet another in vivo assay, the effect on cardiac hypertrophy following experimentally induced myocardial infarction (MI) is measured Acute MI is induced in rats by left coronary artery hgation and confirmed by electrocardiographic examination A sham-operated group of animals is also prepared as control animals Earlier data have shown that cardiac hypertrophy is present in the group of animals with MI. as evidenced by an 18% increase in heart weight-to-body weight ratio Lai et al , supra Treatment of these animals with candidate blockers of cardiac hypertrophy, e , PRO230. PR0216 or PRO302 polypeptide. provides valuable information about the therapeutic potential of the candidates tested One further such assay test for induction of cardiac hypertrophy is disclosed in U S Pat No 5,773,415, using Sprague-Dawley rats

For cancer, a variety of well-known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies and other antagonists of the native PRO230, PR0216 or PRO302 polypeptides, such as small-molecule antagonists The in vivo nature of such models makes them particularly predictive of responses in human patients Animal models of tumors and cancers (e g . breast cancer, colon cancer, prostate cancer, lung cancer, etc ) include both non-recombinant and recombinant (transgenic) animals Non-recombinant animal models include, for example, rodent, e , murine models Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e g , subcutaneous injection tail vein injection, spleen implantation mtraperitoneal implantation, implantation underthe renal capsule, ororthopin implantation, e g colon cancer cells implanted in colonic tissue See, e g PCT publication No WO 97/33551. published September 18, 1997 Probably the most often used animal species in oncological studies are lmmunodeficient mice and, in particular, nude mice The observation that the nude mouse with thymic hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this puφose The autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains ot nude mouse, including, for example, ASW, A/He, AKR, BALB/c. BIO LP. C 17, C3H. C57BL. C57, CBA. DBA DDD. I/st. NC. NFR. NFS, NFS/N, NZB, NZC. NZW, P, RIII, and SJL In addition, a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts r or further details see, e g , The Nude Mouse in Oncology Research, E Boven and B Winograd, eds (CRC Press, Inc , 1991)

The cells introduced into such animals can be derived from known tumor/cancer cell lines, such as any of the above-listed tumor cell lines, and, for example, the B 104-1- 1 cell line (stable NIH-3T3 cell line transfected with the nett protooncogene). rαs-transfected NIH-3T3 cells Caco-2 (ATCC HTB-37). or a moderately weli- differentiated grade II human colon adenocarcinoma cell line. HT-29 (ATCC HTB-38), or from tumors and cancers

Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions involving freezing and storing in liquid nitrogen Karmah et al , Br J Cancer, 48 689-696 (1983)

Tumor cells can be introduced into animals such as nude mice by a variety of procedures The subcutaneous (s c.) space in mice is very suitable for tumor implantation Tumors can be transplanted s c as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions For solid-block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s c space Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously Tumor cells can also be injected as subdermal implants In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s c tissue Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogene was initially isolated), or new-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al Proc Nat Acad Sci USA. 83 9129-9133 ( 1986)

Similarly, animal models of colon cancer can be generated by passaging colon cancer cells in animals, e g , nude mice, leading to the appearance of tumors in these animals An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et al Cancer Research. 54 4726-4728 (1994) and

Too et al , Cancer Research. 55 681-684 (1995) This model is based on the so-called "METAMOUSE"™ sold by AntiCancer, Inc , (San Diego, California)

Tumors that arise in animals can be removed and cultured in vitro Cells from the in vitro cultures can then be passaged to animals Such tumors can serve as targets for further testing or drug screening Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest Such passaging techniques can be performed with any known tumor or cancer cell lines For example, Meth A CMS4. CMS5. CMS21. and WEHI-164 are chemicallv induced fibrosarcomas ot BALB/c female mice (DeLeo et al 146 720 (1977)) which provide a highly controllable model system for studying the anti-tumor activities of various agents Palladino et al J Immunol . 138 4023-4032 ( 1987) Briefly, tumor cells are propagated m vitro in cell culture Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about 10x10" to lOx l O⁷ cells/ml The animals are then infected subcutaneously with 10 to 100 w 1 of the cell suspension, allowing one to three weeks for a tumor to appear

In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most thoroughly studied — experimental tumors, can be used as an investigational tumor model Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small-cell carcinoma of the lung (SCCL) This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or ofcells maintained in culture Zupi er α/ Br J Cancer. 41 suppl 4 30 (1980) Evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive For further information about this tumor model see. Zacharski. Haemostasis. 16. 300-320 (1986)

One way of evaluating the efficacv of a test compound in an animal model with an implanted tumor is to measure the size of the tumor before and after treatment Traditionally, the size ot implanted tumors has been measured with a slide cahper in two or three dimensions The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula However, the measurement of tumor size is very inaccurate The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay Another important variable in the description of tumor growth is the tumor volume doubling time Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc 6th Int Workshop on Immune-Deficient Animals, Wu and Sheng eds (Basel, 1989), p 301 It is noted, however, that necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially Therefore these changes need to be carefully monitored bv a combination of a moφhometπc method and flow cytometπc analysis

Further, recombinant (transgenic) animal models can be engineered by introducing the coding portion of the PRO230, PR0216 or PRO302 genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g , baboons, chimpanzees and monkeys Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (U S Patent No 4.873,191 ), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al Proc Nati Acad Sci USA, 82 6148-615 (1985)), gene targeting in embryonic stem cells (Thompson et al CeH. 56 313-321 ( 1989)), electroporation of embryos (Lo Mol Cell Biol . 3 1803-1814 ( 1983)), and sperm-mediated gene transfer Lavitrano et al Cell, 57 717-73 ( 1989) For a review, see for example, U S Patent No 4.736,866

For the puφose of the present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals") The transgene can be integrated either as a single transgene. or in concatamers, e g , head-to-head or head-to-tail tandems Selective introduction ot a transgene into a particular cell type is also possible by following, for example the technique of Lasko et al Proc Nati Acad Sci USA, 89 6232-636 ( 1992) The expression of the transgene in transgenic animals can be monitored by standard techniques For example. Southern blot analysis or PCR amplification can be used to verify the integration of the transgene The level of mR A expression can then be analyzed using techniques such as in situ hybridization. Northern blot analysis, PCR, or immunocytochemistry The animals are further examined for signs of tumor or cancer development

Alternatively, "knock-out" animals can be constructed that have a defective or altered gene encoding a_ PRO230, PR0216 or PRO302 polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the PRO230, PR0216 or PRO302 polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal For example. cDNA encoding a particular

PRO230, PR0216 or PRO302 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques A portion of the genomic DNA encoding a particular PRO230, PR0216 or PRO302 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker that 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, Cell 51 503 ( 1987) for a description of homologous recombination vectors The vector is introduced into an embryonic stem cell line ( g , by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected See, e g , Li et al , Cell, 69 915 ( 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 Teratocarcmomas and Embryonic Stem Cells A Practical Approach. E J Robertson, ed (IRL Oxford, 1987), pp 1 13-152 A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock-out" animal Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA Knockout animals can be characterized, for instance, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to absence of the PRO230,

PR0216 or PRO302 polypeptide

The efficacy of antibodies specifically binding the PRO230, PR0216 or PRO302 polypeptides identified herein, and other drug candidates, can be tested also in the treatment of spontaneous animal tumors A suitable target for such studies is the feline oral squamous cell carcinoma (SCC) Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity At present, there is no effective treatment for this tumor Prior to entry into the study, each cat undergoes complete clinical examination and biopsy, and is scanned by computed tomography (CT) Cats diagnosed with subhngual oral squamous cell tumors are excluded from the study The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves Each cat is treated repeatedly, over a longer period of time Photographs of the tumors will be taken dailv during the treatment period, and at each subsequent recheck After treatment, each cat undergoes another CT scan CT scans and thoracic radiograms are evaluated every 8 weeks thereafter The data are evaluated for differences in survival, response and toxicitv as compared to control groups Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span

In addition, other spontaneous animal tumors, such as fibrosarcoma.adenocarcinoma, lymphoma. chondroma, or leiomyosarcoma of dogs, cats, and baboons can also be tested Of these, mammary adenocarcinoma in dogs and __ cats is a preferred model as its appearance and behavior are very similar to those in humans However, the use of this model is limited by the rare occurrence of this type of tumoi in animals Other in vitro and in vivo cardiovascular, endothelial, and angiogenic tests known in the art are also suitable herein

ii Tissue Distribution

The results of the cardiovascular, endothelial and angiogenic assavs herein can be verified by further studies, such as by determining mRNA expression in various human tissues

As noted before, gene amplification and/or gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc Nati Acad Sci USA, 77 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 that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or

DNA-protein duplexes

Gene expression in various tissues, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal. and mav be prepared in any mammal Conveniently, the antibodies may be prepared against a native-sequence PRO230, PR0216 or PRO302 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO230, PR0216 or PRO302 DNA and encoding a specific antibody epitope General techniques for generating antibodies, and special protocols for in situ hybridization are provided hereinbelow

in Antibody Binding Studies

The results of the cardiovascular, endothelial, and angiogenic study can be further verified by antibody binding studies, in which the ability of antι-PRO230. antι-PR0216 or antι-PRO302 antibodies to inhibit the effect of the PRO230, PR0216 or PRO302 polypeptides on endothelial cells or other cells used in the cardiovascular, endothelial. and angiogenic assays is tested Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow

Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assavs and immunoprecipitation assavs Zola Monoclonal Antibodies A Manual of Techniques (CRC Press. Inc 1987), pp 147-158

Competitive binding assavs rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody The amount of target protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies To facilitate determining the amount of standard that becomes bound, the antibodies preferably are lnsolubihzed before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and _ analyte that remain unbound

Sandwich assays involve the use of two antibodies, each capable of binding to a different lmmunogenic portion, or epitope, of the protein to be detected In a sandwich assay, the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte. thus forming an insoluble three-part complex See, e g , US Pat No 4,376,1 10 The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobuhn antibody that is labeled with a detectable moiety (indirect sandwich assay) For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme

For immunohistochemistry, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example

iv Cell-Based Tumor Assavs Cell-based assays and animal models for cardiovascular, endothelial, and angiogenic disorders, such as tumors, can be used to verify the findings of a cardiovascular, endothelial, and angiogenic assay herein, and further to understand the relationship between the genes identified herein and the development and pathogenesis of undesirable cardiovascular, endothelial. and angiogenic cell growth The role of gene products identified herein in the development and pathology of undesirable cardiovascular, endothelial. and angiogenic cell growth, e , tumor cells, can be tested by using cells or cells lines that have been identified as being stimulated or inhibited by the PRO230, PR0216 or PRO302 polypeptide herein Such cells include, for example, those set forth in the Examples below

In a different approach, cells of a cell type known to be involved in a particular cardiovascular, endothelial, and angiogenic disorder are transfected with the cDNAs herein, and the ability of these cDN As to induce excessive growth or inhibit growth is analyzed If the cardiovascular, endothelial, and angiogenic disorder is cancer, suitable tumor cells include, for example, stable tumor cells lines such as the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and rαs-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumoπgenic growth Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumoπgenic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC) Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of cardiovascular, endothelial, and angiogenic disorders such as cancer In addition, pπmarv cultures derived from tumors in transgenic animals (as described above) can be used in the cell-based assays herein, although stable cell lines are preferred Techniques to derive continuous cell lines from transgenic animals are well known in the art See. e g , Small et al Mol Cell Biol , 5 642-648 (1985)

v Gene Therapy

The PRO230, PR0216 or PRO302 polypeptide herein and polypeptidvl agonists and antagonists may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often __ referred to as gene therapy

There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells in vivo and ex vivo For in vivo delivery the nucleic acid is injected directly into the patient, usually at the sites where the PRO230, PR0216 or PRO302 polypeptide is required, ; e , the site of synthesis of the PRO230, PR0216 or PRO302 polypeptide. if known, and the site (e g , wound) where biological activity of PRO230. PR0216 or PRO302 polypeptide is needed For ex vivo treatment, the patient's cells are removed the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or. for example encapsulated within porous membranes that are implanted into the patient (see. e . U S Pat Nos 4 892.538 and

5.283,187) There are a variety of techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or transferred in vivo in the cells of the intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction. cell fusion. DEAE-dextran, the calcium phosphate precipitation method, etc Transduction involves the association of a replication-defective, recombinant viral (preferably retroviral) particle with a cellular receptor, followed by introduction of the nucleic acids contained by the particle into the cell A commonly used vector for ex vivo delivery of the gene is a retrovirus

The currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral vectors (such as adenovirus, lentivirus. Heφes simplex I virus, or adeno-associated virus (AAV)) and hpid-based systems (useful lipids for hpid-mediated transfer of the gene are, for example, DOTM A DOPE, and DC-Choi. see. e g , Tonkmson et al , Cancer Investigation. 14( 1 ) 54-65 ( 1996)) The most preferred vectors for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses A viral vector such as a retroviral vector includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger In addition, a viral vector such as a retroviral vector includes a nucleic acid molecule that, when transcribed in the presence of a gene encoding PRO230, PR0216 or PRO302 polypeptide. is operably linked thereto and acts as a translation initiation sequence Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof and positive and negative strand primer binding sites appropriate to the virus used (if these are not already present in the viral vector) In addition, such vector typically includes a signal sequence for secretion of the PRO230, PR0216 or PRO302 polypeptide from a host cell in which it is placed Preferably the signal sequence for this puφose is a mammalian signal sequence, most preferably the native signal sequence for the PRO230, PR0216 or PRO302 polypeptide Optionally, the vector construct may also include a signal that directs polyadenylation. as well as one or more restriction sites and a translation termination sequence By wav of example, such vectors will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendπmers In some situations, it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc Where liposomes are employed, proteins that bind to a cell-surface membrane protein associated— with endocytosis may be used for targeting and/or to facilitate uptake, e , capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo intemahzation in cycling, and proteins that target mtracellular localization and enhance intracellular half-life The technique of receptor-mediated endocytosis is described, for example, by Wu et al . J Biol Chem , 262 4429-4432 (1987), and Wagner et al , Proc Nati Acad Sci USA, 87 3410-3414 (1990) For a review of the currently known gene marking and gene therapy protocols, see, Anderson et al , Science, 256 808-813 (1992) See also WO 93/25673 and the references cited therein

Suitable gene therapy and methods for making retroviral particles and structural proteins can be found in, e g , U S Pat No 5,681,746

vi Use of Gene as Diagnostic This invention is also related to the use of the gene encoding the PRO230, PR0216 or PRO302 polypeptide as a diagnostic Detection of a mutated form of the PRO230, PR0216 or PRO302 polypeptide will allow a diagnosis of a cardiovascular, endothelial, and angiogenic disease or a susceptibilityto a cardiovascular, endothelial, and angiogenic disease, such as a tumor, since mutations in the PRO230, PR0216 or PRO302 polypeptide may cause tumors

Individuals carrying mutations in the genes encoding a human PRO230, PR0216 or PRO302 polypeptide mav be detected at the DNA level by a vaπetv of techniques Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva tissue biopsy, and autopsy material The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al . Nature. 324 163-166 (1986)) prior to analysis RNA or cDNA may also be used for the same puφose As an example, PCR primers complementary to the nucleic acid encoding the PRO230, PR0216 or PRO302 polypeptide can be used to identify and analyze PRO230, PR0216 or PRO302 polypeptide mutations For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA encoding the PRO230. PR0216 or PRO302 polypeptide, or alternatively, radiolabeled antisense DNA sequences encoding the PRO230, PR0216 or PRO302 polypeptide Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures Genetic testing based on DNA sequence differences may be achieved by detection of alteration- in electrophoretic mobility of DNA fragments in gels with or without denaturing agents Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis DNA fragments of different sequences may be distinguished on denaturing formamidine gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures See. e g . Mvers et al , Science. 230 1242 ( 1985)

Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method, for example, Cotton et al , Proc Nati Acad Sci USA. 85 4397-4401 (1985)

Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase — protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes, e g , restriction fragment length polymoφhisms (RFLP), and Southern blotting of genomic DNA

vii Use to Detect PRO230 PRQ216 or PRO302 Polypeptide Levels In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis

Expression of nucleic acid encoding the PRO230 PR0216 or PRO302 polypeptide may be linked to vascular disease or neovasculaπzation associated with tumor formation If the PRO230, PR0216 or PRO302 polypeptide has a signal sequence and the mRNA is highly expressed in endothelial cells and to a lesser extent in smooth muscle cells, this indicates that the PRO230, PR0216 or PRO302 polypeptide is present in serum Accordingly, an anti- PRO230, antι-PR0216 or antι-PRO302 polypeptide antibody could be used to diagnose vascular disease or neovasculaπzation associated with tumor formation, since an altered level of this PRO230, PR0216 or PRO302 polypeptide may be indicative of such disorders

A competition assay may be employed wherein antibodies specific to the PRO230, PR0216 or PRO302 polypeptide are attached to a solid support and the labeled PRO230. PR0216 or PRO302 polypeptide and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of PRO230, PR0216 or PRO302 polypeptide in the sample

viu Chromosome Mapping

The sequences of the present invention are also valuable for chromosome identification The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome Moreover, there is a current need for identifying particular sites on the chromosome Few chromosome marking reagents based on actual sequence data (repeat polymoφhisms) are presently available for marking chromosomal location The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA Computer analysis for the 3'- untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process These primers are then usedfor PCR screening of somatic cell hybrids containing individual human chromosomes Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner Other mapping strategies that can similarly be used to map to its chromosome include m situ hybridization prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome- specific cDNA libraries

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used__ to provide a precise chromosomal location in one step This technique can be used with cDNA as short as 500 or 600 bases, however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection FISH requires use of the clones from which the gene encoding the PR0230, PR0216 or PRO302 polypeptide was derived, and the longer the better For example, 2,000 bp is good, 4,000 bp is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time For a review of this technique, see, Verma e/ α/ Human Chromosomes a Manual of Basic Techniques (Pergamon Press, New York. 1988) Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data are found, for example, in V McKusick, Mende an Inheritance in Man (available online through Johns Hopkins University Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region is then identified through linkage analysis (coinheπtance of physically adjacent genes) Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals If a mutation is observed m some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes (This assumes 1 megabase mapping resolution and one gene per 20 kb)

lx Screening Assays for Drug Candidates

This invention encompasses methods of screening compounds to identify those that mimic the PRO230 PR0216 or PRO302 polypeptide (agonists) or prevent the effect of the PRO230, PR0216 or PRO302 polypeptide (antagonists) Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO230, PR0216 or PRO302 polypeptide encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art

All assays for antagonists are common in that they call for contacting the drug candidate with a PRO230. PR0216 or PRO302 polypeptide encoded bv a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact

In binding assavs the interaction is binding and the complex formed can be isolated or detected in the reaction mixture In a particular embodiment the PRO230 PR0216 or PRO302 polypeptide encoded bv the gene identified herein or the drug candidate is immobilized on a solid phase, e , on a microtiter plate by covalent or non-covalent attachments Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the PRO230 PR0216 or PRO302 polypeptide and drying Alternatively, an immobilized antibody, eg , a _ monoclonal antibody, specific for the PRO230 PR0216 or PRO302 polypeptide to be immobilized can be used to anchor it to a solid surface The assay is performed bv adding the non-immobi zed component which may be labeled by a detectable label, to the immobilized component, e g the coated surface containing the anchored component When the reaction is complete, the non-reacted components are removed, g bv washing, and complexes anchored on the solid surface are detected When the originally non-immobihzed component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred Where the originally non-immobilized component does not carry a label, complexing can be detected for example, by using a labeled antibody specifically binding the immobilized complex

If the candidate compound interacts with but does not bind to a particular PRO230 PR0216 or PRO302 polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions Such assays include traditional approaches, such as, e g , cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London).340 245-246 ( 1989), Chien et al , Proc Nati Acad Sci USA, 88 9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc Nati Acad Sci USA. 89 5789-5793 (1991) Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain The yeast expression system described in the foregoing publications (generally referred to as the ' two-hvbπd svstem") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA- bindmg domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain The expression of a GAL l-/αcZ reporter gene under control of a GAL4-actιvated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase A complete kit (MATCHMAKER™) for identifying protein-protein lnteractionsbetweentwo specific proteins using the two-hybrid technique is commercially available from Clontech This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions

Compounds that interfere with the interaction of a gene encoding a PRO230, PR0216 or PRO302 polypeptide identified herein and other intra- or extracellular components can be tested as follows usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence ot the test compound In addition, a placebo may be added to a third reaction mixture, to serve as positive control The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove The formation of a complex in the control reactιon(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction ot the test compound and its reaction partner

If the PRO230, PR0216 or PRO302 polypeptide has the ability to stimulate the proliferation of endothelial __ cells in the presence of the co-mitogen ConA, then one example of a screening method takes advantage of this ability Specifically, in the proliferation assay, human umbilical vein endothelial cells are obtained and cultured in 96-well flat-bottomed culture plates (Costar. Cambridge, MA) and supplemented with a reaction mixture appropriate for facilitating proliferation of the cells, the mixture containing Con-A (Caibiochem, La Jolla, CA) Con-A and the compound to be screened are added and after incubation at 37°C, cultures are pulsed with H- thvmidine and harvested onto glass fiber filters (phD, Cambridge Technology, Watertown, MA) Mean ' H- thymidme incoφoration (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments. Irvine CA) Significant ^J (H)thymιdιne incoφoration indicates stimulation of endothelial cell proliferation

To assay for antagonists, the assay described above is performed, however, in this assay the PRO230, PR0216 or PRO302 polypeptide is added along with the compound to be screened and the ability of the compound to inhibit ' (H)thymιdιne incoφoration in the presence of the PRO230, PR0216 or PRO302 polypeptide indicates that the compound is an antagonist to the PRO230, PR0216 or PRO302 polypeptide Alternatively, antagonists may be detected by combining the PRO230, PR0216 or PRO302 polypeptide and a potential antagonist with membrane- bound PRO230, PR0216 or PRO302 polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay The PRO230, PR0216 or PRO302 polypeptide can be labeled, such as by radioactivity, such that the number of PRO230. PR0216 or PRO302 polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting Cohgan et al , Current Protocols in Immun . 1 (2) Chapter 5 (1991 ) Preferably, expression cloning is employed wherein polyadeny lated RNA is prepared from a cell responsive to the PRO230 PR0216 or PRO302 polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the PRO230, PR0216 or PRO302 polypeptide Transfected cells that are grown on glass slides are exposed to the labeled PRO230, PR0216 or PRO302 polypeptide The PRO230. PR0216 or PRO302 polypeptide can be labeled by a variety of means including lodination or inclusion of a recognition site for a site- specific protein kinase Following fixation and incubation, the slides are subjected to autoradiographic analysis Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re- screening process, eventually yielding a single clone that encodes the putative receptor

As an alternative approach for receptor identification, labeled PRO230. PR0216 or PRO302 polypeptide can be photoaffinity- nked with cell membrane or extract preparations that express the receptor molecule Cross-linked material is resolved bv PAGE and exposed to X-rav film The labeled complex containing the receptor can be excised, resolved into peptide fragments and subjected to protein micro-sequencing The amino acid sequence obtained from micro-sequencing would be used to design a set ot degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor In another assay for antagonists mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO230 PR0216 or PRO302 polypeptide in the presence of the candidate compound The ability of the compound to enhance or block this interaction could then be measured —

The compositions useful in the treatment of cardiovascular, endothelial, and angiogenic disorders include, without limitation, antibodies small organic and inorganic molecules, peptides. phosphopeptides antisense and ribozyme molecules, tπple-hehx molecules etc that inhibit the expression and/or activity of the target gene product

More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with a PRO230 PR0216 or PRO302 polypeptide. and in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments single-chain antibodies anti-idiotvpic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the PRO230, PR0216 or PRO302 polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO230, PR0216 or PRO302 polypeptide

Another potential PRO230, PR0216 or PRO302 polypeptide antagonist or agonist is an antisense RNA or DNA construct prepared using antisense technology, where, e , an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation Antisense technology can be used to control gene expression through tπple-hehx formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA For example, the 5' coding portion of the polynucleotide sequence which encodes the mature PRO230 PR0216 or PRO302 polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see, Lee et al , Nucl Acids Res , 6 3073 ( 1979) Cooney et al . Science. 241 456 (1988), Dervan et al , Science, 251 1360 ( 1991)), thereby preventing transcription and the production of the PRO230, PR0216 or PRO302 polypeptide The antisense RNA oligonucleotide hybridizes to the mRNA m vivo and blocks translation of the mRNA molecule into the PRO230 PR0216 or PRO302 polypeptide (antisense - Okano, Neurochem , 56 560

(1991), Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press Boca Raton, FL, 1988) The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO230, PR0216 or PRO302 polypeptide When antisense DNA is used oligodeoxyπbonucleotides derived from the translation-initiation site, e g , between about -10 and +10 positions of the target gene nucleotide sequence are preferred

Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases m length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length about 80 bases length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more Potential antagonists include small molecules that bind to the active site the receptor binding site, or growth factor or other relevant binding site of the PRO230, PR0216 or PRO302 polypeptide. thereby blocking the normal biological activity of the PRO230, PR0216 or PRO302 polypeptide Examples of small molecules include, but __ are not limited to, small peptides or peptide-like molecules, preferably soluble peptides. and synthetic non-peptidyl organic or inorganic compounds Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g Rossi, Current Biology, 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18 1997) Nucleic acid molecules in tπple-hehx formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairmg rules, which generally require sizeable stretches of purines or pyπmidines on one strand of a duplex For further details see, e , PCT publication No WO 97/33551, supra

These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art

x Types of Cardiovascular. Endothelial. and Angiogenic Disorders to be Treated

The PRO230. PR0216 or PRO302 polypeptides, or agonists or antagonists thereto, that have activity in the cardiovascular, angiogenic, and endothelial assays described herein, and/or whose gene product has been found to be localized to the cardiovascular system, are likely to have therapeutic uses in a variety of cardiovascular, endothelial, and angiogenic disorders, including systemic disorders that affect vessels, such as diabetes mel tus Their therapeutic utility could include diseases of the arteries, capillaries, veins, and/or lymphatics Examples of treatments hereunder include treating muscle wasting disease, treating osteoporosis aiding in implant fixation to stimulate the growth ofcells around the implant and therefore facilitate its attachment to its intended site, increasing IGF stability in tissues or in serum, if applicable, and increasing binding to the IGF receptor (since IGF has been shown m vitro to enhance human marrow erythroid and granulocytic progenitor cell growth)

The PRO230, PR0216 or PRO302 polypeptides or agonists or antagonists thereto may also be employed to stimulate erythropoiesis or granulopoiesis to stimulate wound healing or tissue regeneration and associated therapies concerned with re-growth of tissue, such as connective tissue, skin, bone, cartilage, muscle, lung, or kidney, to promote angiogenesis, to stimulate or inhibit migration of endothelial cells, and to proliferate the growth of vascular smooth muscle and endothelial cell production The increase in angiogenesis mediated by the PRO230 PR0216 or PRO302 polypeptide or antagonist would be beneficial to lschemic tissues and to collateral coronary development in the heart subsequent to coronarv stenosis Antagonists are used to inhibit the action ot such polypeptides, for example, to limit the production of excess connective tissue during wound healing or pulmonary fibrosis if the PRO230, PR0216 or PRO302 polypeptide promotes such production This would include treatment of acute myocardial infarction and heart failure Moreover, the present invention concerns the treatment ofcardiac hypertrophy, regardless of the underlying cause, by administering a therapeutically effective dose of the PRO230, PR0216 or PRO302 polypeptide, or agonist or antagonist thereto If the objective is the treatment of human patients, the PRO230, PR0216 or PRO302 __ polypeptide preferably is recombinant human PRO230. PR0216 or PRO302 polypeptide (rhPRO230. rhPR0216 or rhPRO302 polypeptide) The treatment for cardiac hypertrophy can be performed at any of its various stages, which may result from a variety of diverse pathologic conditions, including myocardial infarction, hypertension, hypertrophic cardiomyopathy, and valvular regurgitation The treatment extends to all stages of the progression ofcardiac hypertrophy, with or without structural damage of the heart muscle, regardless of the underlying cardiac disorder

The decision of whether to use the molecule itself or an agonist thereof for any particular indication, as opposed to an antagonist to the molecule would depend mainly on whether the molecule herein promotes cardiovascularization. genesis of endothelial cells, or angiogenesis or inhibits these conditions For example, if the molecule promotes angiogenesis, an antagonist thereof would be useful for treatment of disorders where it is desired to limit or prevent angiogenesis Examples of such disorders include vascular tumors such as haemangioma, tumor angiogenesis, neovasculaπzation in the retina, choroid, or cornea, associated with diabetic retinopathy or premature infant retinopathy or macular degeneration and prohferative vitreoretinopathy, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation, psoriasis, endometπosis associated with neovasculaπzation, restenosis subsequent to balloon angioplasty, scar tissue oveφroduction, for example, that seen in a keloid that forms after surgery, fibrosis after myocardial infarction, or fibrotic lesions associated with pulmonary fibrosis

If. however, the molecule inhibits angiogenesis, it would be expected to be used directly for treatment of the above conditions

On the other hand, if the molecule stimulates angiogenesis it would be used itself (or an agonist thereof) for indications where angiogenesis is desired such as peripheral vascular disease, hypertension, inflammatory vascu tides, Reynaud's disease and Reynaud's phenomenon, aneurysms, arterial restenosis, thrombophlebitis, lymphangitis, lymphedema, wound healing and tissue repair, ischemia reperfusion injury, angina, myocardial infarctions such as acute myocardial infarctions, chronic heart conditions heart failure such as congestive heart failure, and osteoporosis

If, however, the molecule inhibits angiogenesis. an antagonist thereof would be used for treatment of those conditions where angiogenesis is desired

Specific types of diseases are described below, where the PRO230, PR0216 or PRO302 polypeptide herein or antagonists thereof may serve as useful for vascular-related drug targeting or as therapeutic targets for,the treatment or prevention of the disorders Atherosclerosis is a disease characterized by accumulation of plaques of intimal thickening in arteries, due to accumulation of lipids, proliferation ot smooth muscle cells, and formation of fibrous tissue within the arterial wall The disease can affect large, medium, and small arteries in any organ Changes in endothelial and vascular smooth muscle cell function are known to play an important role in modulating the accumulation and regression of these plaques

Hypertension is characterized by raised vascular pressure in the systemic arterial, pulmonary arterial, or portal venous systems Elevated pressure may result from or result in impaired endothelial function and/or vascular disease

Inflammatory vascu tides include giant cell arteπtis, Takayasu's arteπtis, polyarteπtis nodosa (including the__ microangiopathic form), Kawasaki's disease, microscopic polyangntis, Wegener's granuiomatosis, and a variety of infectious-related vascular disorders (including Henoch-Schonlein prupura) Altered endothelial cell function has been shown to be important in these diseases

Reynaud's disease and Reynaud's phenomenon are characterized by intermittent abnormal impairment of the circulation through the extremities on exposure to cold Altered endothelial cell function has been shown to be important in this disease

Aneurysms are saccular or fusiform dilatations of the arterial or venous tree that are associated with altered endothelial cell and/or vascular smooth muscle cells

Arterial restenosis (restenosis of the arterial wall) may occur following angioplasty as a result of alteration in the function and proliferation of endothelial and vascular smooth muscle cells

Thrombophlebitis and lymphangitis are inflammatory disorders of veins and lymphatics, respectively, that may result from, and/or in, altered endothelial cell function Similarly, lymphedema is a condition involving impaired lymphatic vessels resulting from endothelial cell function

The family of benign and malignant vascular tumors are characterized by abnormal proliferation and growth of cellular elements of the vascular system For example, lymphangiomas are benign tumors of the lymphatic system that are congenital, often cystic, malformations of the lymphatics that usually occur in newborns Cystic tumors tend to grow into the adjacent tissue Cystic tumors usually occur in the cervical and axillary region They can also occur in the soft tissue of the extremities The main symptoms are dilated, sometimes reticular, structured lymphatics and lymphocysts surrounded by connective tissue Lymphangiomas are assumed to be caused by improperly connected embryonic lymphatics or their deficiency The result is impaired local lymph drainage Gnener et al , Lymphology, 4 140-144 (1971)

Another use for the PRO230, PR0216 or PRO302 polypeptides herein or antagonists thereto is in the prevention of tumor angiogenesis, which involves vascularization of a tumor to enable it to growth and/or metastasize This process is dependent on the growth of new blood vessels Examples of neoplasms and related conditions that involve tumor angiogenesis include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endometπal carcinoma, endometπal hypeφlasia, endometπosis, fibrosarcomas, choπocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma,

Kaposι'ssarcoma,melanoma,skmcarcιnomas,hemangιoma,cavernoushemangιoma, hemangιoblastoma pancreas carcinomas, retinoblastoma, astrocytoma. ghoblastoma, Schwannoma, o godendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma, abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

Age-related macular degeneration (AMD) is a leading cause of severe visual loss in the elderly population. The exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment. Because choroidal neovascularization is associated with a dramatic worsening in prognosis, the PRO230, PR0216 or PRO302 polypeptides or antagonist thereto is expected to be useful in reducing the severity__ of AMD.

Healing of trauma such as wound healing and tissue repair is also a targeted use for the PRO230, PR0216 or PRO302 polypeptides herein or their antagonists. Formation and regression of new blood vessels is essential for tissue healing and repair. This category includes bone, cartilage, tendon, ligament, and/or nerve tissue growth or regeneration, as well as wound healing and tissue repair and replacement, and in the treatment of burns, incisions, and ulcers. A PRO230, PR0216 or PRO302 polypeptide or antagonist thereof that induces cartilage and or bone growth in circumstances where bone is not normally formed has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a PRO230, PR0216 or

PRO302 polypeptide or antagonist thereof may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma-induced, or oncologic, resection-induced craniofacial defects, and also is useful in cosmetic plastic surgery. PRO230, PR0216 or PRO302 polypeptides or antagonists thereto may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.

It is expected that a PRO230, PR0216 or PRO302 polypeptide or antagonist thereto may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, or endothehum), muscle (smooth, skeletal, or cardiac), and vascular (including vascular endothehum) tissue, or for promoting the growth ofcells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate.

A PRO230, PR0216 or PRO302 polypeptide herein or antagonist thereto may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage. Also, the PRO230, PR0216 or PRO302 polypeptide or antagonist thereto may be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells, or for inhibiting the growth of tissues described above.

A PRO230, PR0216 or PRO302 polypeptide or antagonist thereto may also be used in the treatment of periodontal diseases and in other tooth-repair processes. Such agents may provide an environment to attract bone- forming cells, stimulate growth of bone-forming cells, or induce differentiation of progenitors of bone-forr ng cells. A PRO230, PR0216 or PRO302 polypeptide herein or an antagonist thereto may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity osteoclast activity, etc ) mediated by inflammatory processes, since blood vessels play an important role in the regulation of bone turnover and growth Another category of tissue regeneration activity that may be attributable to the PRO230, PR0216 or PRO302 polypeptide herein or antagonist thereto is tendon/ligament formation A protein that induces tendon/hgament- ke tissue or other tissue formation in circumstances where such tissue is not normally formed has application in the healing of tendon or ligament tears, deformities, and other tendon or ligament defects in humans and other animals Such a preparation may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use __ in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue De novo tendon/ligament-like tissue formation induced by a composition of the PRO230, PR0216 or PRO302 polypeptide herein or antagonist thereto contributes to the repair of congenital, trauma-induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments The compositions herein may provide an environment to attract tendon- or ligament- forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair The compositions herein may also be useful in the treatment of tendinitis, caφal tunnel syndrome, and other tendon or ligament defects The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art

The PRO230, PR0216 or PRO302 polypeptide or its antagonist may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, / e , for the treatment of central and peripheral nervous system disease and neuropathies, as well as mechanical and traumatic disorders, that involve degeneration, death, ortrauma to neural cells or nerve tissue More specifically, a PRO230, PR0216 or PRO302 polypeptide or its antagonist may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosιs,and Shy-Dragersyndrome Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma, and cerebrovascular diseases such as stroke Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a PRO230, PR0216 or PRO302 polypeptide herein or antagonist thereto

Ischemia-reperfusion injury is another indication Endothelial cell dysfunction may be important in both the initiation of, and in regulation of the sequelae of events that occur following ischemia-reperfusion injury

Rheumatoid arthritis is a further indication Blood vessel growth and targeting of inflammatory cells through the vasculature is an important component in the pathogenesis of rheumatoid and sero-negative forms of arthritis

PRO230, PR0216 or PRO302 polypeptide or its antagonist may also be administered prophylactically to patients with cardiac hypertrophy, to prevent the progression of the condition, and avoid sudden death, including death of asymptomatic patients Such preventative therapy is particularly warranted in the case of patients diagnosed with massive left ventricular cardiac hypertrophy (a maximal wall thickness of 35 mm or more-in adults, or a comparable value in children), or in instances when the hemodynamic burden on the heart is particularly strong PRO230. PR0216 or PRO302 polypeptide or its antagonist may also be useful in the management of atπal fibrillation, which develops in a substantial portion of patients diagnosed with hypertrophic cardiomyopathy

Further indications include angina, myocardial infarctions such as acute myocardial infarctions, and heart failure such as congestive heart failure Additional non-neoplastic conditions include psoriasis, diabetic and other prohferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, thyroid hypeφlasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, peπcardial effusion (such as that associated wιth__ pericarditis), and pleural effusion

In view of the above, the PRO230. PR0216 or PRO302 polypeptides or agonists or antagonists thereof described herein, which are shown to alter or impact endothelial cell function, proliferation, and/or form, are likely to play an important role in the etiology and pathogenesis of many or all of the disorders noted above, and as such can serve as therapeutic targets to augment or inhibit these processes or for vascular-related drug targeting in these disorders

xi Administration Protocols. Schedules. Doses, and Formulations

The molecules herein and agonists and antagonists thereto are pharmaceutically useful as a prophylactic and therapeutic agent for various disorders and diseases as set forth above

Therapeutic compositions of the PRO230, PR0216 or PRO302 polypeptides or agonists or antagonists are prepared for storage by mixing the desired molecule having the appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition,

Osol, A ed ( 1980)), in the form of lyophihzed formulations or aqueous solutions Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidantsincludmg ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben. catechol, resorcinol. cyclohexanol,3-pentanol. and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, or immunoglobuhns, hydrophilic polymers such as polyvinylpyrrohdone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosacchaπdes, disacchaπdes, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA. sugars such as sucrose, mannitol. trehalose or sorbitol. salt-forming counter-ions such as sodium, metal complexes (e g , Zn- protein complexes), and/or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG)

Additional examples of such carriers include ion exchangers, alumina, aluminum stearate. lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine. sorbic acid, potassium sorbate, partial glyceπde mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate. disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium tπsihcate, polyvinyl pyrrohdone. cellulose-based substances, and polyethylene glycol Carriers for topical or gel-based forms of antagonist include polysacchaπdes such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrroiidone. polyacrylates polyoxyethvlene-poiyoxypropylene-block polymers, polyethylene glycol. and wood wax alcohols For all administrations, conventional depot forms are suitably used Such forms include, for example, microcapsules, nano-capsules, liposomes piasters, inhalation forms, nose sprays, subhngual tablets, and sustained-release preparations The PRO230, PR0216 or PRO302 polypeptides or agonists or antagonists will typically be formulated in such vehicles at a concentration of about 0 1 mg/ml to 100 mg/ml

Another formulation comprises incoφorating a PRO230, PR0216 or PRO302 polypeptide or antagonist thereof into formed articles Such articles can be used in modulating endothelial cell growth and angiogenesis In — addition, tumor invasion and metastasis may be modulated with these articles

PRO230, PR0216 or PRO302 polypeptide or antagonist to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution PRO230, PR0216 or PRO302 polypeptide ordinarily will be stored in lyophi zed form or in solution if administered systemically If in lyophihzed form. PRO230, PR0216 or PRO302 polypeptide or antagonist thereto is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use An example of a liquid formulation of PRO230, PR0216 or PRO302 polypeptide or antagonist is a sterile, clear, colorless unpreserved solution filled in a single-dose vial for subcutaneous injection Preserved pharmaceutical compositions suitable for repeated use may contain, for example, depending mainly on the indication and type of polypeptide a) PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto, b) a buffer capable of maintaining the pH in a range of maximum stability of the polypeptide or other molecule in solution, preferably about 4-8, c) a detergent/surfactant primarily to stabilize the polypeptide or molecule against agitation-induced aggregation, d) an isotonifier, e) a preservative selected from the group of phenol, benzyl alcohol and a benzethonium ha de, e g , chloride, and f) water

If the detergent employed is non-ionic, it may, for example, be polysorbates (e , POLYSORBATE™

(TWEEN™) 20, 80, etc ) or poloxamers (e g , POLOXAMER™ 188) The use of non-ionic surfactants permits the formulation to be exposed to shear surface stresses without causing denaturation of the polypeptide Further, such surfactant-containing formulations may be employed in aerosol devices such as those used in a pulmonary dosing, and needleless jet injector guns (see, e g , EP 257,956)

An isotonifier may be present to ensure isotonicity of a liquid composition of the PRO230, PR0216 or

PRO302 polypeptide or antagonist thereto, and includes polyhydπc sugar alcohols, preferably tπhydπc or higher sugar alcohols, such as glycerin, erythπtol, arabitol, xyhtol, sorbitol, and mannitol These sugar alcohols can be used alone or in combination Alternatively, sodium chloride or other appropriate inorganic salts may be used to render the solutions isotonic

The buffer may, for example, be an acetate, citrate, succinate. or phosphate buffer depending on the pH desired The pH of one type of liquid formulation of this invention is buffered in the range of about 4 to 8, preferably about physiological pH

The preservatives phenol, benzyl alcohol and benzethonium halides. e g , chloride, are known antimicrobial agents that may be employed Therapeutic PRO230, PR0216 or PRO302 polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle The formulations are preferably administered as repeated intravenous (I v ),__ subcutaneous (s c ), or intramuscular (I m ) injections, or as aerosol formulations suitable for intranasal or mtrapulmonary delivery (for intrapulmonary delivery see, e g , EP 257,956) PRO230, PR0216 or PRO302 polypeptide can also be administered in the form of sustained-released preparations Suitable examples of sustained-release preparations include semφermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e g , films, or microcapsules Examples of sustained-release matrices include polyesters, hydrogels (e g , poly(2-hydroxyethyl- methacrylate) as described by Langer et al , J Biomed Mater Res . j_5 167-277 (1981) and Langer, Chem Tech . 12 98-105 (1982) or poly(vmylalcohol)), polylactides (U S Patent No 3,773,919, EP 58,481), copolymers of L- glutamic acid and gamma ethyl-L-glutamate (Sidman et al , Biopolymers. 22 547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer et al , supra), degradable lactic acid-glyco c acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycohc acid copolymer and leupro de acetate), and poly-D-(-)-3-hydroxybutyπc acid (EP 133,988) While polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised for protein stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be lntermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophi zing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions

Sustained-release PRO230, PR0216 or PRO302 polypeptide compositionsalso lnclude posomailyentrapped PRO230, PR0216 or PRO302 polypeptides Liposomes containing the PRO230. PR0216 or PRO302 polypeptide are prepared by methods known per se DE 3.218.121, Epstein et al . Proc Nati Acad Sci USA, 82 3688-3692

(1985), Hwang et al , Proc Nati Acad Sci USA. 77 4030-4034 (1980), EP 52,322. EP 36,676, EP 88,046, EP 143,949, EP 142,641 , Japanese patent application 83-1 18008, U S Patent Nos 4,485,045 and 4 544,545, and EP 102,324 Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy The therapeutically effective dose of PRO230, PR0216 or PRO302 polypeptide or antagonist thereto will. of course, vary depending on such factors as the pathological condition to be treated (including prevention), the method of administration, the type of compound being used for treatment, any co-therapy involved, the patient's age, weight, general medical condition, medical history, etc , and its determination is well with the skill of a practicing physician Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the maximal therapeutic effect If the PRO230, PR0216 or PRO302 polypeptide has a narrow host range, for the treatment of human patients formulations comprising human PRO230, PR0216 or PRO302 polypeptide, more preferably native-sequence human PRO230, PR0216 or PRO302 polypeptide, are preferred The clinician will administer PRO230, PR0216 or PRO302 polypeptide until a dosage is reached that achieves the desired effect for treatment of the condition in question For example, if the objective is the treatment of CHF, the amount would be one that inhibits the progressive cardiac hypertrophy associated with this condition The progress of this therapy is easily monitored by echo cardiography Similarly, in patients with hypertrophic cardiomyopathy, PRO230, PR0216 or PRO302 polypeptide can be administered on an empirical basis

With the above guidelines, the effective dose generally is within the range of from about 0 001 to about 1 0 mg/kg, more preferably about 0 01-1 0 mg/kg, most preferably about 0 01-0 1 mg/kg

For non-oral use in treating human adult hypertension, it is advantageous to administer PRO230, PR0216 or PRO302 polypeptide in the form of an injection at about 0 01 to 50 mg, preferably about 0 05 to 20 mg, most preferably 1 to 20 mg, per kg body weight, 1 to 3 times daily by intravenous injection For oral administration, a molecule based on the PRO230, PR0216 or PRO302 polypeptide is preferably administered at about 5 mg to 1 g, preferably about 10 to 100 mg, per kg body weight, 1 to 3 times daily It should be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0 5 ng/mg protein Moreover, for human administration, the formulationspreferably meet sterility, pyrogenicity, general safety, and purity as required by FDA Office and Biologies standards

The dosage regimen of a pharmaceutical composition containing PRO230, PR0216 or PRO302 polypeptide to be used in tissue regeneration will be determined by the attending physician considering various factors that modify the action of the polypeptides, e g , amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e g , bone), the patient's age, sex, and diet, the severity of any infection, time of administration, and other clinical factors The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition For example, the addition of other known growth factors, such as IGF-I, to the final composition may also affect the dosage Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomoφhometπc determinations, and tetracyclme labeling

The route of PRO230, PR0216 or PRO302 polypeptide or antagonist or agonist administration is in accord with known methods, e , by injection or infusion by intravenous, intramuscular, lntracerebral, mtraperitoneal, intracerobrospinal, subcutaneous, intraocular, lntraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes, or by sustained-release systems as noted below The PRO230, PR0216 or PRO302 polypeptide or antagonists thereof also are suitably administered by intratumoral, peπtumoral, intralesional, or peπlesional routes, to exert local as well as systemic therapeutic effects The mtraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors If a peptide or small molecule is employed as an antagonist or agonist, it is preferably administered orally or non-orally in the form of a liquid or solid to mammals.

Examples of pharmacologically acceptable salts of molecules that form salts and are useful hereunder include alkali metal salts (e.g., sodium salt, potassium salt), alkaline earth metal salts (e.g., calcium salt, magnesium salt). ammonium salts, organic base salts (e.g. , pyridine salt, triethylamine salt), inorganic acid salts (e.g., hydrochloride, sulfate, nitrate), and salts of organic acid (e.g., acetate, oxalate, p-toluenesulfonate).

For compositions herein that are useful for bone, cartilage, tendon, or ligament regeneration, the therapeutic method includes administering the composition topically, systemically, or locally as an implant or device. When administered, the therapeutic composition for use is in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage, ortissue damage. Topical administration may be suitable for wound healing and tissue repair. Preferably, for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein- containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and preferably capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance, and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices forthe compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid, and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradabie and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.

One specific embodiment is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the polypeptide compositions from disassociating from the matrix. One suitable family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,hydoxyethylcelluloseJιydroxypropylcellulose. hydroxypropylmethylcellulose, and carboxymethylcellulose, one preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate. poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1 - 10 wt%, based on total formulation weight, which represents the amount necessary to prevent desoφtion of the polypeptide (or its antagonist) from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide (or its antagonist) the opportunity to assist the osteogenic activity of the progenitor cells

xu Combination Therapies The effectiveness of the PRO230. PR0216 or PRO302 polypeptide or an agonist or antagonist thereof in preventing or treating the disorder in question may be improved by administering the active agent serially or in combination with another agent that is effective for those puφoses, either in the same composition or as separate compositions

For example, for treatment of cardiac hypertrophy, PRO230, PR0216 or PRO302 polypeptide therapy can be combined with the administration of inhibitors of known cardiac myocyte hypertrophy factors, e , inhibitors of α-adrenergic agonists such as phenylephrine, endothehn- 1 inhibitors such as BOSENTAN™ and MOXONODIN™, inhibitors to CT-1 (US Pat No 5,679,545), inhibitors to LIF, ACE inhibitors, des-aspartate- angiotensm I inhibitors (U S Pat No 5,773,415), and angiotensin II inhibitors

For treatment ofcardiac hypertrophy associated with hypertensιon,PRO230, PR0216 or PRO302 polypeptide can be administered in combination with β-adrenergic receptor blocking agents, e g , propranolol, timolol.tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol, ACE inhibitors, e g , qumapπl, captopπl, enalapπl, ramipπl, benazepπl, fosinopπl, or lisinopπl, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichloφhenamide, acetazolamide, or indapamide, and/or calcium channel blockers, e g , diltiazem, nifedipine, verapamil, or nicardipine Pharmaceutical compositions compπsingthe therapeutic agents identified herein by their generic names are commercially available, and are to be administered following the manufacturers' instructions for dosage, administration, adverse effects, contraindications, etc See, e g , Physicians' Desk Reference (Medical Economics Data Production Co Montvale, N J , 1997), 51th Edition

Preferred candidates for combination therapy in the treatment of hypertrophic cardiomyopathy are β- adrenergic-blocking drugs (e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol), verapamil, difedipine, or diltiazem Treatment of hypertrophy associated with high blood pressure may require the use of antihypertensive drug therapy, using calcium channel blockers, e g , diltiazem, nifedipine, verapamil, or nicardipine, β-adrenergic blocking agents, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichloφhenamide, acetazolamide, or indapamide, and/or ACE-inhibitors, e g , quinapπl. captopπl, enalapπl, ramipπl, benazepπl, fosinopπl, or sinopπl

For other indications. PRO230, PR0216 or PRO302 polypeptides or their antagonists may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question These agents include various growth factors such as EGF, PDGF, TGF-α or TGF-β, IGF, FGF, and CTGF In addition, PRO230, PR0216 or PRO302 polypeptides or their antagonists used to treat cancer may be combined with cytotoxic, chemotherapeutic. or growth-inhibitory agents as identified above Also, for cancer treatment, the PRO230, PR0216 or PRO302 polypeptide or antagonist thereof is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances

The effective amounts of the therapeutic agents administered in combination with the PRO230, PR0216 or PRO302 polypeptide or antagonist thereof will be at the physician s or veterinarian's discretion Dosage administration and adjustment is done to achieve maximal management of the conditions to be treated For example, for treating hypertension, these amounts ideally take into account use of diuretics or digitalis, and conditions such as hyper- or hypotension, renal impairment, etc The dose will additionally depend on such factors as the type of the therapeutic agent to be used and the specific patient being treated Typically, the amount employed will be the same dose as that used, if the given therapeutic agent is administered without PRO230, PR0216 or PR0302 polypeptide

xin Articles of Manufacture An article of manufacture such as a kit containing PRO230, PR0216 or PRO302 polypeptide or antagonists thereof useful for the diagnosis or treatment of the disorders described above comprises at least a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes The containers may be formed from a variety of materials such as glass or plastic The container holds a composition that is effective for diagnosing or treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) The active agent in the composition is the PRO230, PR0216 or PRO302 polypeptide or an agonist or antagonist thereto The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice The article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution It may further include other materials desirable from a commercial and user standpoint, including other buffers diluents, filters, needles, syringes, and package inserts with instructions for use The article of manufacture may also comprise a second or third container with another active agent as described above

C Antibodies

Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments that may inhibit the production or the gene product of the genes identified herein and/or reduce the activity of the gene products

l 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 will be injected in the mammal by multiple subcutaneous or mtraperitoneal injections The immunizing agent may include the PRO230, PR0216 or PRO302 polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be lmmunogenic in the mammal being immunized Examples of such lmmunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobuhn, and soybean trypsin inhibitor Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A or synthetic trehalose dicorynomycolate) The immunization protocol may be selected by one skilled in the art without undue experimentation

11 Monoclonal Antibodies

The antι-PRO230, antι-PR0216 or antι-PRO302 antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybridoma methods, 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 immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro

The immunizing agent will typically include the PRO230, PR0216 or PRO302 polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell Goding, Monoclonal Antibodies Principles and Practice (New York Academic Press, 1986), pp 59- 103 Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin Usually, rat or mouse myeloma cell lines are 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 phosphoπbosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically will include hypoxanthine, aminopteπn, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT- deficient cells Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of 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, Manassas, Virginia 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 the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the PRO230, PR0216 or PRO302 polypeptide Preferably, the binding specificity of monoclonal antibodies produced by the 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 the art The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem , 07 220 ( 1980) After the desired hybridoma cells are identified, the clones may be subcloned bv limiting dilution procedures and grown by standard methods Goding supra Suitable culture media for this puφose 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 the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures ( g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) The hybridoma cells of the invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which 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 cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (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-immunoglobulin polypeptide Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody

The antibodies may be monovalent antibodies Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosslinking Alternatively, the relevant cysteme residues are substituted with another 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

in Human and Humanized Antibodies The antι-PRO230, antι-PR0216 or antι-PRO302 antibodies may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobuhns, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin Humanized antibodies include human immunoglobuhns (recipient antibody) in which residues from a 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 that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody preferably also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al. , Nature, 321 : 522-525 ( 1986); Riechmann et al. , Nature, 332: 323-329 (1988); 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 that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.

Humanization can be essentially performed following the method of Winter and co-workers (Jones et at. 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 the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol.. 227: 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 Therapy. Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol., 147(1): 86-95 (1991). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g. , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807: 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661 ,016, and in the following scientific publications: Marks et al. , Bio/Technology, K): 779-783 ( 1992); Lonberg etal.. Nature, 368: 856-859( 1994); Morrison, Nature, 368: 812-813 1994): Fishwild et al.. Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, F4: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol.. 13: 65-93 (1995).

iv. 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 PRO230, PR0216 or PRO302 polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/hght-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 hybπdomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure The purification of the correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 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 the first heavy-chain constant region (CHI) containing the 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)

v Heteroconiugate Antibodies

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, lmmunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond Examples of suitable reagents for this puφose include lminofhiolate and methyl-4- mercaptobutyπmidate and those disclosed, for example, in U S Patent No 4,676,980

vi Effector Function Engineering

It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e g , the effectiveness of the antibody in treating cancer For example, cysteine resιdue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region The homodimeπc antibody thus generated may have improved intemahzation capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See, Caron et al , J Exp Med , 176 1 191-1 195 (1992) and

Shopes, J Immunol , 148 2918-2922 ( 1992) Homodimeπc antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al Cancer Research, 53 2560-2565 (1993) Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See, Stevenson et al Anti-Cancer Drug Design, 3 219-230 (1989)

vn Immunoconiugates

The invention also pertains to lmmunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (t e a radioconjugate)

Chemotherapeutic agents useful in the generation of such lmmunoconjugates have been described above Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxm A chain (from Pseudomonas aeruginosa), πcin A chain, abπn A chain, modeccin A chain, alpha-sarcin, Aleurttes fordu proteins, dianthin proteins Phytolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotm, sapaonaπa officinalis inhibitor, gelonin, mitogelhn, restrictocin, phenomycm, enomycin, and the tricothecenes A variety of radionuclides are available for the production of radioconjugated antibodies Examples include ²¹ Bι, ^lj,I, ^13lIn, ⁹⁰Y, and ^l86Re Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succιnιmιdyl-3-(2-pyπdyldιthιol) propιonate(SPDP), lmmothiolane (IT), bifunctional derivatives of lmidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bιs-(p-dιazonιumbenzoyl)-ethylenedιamιne), dnsocyanates (such as tolyene 2,6-dnsocyanate), and bis- active fluorine compounds (such as l,5-dιfluoro-2,4-dιnιtrobenzene) For example, a πcin lmmunotoxin can be prepared as described in Vitetta et al , Science. 238 1098 ( 1987) Carbon- 14-labeled 1 -ιsothιocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelatmg agent for conjugation of radionucleotide to the antibody See, W094/ 1 1026

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e , avidin) that is conjugated to a cytotoxic agent (e g , a radionucleotide)

vin Immunohposomes The antibodies disclosed herein may also be formulated as immunohposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et αl Proc Nati Acad Sci USA, 82 3688 (1985), Hwang et αl , Proc Nati Acad Sci USA 77 4030 (1980), and U S Pat Nos 4,485 045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5,013,556

Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcho ne, cholesterol, and PEG-deπvatized phosphatidylethanolamine (PEG-

PE) Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et αl J Biol Chem , 257 286-288 (1982) via a disulfide-interchange reaction A chemotherapeutic agent (such as Doxorubicin) is optionally contained withm the liposome See, Gabizon et αl , J National Cancer Inst , 81(19) 1484 (1989) lx Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO230, PR0216 or PRO302 polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders as noted above and below in the form of pharmaceutical compositions If the PRO230, PR0216 or PRO302 polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred However, pofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred For example, based upon the variable- region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence Such peptides can be synthesized chemically and/or produced by recombinant DNA technology See, e g, Marasco et al , Proc Nati Acad Sci USA, 90 7889-7893 (1993)

The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the puφose intended

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by mterfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-( ethylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra

The formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes

Sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g films, or microcapsules Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vιnylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycohc acid copolymer and leupro de acetate) and poly-D-(-)-3-hydroxybutyπc acid

While polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised for stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to he lntermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophi zing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

x. Methods of Treatment using the Antibody

It is contemplated that the antibodies to a PRO230, PR0216 or PRO302 polypeptide may be used to treat various cardiovascular, endothelial, and angiogenic conditions as noted above.

The antibodies are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred. Other therapeutic regimens may be combined with the administration of the antibodies of the instant invention as noted above. For example, if the antibodies are to treat cancer, the patient to be treated with such antibodies may also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, Ed., M.C. Perry (Williams &

Wilkins: Baltimore, MD, 1992). The chemotherapeutic agent may precede, or follow administration of the antibody, or may be given simultaneously therewith. The antibody may be combined with an anti-estrogen compound such as tamoxifen or EVISTA™ or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules. If the antibodies are used for treating cancer, it may be desirable also to administer antibodies against other tumor-associated antigens, such as antibodies that bind to one or more of the ErbB2, EGFR, ErbB3, ErbB4, or VEGF receptor(s). These also include the agents set forth above. Also, the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances. Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to the patient. Sometimes, it may be beneficial also to administer one or more cytokines to the patient. In a preferred embodiment, the antibodies herein are co- administered with a growth-inhibitory agent. For example, the growth-inhibitory agent may be administered first, followed by an antibody of the present invention. However, simultaneous administration or administration of the antibody of the present invention first is also contemplated. Suitable dosages for the growth-inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth-inhibitory agent and the antibody herein.

In one embodiment, vascularization of tumors is attacked in combination therapy. The anti-PRO230, anti- PR0216 or anti-PRO302 polypeptide and another antibody (e.g., anti-VEGF) are administered to tumor-bearing patients at therapeutically effective doses as determined, for example, by observing necrosis of the tumor or its metastatic foci, if any. This therapy is continued until such time as no further beneficial effect is observed or clinical examination shows no trace of the tumor or any metastatic foci. Then TNF is administered, alone or in combination with an auxiliary agent such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin, anti-heregulin antibody, D-factor, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), or agents that promote microvascular coagulation in tumors, such as anti-protein C antibody, anti-protein S antibody, or C4b binding protein (see. WO 91/01753, published 21 February 1991 ), or heat or radiation. Since the auxiliary agents will vary in their effectiveness, it is desirable to compare their impact on the tumor by matrix screening in conventional fashion. The administration of anti-PRO230, anti-PR0216 or anti-PRO302 polypeptide antibody and TNF is repeated until the desired clinical effect is achieved. Alternatively, the anti- — PRO230, anti-PR0216 or anti-PRO302 polypeptide antibody is administered together with TNF and, optionally, auxiliary agent(s). In instances where solid tumors are found in the limbs or in other locations susceptible to isolation from the general circulation, the therapeutic agents described herein are administered to the isolated tumor or organ. In other embodiments, a FGF or PDGF antagonist, such as an anti-FGF or an anti-PDGF neutralizing antibody, is administered to the patient in conjunction with the anti-PRO230, anti-PR0216 or anti-PRO302 polypeptide antibody. Treatment with anti-PRO230, anti-PR0216 or anti-PRO302 polypeptide antibodies preferably may be suspended during periods of wound healing or desirable neovascularization. Forthe prevention ortreatment of cardiovascular, endothelial, and angiogenicdisorder, the appropriate dosage of an antibody herein will depend on the type of disorder to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic puφoses, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. For example, depending on the type and severity of the disorder, about 1 g/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily or weekly dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated or sustained until a desired suppression of disorder symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging.

xi. Articles of Manufacture with Antibodies

An article of manufacture containing a container with the antibody and a label is also provided. Such articles are described above, wherein the active agent is an anti-PRO230, anti-PR0216 or anti-PRO302 antibody.

xii. Diagnosis and Prognosis of Tumors using Antibodies

If the indication for which the antibodies are used is cancer, while cell-surface proteins, such as growth receptors over expressed in certain tumors, are excellent targets for drug candidates or tumor (e.g., cancer) treatment, the same proteins along with PRO230, PR0216 or PRO302 polypeptides find additional use in the diagnosis and prognosis of tumors. For example, antibodies directed against the PRO230, PR0216 or PRO302 polypeptides may be used as tumor diagnostics or prognostics. For example, antibodies, including antibody fragments, can be used qualitatively or quantitatively to detect the expression of genes including the gene encoding the PRO230, PR0216 or PRO302 polypeptide. The antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. Such binding assays are performed essentially as described above.

In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this puφose, a histological specimen is removed from _ the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent to those skilled in the art that a wide variety of histological methods are readily available for in situ detection.

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

The disclosures of all patent and literature references cited in the present specification are hereby incoφorated by reference in their entirety.

EXAMPLES Commercially available reagents referred to in the Examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following Examples, and throughoutthe specification, by ATCC accession numbers is the American Type Culture Collection,Manassas, VA.

Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks: Sambrook et al, supra; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N.Y., 1989); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.: N.Y., 1990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide

Synthesis (IRL Press: Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al, Current Protocols in Immunology, 1991.

EXAMPLE 1 Isolation of cDNA Clones Encoding Human PRO230 (a tubulointerstitial nephritis antigen homolog)

The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al. , Methods in Enzymology. 266:460-480 ( 1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. 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).

A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above.

This consensus sequence is herein designated DNA30857. An EST proprietary to Genentech was employed in the consensus assembly and is herein designated DNA20088. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above.

Based on the DNA30857 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 PRO230. 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 1-1.5 kbp. 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, supra, 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.

PCR primers (forward and reverse) were synthesized: forward PCR primer:

5'-TTCGAGGCCTCTGAGAAGTGGCCC-3' (SEQ ID NO:3) reverse PCR primer: 5'-GGCGGTATCTCTCTGGCCTCCC-3' (SEQ ID NO:4)

Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30857 sequence which had the following nucleotide sequence: hybridization probe:

5'-TTCTCCACAGCAGCTGTGGCATCCGATCGTGTCTCAATCCATTCTCTGGG-3' (SEQ ID NO:5) RNA for construction of the cDNA libraries was isolated from human fetal lung tissue. The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDN A 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 the clones isolated as described above gave the full-length DNA sequence for a full-length PRO230 polypeptide (designated herein as DNA33223-1136 [Figures 1 A- IB, SEQ ID NO: l]) and the derived protein sequence for that PRO230 polypeptide.

The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 100-102 and a stop signal at nucleotide positions 592-594 (Figures 1A-1B,

SEQ ID NO: 1 ). The predicted polypeptide precursor is 164 amino acids long [Figure 2; (SEQ ID NO:2)] and has a calculated molecular weight of approximately 18,359 daltons and an estimated pi of about 7.45. Analysis of the full-length PRO230 sequence shown in Figure 2 (SEQ ID NO 2) evidences the presence of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PRO230 sequence (Figure 2, SEQ ID NO 2) evidences the following a signal peptide from about amino acid 1 to about amino acid 21 , an N-hnked glycosylation site from about amino acid 78 to about ammo acid 82, casein kinase II phosphorylation sites from about amino acid 80 to about amino acid 84, from about am o acid 1 17 to about ammo acid 121 , from about amino acid 126 to about am o acid 130 and from about amino acid 137 to about amino acid 141 , N-myπstoylation sites from about am o ^~~ acid 21 to about ammo acid 27, from about amino acid 39 to about amino acid 45, from about amino acid 44 to about amino acid 50 and from about ammo acid 104 to about amino acid 1 10, and an amidation site from about amino acid 26 to about amino acid 30 Clone DNA33223- 1 136 has been deposited with ATCC on September 16,

1997 and is assigned ATCC deposit no 209264

An analysis of the Dayhoff database (version 35 45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 2 (SEQ ID NO 2), evidenced sequence identity between the PRO230 amino acid sequence and tubulointerstitial nephritis antigen

EXAMPLE 2 Isolation of cDNA Clones Encoding Human PRQ216 (an osteomodulin/fibromoduhn homolog) The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases The EST databases included public EST databases (e g , GenBank) and a proprietary EST database (LIFESEQ®, Incyte

Pharmaceuticals, Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2 [Altschul et al , Methods in Enzymology, 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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)

The complete cDNA sequenceof DN A33087 is disclosed in GenBank under accession numbers AB0001 14 1 and AB009589 1 (human osteomoduhn) A related, but probably different protein, corneal keratin sulfate, is disclosed in Funderburgh et al , J Biol Chem , 27 .31431 -31436 ( 1996)

A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA28754 In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above

Based on the DNA28754 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 PR0216 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 5 kbp In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel etal , Current Protocols in Molecular Biology, supra, 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 PCR primers (forward and reverse) were synthesized forward PCR primer

5'-TCACGATGATCCTGACAATGC-3' (SEQ ID NO 8) - reverse PCR primer

5'-AATAATGAAGGTCAAAGTGCCCTT-3' (SEQ ID NO 9) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28754 sequence which had the following nucleotide sequence hybridization probe

5'-TGCTCCTTCTTGTTCTGGGCTCTCATG-3' (SEQ ID NO 10)

RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue The cDN A libraries used to isolate the cDN A clones were constructed bv standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDN A was primed with ohgo 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 the clones isolated as described above gave the full-length DNA sequence for a full-length PR0216 polypeptide (designated herein as DNA33087 [Figure 3, SEQ ID NO 6]) and the derived protein sequence for that PR0216 polypeptide

The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 268-270 and a stop signal at nucleotide positions 1531-1533 (Figure 3, SEQ ID NO 6) The predicted polypeptide precursor is 421 am o acids long [Figure 4, (SEQ ID NO 7)] and has a calulated molecular weight of approximately 49,492 daltons and an estimated pi of about 5 51 Analysis of the full-length PR0216 sequence shown in Figure 4 (SEQ ID NO 7) evidences the presence of important polypeptide domains as shown in Figure 4, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PR0216 sequence (Figure 4, SEQ ID NO 7) evidences the following N-hnked glycosylation sites from about amino acid 1 13 to about amino acid 1 17, from about amino acid 121 to about amino acid 125, from about amino acid 187 to about amino acid 191, from about amino acid 242 to about amino acid 246 and from about ammo acid 316 to about am o acid 320, casein kinase II phosphorylation sites from about amino acid 189 to about ammo acid 193 and from about amino acid 247 to about amino acid 251 , tyrosine kinase phosphorylation sites from about amino acid 268 to about amino acid 275 and from about ammo acid 300 to about am o acid 307, an N-myπstoylation site from about amino acid 230 to about am o acid 236, and leucine zipper patterns from about am o acid 146 to about ammo acid 168 and from about amino acid 217 to about ammo acid 239 Clone DNA33087 has been deposited with ATCC on October 16, 1997 and is assigned ATCC deposit no 209381

This clone is the same as human osteomodulm submitted by I Ohno to GenBank on December 26, 1996 (AB0001 14_1) and submitted by I Ohno et al , to GenBank on December 5, 1997 (AB009589-1)

EXAMPLE 3

Isolation of cDNA Clones Encoding Human PRO302 (vitellogenic carboxypeptidase homolog) The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about ^~~ 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases The EST databases included public EST databases (e g , GenBank) and a proprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2

[Altschul et al , Methods in Enzymology, 266 460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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) A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above

This consensus sequence is herein designated DNA35953 In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above Based on the DNA35953 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 PRO302 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 5 kbp 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, supra, 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

PCR primers (forward and reverse) were synthesized forward PCR primer 1 5'-GTCCGCAAGGATGCCTACATGTTC-3' (SEQ ID NO 13) forward PCR primer 2

5'-GCAGAGGTGTCTAAGGTTG-3' (SEQ ID NO 14) reverse PCR primer

5'-AGCTCTAGACCAATGCCAGCTTCC-3' (SEQ ID NO 15) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA35953 sequence which had the following nucleotide sequence hybridization probe

5'-GCCACCAACTCCTGCAAGAACTTCTCAGAACTGCCCCTGGTCATG-3' (SEQ ID NO 16) RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228) The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with ohgo dT containing a Notl site, linked with blunt to Sail hemik ased 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 ofpRK5D 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 the clones isolated as described above gave the full-length DNA sequence for a full-length PRO302 polypeptide (designated herein as DNA40370- 1217 [Figure 5, SEQ ID NO 1 1 ]) and the derived protein sequence for that PRO302 polypeptide

The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 34-36 and a stop signal at nucleotide positions 1390-1392 (Figure 5, SEQ ID NO 1 1) The predicted polypeptide precursor is 452 amino acids long [Figure 6, (SEQ ID NO 12)] and has a calculated molecular weight of approximately 50,831 daltons and an estimated pi of about 5 74 Analysis of the full-length PRO302 sequence shown in Figure 6 (SEQ IDNO 12) evidences the presence of important polypeptide domains as shown in Figure 6, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PRO302 sequence (Figure 6, SEQ ID NO 12) evidences the following a signal peptide from about amino acid 1 to about amino acid 25, N-hnked glycosylation sites from about amino acid 64 to about amino acid 68, from about am o acid 126 to about ammo acid 130 and from about amino acid 362 to about ammo acid 366, a c AMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 101 to about amino acid 105. casein kinase II phosphorylation sites from about ammo acid 204 to about ammo acid 208, from about amino acid 220 to about amino acid 224, from about amino acid 280 to about ammo acid 284, from about amino acid 284 to about amino acid 288, from about amino acid 351 to about amino acid 355 and from about ammo acid 449 to about amino acid 453, and N-myπstoylation sites from about amino acid 22 to about amino acid 28, from about amino acid 76 to about amino acid 82, from about amino acid 79 to about amino acid 85, from about ammo acid 80 to about amino acid 86, from about amino acid 1 19 to about amino acid 125, from about amino acid 169 to about amino acid 175, from about amino acid 187 to about ammo acid 193, from about amino acid 195 to about amino acid 201 , from about amino acid 331 to about amino acid 337, from about amino acid 332 to about amino acid 338 and from about amino acid 360 to about amino acid 366 Clone DNA40370-1217 has been deposited with ATCC on November 21, 1997 and is assigned ATCC deposit no 209485

An analysis of the Dayhoff database (version 35 45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 6 (SEQ ID NO 12), evidenced sequence identity between the

PRO302 ammo acid sequence and vitellogenic carboxvpeptidase EXAMPLE 4 Stimulation of Endothelial Tube Formation This assay follows the assay described in Davis and Camaπllo, Experimental Cell Research, 224 39-51 (1996), or one modified from it as follows Protocol Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) (passage number less than 8 from primary) are mixed with type I rat tail collagen, final concentration 2 6 mg/ml at a density of 6 x 10⁵ cells/ml and plated at 50 l per well on a 96-well plate The gel is allowed to solidify for 1 hr at 37°C, then 50 μl per well of ^~~ Ml 99 culture media supplemented with 1% FBS and a PRO230 polypeptide sample (at dilutions of 1%, 0 1%, and

0 01%, respectively) is added along with 1 μM 6-FAM-FITC dye to stain vacuoles while they are forming Cells are incubated at 37°C/5% CO, for 48 hrs, fixed with 3 7% formalin at room temperature for 10 minutes, washed with PBS five times, then stained with Rh-Phalloidin at 4°C overnight followed by nuclear staining with 4 μM DAPI

1 Apoptosis Assay

This assay will identify factors that facilitate cell survival in a 3-dιmensιonal matrix in the presence of exogenous growth factors (VEGF, bFGF without PMA)

A positive result is equal to or less than 1 0 = no apoptosis, 1 = less than 20% cells are apoptotic, 2 = less than 50% cells are apoptotic, 3 = greater than 50% cells are apoptic Stimulators of apoptosis in this system are expected to be apoptotic factors, and inhibitors are expected to prevent or lessen apoptosis

2 Vacuoles Assay This assay will identify factors that stimulate endothelial vacuole formation and lumen formation in the presence of bFGF and VEGF (40ng/ml)

A positive result is equal to or greater than 2 1 = vacuoles present in less than 20% of cells, 2 = vacuoles present in 20-50% of cells, 3 = vacuoles present in greater than 50% of cells This assay is designed to identify factors that are involved in stimulating pinocytosis, ion pumping, permeability, and junction formation 3 Tube Formation Assay

This assay is to identify factors that stimulate endothelial tube formation in a 3-dιmensιonal matrix This assay will identify factors that stimulate endothelial cells to differentiate into a tube-like structure in a 3-dιmensιonal matrix in the presence of exogenous growth factors (VEGF, bFGF)

A positive result is equal to or greater than 2 1 = cells are all round, 2 = cells are elongated, 3 = cells are forming tubes with some connections, 4 = cells are forming complex tubular networks This assay would identify factors that may be involved in stimulating tracking, chemotaxis, or endothelial shape change

The results clearly demonstrate that more complex tube formation occurs with PRO230-IgG and PRO230- poly-His samples at 1 % dilution compared with buffer controls ( 10 mM HEPES/0 14M NaCl/4% mannitol, pH 6 8) at 1% dilution EXAMPLE 5 Induction of c-fos m Endothelial Cells This assay is designed to determine whether PR0216 (human osteomodu n) polypeptide shows the ability to induce c-fos in endothelial cells Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) in growth media (50% Ham's F12 w/o GHT low glucose, and 50% DMEM without glycine with NaHC03, 1% glutamine, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF) were plated on 96-well microtiter plates at a cell density of lxlO^"4 cells/well The day after ^~~ plating, the cells were starved by removing the growth media and treating the cells with 100 μl/well test samples and controls (positive control growth media, negative control 10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6 8) The cells were incubated for 30 minutes at 37°C, in 5% CO, The samples were removed, and the first part of the bDNA kit protocol (Chiron Diagnostics, cat #6005-037) was followed where each capitalized reagent/buffer listed below was available from the kit

Briefly, the amounts of the TM Lysis Buffer and Probes needed for the tests were calculated based on information provided by the manufacturer The appropπate amounts of thawed Probes were added to the TM Lysis Buffer The Capture Hybridization Buffer was warmed to room temperature The bDNA strips were set up in the metal strip holders, and 100 μl of Capture Hybridization Buffer was added to each b-DNA well needed, followed by incubation for at least 30 minutes The test plates with the cells were removed from the incubator, and the media was gently removed using the vacuum manifold 100 μl of Lysis Hybridization Buffer with Probes were quickly pipetted into each well of the microtiter plates The plates were then incubated at 55 °C for 15 minutes Upon removal from the incubator, the plates were placed on the vortex mixer with the microtiter adapter head and vortexed on the #2 setting for one minute 80 μl of the lysate was removed and added to the bDNA wells containing the Capture Hybridization Buffer, and pipetted up and down to mix The plates were incubated at 53 °C for at least 16 hours

On the next day, the second part of the bD A kit protocol was followed Specifically, the plates were removed from the incubator and placed on the bench to cool for 10 minutes The volumes of additions needed were calculated based upon information provided by the manufacturer An Amplifier Working Solution was prepared by making a 1 100 dilution of the Amplifier Concentrate (20 frn/μl) in AL Hybridization Buffer The hybridization mixture was removed from the plates and washed twice with Wash A 50 μl of Amplifier Working Solution was added to each well and the wells were incubated at 53 °C for 30 minutes The plates were then removed from the incubator and allowed to cool for 10 minutes The Label Probe Working Solution was prepared by making a 1 100 dilution of Label Concentrate (40 pmoles/μl) in AL Hybridization Buffer After the 10-mιnute cool-down period, the amplifier hybridization mixture was removed and the plates were washed twice with Wash A 50 μl of Label Probe Working Solution was added to each well and the wells were incubated at 53 °C for 15 minutes After cooling for 10 minutes, the Substrate was warmed to room temperature Upon addition of 3 μl of Substrate Enhancer to each ml of Substrate needed for the assay, the plates were allowed to cool for 10 minutes, the label hybridization mixture was removed, and the plates were washed twice with Wash A and three times with Wash D 50 μl of the Substrate Solution with Enhancer was added to each well The plates were incubated for 30 minutes at 37°C and RLU was read in an appropπate luminometer

The replicates were averaged and the coefficient of variation was determined The measure of activity of the fold increase over the negative control (HEPES buffer described above) value was indicated by chemiluminescence units (RLU) Samples that show an at least two-fold value over the negative control value were considered positive

PR0216 assayed "positive" two times (1) Negative Control = 4 18 RLU -

Positive control = 14 95 RLU PRO216 at 0 01% = 10 33 RLU (2) Negative control = 6 35 RLU

Positive control = 30 46 RLU PRO216 at 0 01% = 13 37 RLU

EXAMPLE 6 Guinea Pig Vascular Leak

This assay is designed to determine whether PRO302 polypeptide shows the ability to induce vascular permeability

Hairless guinea pigs weighing 350 grams or more were anesthetized with Ketamme (75-80 mg/kg) and 5 mg/kg Xylazine intramuscularly Test samples containing the PRO302 polypeptide or a physiological buffer without the test polypeptide are injected into skin on the back of the test animals with 100 μl per injection site intradermally There were approximately 16-24 injection sites per animal One ml of Evans blue dye (1% in PBS) is then injected intracardially Skin vascular permeability responses to the compounds (/ e , blemishes at the injection sites of injection) are visually scored by measuring the diameter (in mm) of blue-colored leaks from the site of injection at 1 and 6 hours post administration of the test materials The mm diameter of blueness at the site of injection is observed and recorded as well as the severity of the vascular leakage Blemishes of at least 5 mm in diameter are considered positive for the assay when testing purified proteins, being indicative of the ability to induce vascular leakage or permeability A response greater than 7 mm diameter is considered positive for conditioned media samples Human VEGF at 0 1 μg/100 μl is used as a positive control, inducing a response of 15-23 mm diameter The results are shown in Table 2 below

Table 2 PRO Name Hours Post-Iniection mm Blueness (diameter)

PRO302 1 0 9 0

PRO302 6 0 9 0 EXAMPLE 7 In situ Hybridization In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations It may be useful, for example, to identify sites of gene expression, analyze the 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 ^3jP-labeled πboprobes 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 πboprobe 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 — P-Riboprobe synthesis

6 0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed-vacuum dried To each tube containing dried ³³P-UTP, the following ingredients were added

2 0 μl 5x transcription buffer

1 O μl DTT (lOO mM)

2 0 μl NTP mix (2 5 mM 10 μl each of 10 mM GTP, CTP & ATP + 10 μl H₂0) 1 0 μl UTP (50 μM) 1 0 μl RNAsm

1 0 μl DNA template (1 μg) 1 0 μl H₂0

1 0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually)

The tubes were incubated at 37 °C for one hour A total of 1 0 μl RQ1 DNase was added, followed by incubation at 37°C for 15 minutes A total of 90 μl TE ( 10 mM Tris pH 7 6/1 M EDTA pH 8 0) was 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, a total of 100 μl TE was added, then 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 A total of 1-3 μl of the probe or 5 μl of RNA Mrk III was 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 wells of gel were flushed, and the sample was loaded and run at 180-250 volts for 45 minutes The gel was wrapped in plastic wrap (SARAN™ brand) and exposed to XAR film with an intensifying screen in a -70°C freezer one hour to overnight ^P-Hvbπdιzatιon

A Pretreatment of frozen sections

The slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes The trays were placed in a 55 °C incubator for five minutes to reduce condensation The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0 5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H₂0) After deprote ation 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 temperature The sections were dehydrated in 70%, 95%, and 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) for human embryo tissue, or 8 x proteinase K ( 100 μl in 250 ml Rnase buffer, 37°C, 30 minutes) for formalin tissues Subsequent rinsing in 0 5 x SSC and dehydration were performed as described above

C Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper The tissue was covered with 50 μl of hybridization buffer (3 75 g 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, and the tissue was vortexed well and incubated at 42 ^αC 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 slides were cooled on ice, and 48 μl hybridization buffer was added per slide After vortexing, 50 μl ³³P mix was added to 50 μl prehybridization on the slide The slides were incubated overnight at 55 °C

E Washes

Washing was done for 2x 10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0 25

M EDTA, V_f=4L), followed 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 x10 minutes with 2 x SSC, EDTA at room temperature 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.- L)

F Oligonucleotides

In situ analysis was performed on two of the DNA sequences disclosed herein The oligonucleotides employed for these analyses are as follows

(1) DNA33223-1 136 (PRO230) 33223pl

5'-GGATTCTAATACGACTCACTATAGGGCGGCGATGTCCACTGGGGCTAC-3' (SEQ ID NO 17) 33223p2 5'- CTATGAAATTAACCCTCACTAAAGGGACGAGG AAGATGGGCGGATGGT-3' (SEQ ID NO 18) (2) DNA33087 (PRO216-) 33087pl :

5'-GGATTCTAATACGACTCACTATAGGGCTGGATGGGCTAGTAAACTTGA-3' (SEQ ID NO: 19) 33087p2: 5'-CTATGAAATTAACCCTCACTAAAGGGACCCTTCTGCTCCTTCTTGTT-3' (SEQ ID NO:20)

G. Results ^~~

In situ analysis was performed on the above two DNA sequences disclosed herein. The results from these analyses are as follows: (1) DNA33223-1 136 (PRO230)

Sections showed an intense signal associated with arterial and venous vessels in the fetus. In arteries, the signal appeared to be confined to smooth-muscle/pericyte cells. The signal was also seen in capillary vessels and in glomeruli. It was not clear whether endothelial cells were expressing this mRNA. Expression was also observed in epithelial cells in the fetal lens. Strong expression was also seen in cells within placental trophoblastic villi; these cells lie between the trophoblast and the fibroblast-like cells that express HGF-uncertain histogenesis. In the adult, there was no evidence of expression and the wall of the aorta and most vessels appeared to be negative. However, expression was seen over vascular channels in the normal prostate and in the epithelium lining the gallbladder. Insurers expression was seen in the vessels of the soft-tissue sarcoma and a renal cell carcinoma.

In summary, PRO230 is a molecule that shows relatively specific vascular expression in the fetus as well as in some adult organs. Expression was also observed in the fetal lens and the adult gallbladder.

In a secondary screen, vascular expression was observed in fetal blocks, similar to that observed above. Expression was on vascular smooth muscle, rather than endothehum. Expression was also seen in smooth muscle of the developing oesophagus, so this molecule is not vascular specific. Expression was examined in four lung and four breast carcinomas. Substantial expression was seen in vascular smooth muscle of at least three out of four lung cancers and two out of four breast cancers. In addition, in one breast carcinoma, expression was observed in peritumoral stromal cells of uncertain histogenesis (possibly myofibroblasts). No endothelial cell expression was observed in this study. (2) DNA33087 (PRO216)

Sections showed strong specific expression in osteoblasts at all sites of enchondral and periosteal new bone formation. Additional sites of expression included the developing pulmonary arterial and aortic trunks. Otherwise, all fetal tissues tested negative. The fetal tissues examined included: placenta, umbilical cord, brain, spinal cord, eye, optic nerve, trachea, lung, heart, thymus, liver, spleen, esophagus, small intestine, pancreas, adrenal, thyroid, body wall, and lower limb. All of the adult tissues were negative. The adult tissues examined included: liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung and skin. PR0216 has a probable role in control of bone matrix deposition and/or osteoblast growth. All adult tissues in the multiblock were positive for beta-actin. EXAMPLE 8 Use of PRO230, PRQ216 or PRO302 as a Hybridization Probe The following method describes use of a nucleotide sequence encoding PRO230, PR0216 or PRO302 as a hybridization probe DNA comprising the coding sequence of full-length or mature PRO230, PR0216 or PRO302 (as shown in

Figures 1 A- 1 B, 3 and 5, respectively, SEQ ID NOS 1 , 6 and 1 1 , respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO230, PR0216 or PRO302) in human tissue cDNA libraries or human tissue genomic libraries

Hybridization and washing of filters containing either library DNAs is performed under the following high- stringency conditions Hybridization of radiolabeled probe derived from the gene encoding a PRO230, PR0216 or PRO302 polypeptide 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 I x SSC and 0 1% SDS at 42°C

DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art

EXAMPLE 9 Expression of Nucleic Acid Encoding PRO230. PRQ216 or PRO302 in E coli This Example illustrates preparation of an unglycosylated form of PRO230, PR0216 or PRO302 by recombinant expression in E coli

The DNA sequence encoding PRO230, PR0216 or PRO302 (SEQ ID NOS 1, 6 and 1 1, respectively) is initially amplified using selected PCR primers The primers should contain restriction enzyme sites that correspond to the restriction enzyme sites on the selected expression vector A variety of expression vectors may be employed An example of a suitable vector is pBR322 (derived from E coli, see Bolivar et al , Gene, 2 95 (1977)), which contains genes for ampicilhn and tetracychne resistance The vector is digested with restriction enzyme and dephosphorylated The PCR-amphfied sequences are then hgated into the vector The vector will preferably include sequences that encode an antibiotic-resistance gene, a tφ promoter, a poly-His leader (including the first six STII codons, poly-His sequence, and enterokinase cleavage site), the region encoding PRO230, PR0216 or PRO302, lambda transcriptional terminator, and an argU gene The hgation 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 with antibiotics The overnight culture may subsequently be used to inoculate a larger-scale culture The cells are then grown to a desired optical density, during which the expression promoter is turned on

After culturing the cells for several more hours, the cells can be harvested by centπfugation The cell pellet obtained by the centrifiigation can be solubihzed using various agents known in the art, and the solubilized PRO230, PR0216 or PRO302 polypeptide can then be purified using a metal-chelating column under conditions that allow tight binding of the polypeptide

EXAMPLE 10

Expression of Nucleic Acid Encoding PRO230. PRQ216 or PRO302 in Mammalian Cells This Example illustrates preparation of a potentially glycosylated form of PRO230, PR0216 or PRO302 by ^~~ recombinant expression m mammalian cells

The vector, pRK5 (see, EP 307,247, published March 15, 1989), is employed as the expression vector Optionally, the PRO230, PR0216 or PRO302 DNA is hgated into pRK5 with selected restriction enzymes to allow insertion of the DNA encoding PRO230, PR0216 or PRO302 using gation methods such as described in Sambrook et al , supra The resulting vector is called pRK5-(DNA encoding PRO230, PR0216 or PRO302)

In one embodiment, the selected host cells are 293 cells Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics About 10 μg DNA of pRK5-(DNA encoding PRO230, PR0216 or

PRO302) is mixed with about 1 μg DNA encoding the VA RNA gene (Thimmappaya et al , Cell, 3 . 543 (1982)) and dissolved in 500 μl of 1 mM Tπs-HCl, 0 1 mM EDTA, 0 227 M CaCl, 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 the 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 the cells are incubated for about 5 days

Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml ³⁵S-cysteιne and 200 μCi/ml ³⁵S-methιomne 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 the

PRO230, PR0216 or PRO302 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, the gene encoding PRO230, PR0216 or PRO302 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Nati Acad Sci . 12 7575 ( 1981 ) 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-(DNA encoding PRO230, PR0216 or PRO302) is added The cells are first concentrated from the spinner flask by centπfugation and washed with 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 the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin, and 0 1 μg/ml bovine transferπn After about four days, the conditioned media is centrifuged and filtered to remove cells and debris The sample containing the expressed gene encoding the PRO230, PR0216 or PRO302 polypeptide can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography In another embodiment the gene encoding PRO230, PR0216 or PRO302 can be expressed in CHO cells The pRK5-(DNA encoding PRO230, PR0216 or PRO302) nucleic acid can be transfected into CHO cells using known reagents such as CaP0₄ or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as ³⁵S-methιonιne After determining the presence of PRO230, PR0216 or PRO302 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 PRO230, PR0216 or PRO302 can then be concentrated and purified by any selected method

PRO230 and PR0216 were stably expressed in CHO cells by the above described method Epitope-tagged gene encoding the PRO230, PR0216 or PRO302 polypeptide may also be expressed in host

CHO cells The gene encoding PRO230. PR0216 or PRO302 may be subcloned out of the pRK5 vector The subclone insert can undergo PCR amplification to fuse in frame with a selected epitope tag such as a poly-His tag into a baculovirus expression vector The gene insert encoding the polv-Hιs-tagged-PRO230, -PR0216 or - PRO302 can then be subcloned into a SV40- driven vector containing a selection marker such as DHFR for selection of stable clones Finally, the CHO cells can be transfected (as described above) with the SV40-dπven vector Labeling may be performed, as described above, to verify expression The culture medium containing the expressed gene encoding the poly-Hιs-tagged-PRO230, -PR0216 or -PRO302 can then be concentrated and purified by any selected method, such as by Nr⁺-chelate affinity chromatography

EXAMPLE 1 1

Expression of Nucleic Acid Encoding PRO230, PRQ216 or PRO302 in Yeast The following method describes recombinant expression of the gene encoding PRO230, PR0216 or PRO302 in yeast

First, yeast expression vectors are constructed for intracellular production or secretion of PRO230 PR0216 or PRO302 from the ADH2'GAPDH promoter DNA encoding PRO230 PR0216 or PRO302 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the gene encoding PRO230, PR0216 or PRO302 For secretion, DNA encoding PRO230, PR0216 or PRO302 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO230, PR0216 or PRO302 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of the gene encoding

PRO230, PR0216 or PRO302

Yeast cells, such as yeast strain AB 1 10, can then be transformed with the expression plasmids described above and cultured in selected fermentation media The transformed yeast supernatants can be analyzed by precipitation with 10% tπchloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain

Recombinant PRO230, PR0216 or PRO302 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centπfugation and then concentrating the medium using selected cartridge filters The concentrate containing PRO230, PR0216 or PRO302 may further be purified using selected column- chromatography resins

EXAMPLE 12 Expression of Nucleic Acid Encoding PRO230 PRQ216 or PRO302 in Baculovirus-Infected Insect Cells

The following method describes recombinant expression in Baculovirus-infected insect cells The sequence coding for PRO230, PR0216 or PRO302 is fused upstream of an epitope tag contained within ^~~ a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding PRO230, PR0216 or PRO302 or the desired portion of the coding sequence of PRO230, PR0216 or PRO302 [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incoφorate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector

Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA

(Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 171 1 ) using hpofectιn(commercιaily available from GIBCO-BRL) After 4 - 5 days of incubation at28°C, the released viruses are harvested and used for further amplifications Viral infection and protein expression are performed as described by O'Reilley et al , Baculovirus Expression Vectors A Laboratory Manual (Oxford Oxford University Press ( 1994))

Expressed poly-Hιstagged-PRO230, -PRO216or-PRO302canthen be purified, for example, byNi "-chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al , Nature, 362 175- 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 l% NP-40, 0 4 M KC1), and sonicated twice for 20 seconds on ice The sonicates are cleared by centrifiigation, 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 Ni

^'"-NT A agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with

25 ml of water and equilibrated with 25 ml of loading buffer The filtered cell extract is loaded onto the column at 0 5 ml per minute The column is washed to baseline A₂₈₀ with loading buffer, at which point fraction collection is started Next, the column is washed with a secondary wash buffer (50 mM phosphate 300 mM NaCl, 10% glycerol, pH 6 0), which elutes non-specifically- bound protein After reaching A₂₈₀ baseline again, the column is developed with a 0 to 500 mM lmidazole gradient in the secondary wash buffer One ml fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni ^:+-NTA-conjugated to alkaline phosphatase

(Qiagen) Fractions containing the eluted Hιs_!0-tagged-PRO230 -PR0216 or -PRO302, respectively, are pooled and dialyzed against loading buffer

Alternatively, purification of the IgG-tagged (or Fc tagged)-PRO230, -PR0216 or -PRO302 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography PRO230 and PR0216 were expressed in baculovirus infected Sf9 insect cells While 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 (lmmunoadhesin), 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 Following PCR amplification, the respective coding sequences were subcloned into a baculovirus expression vector (pb PH IgG for IgG fusions and pb PH His c for poly-His tagged proteins), and the vector and Baculogold® baculovirus DNA (Pharmingen) were co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL ~~ 171 1), using Lipofectin (Gibco BRL) pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified poly nker regions to include the His or Fc tag sequences The cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells were incubated for 5 days at 28°C The supernatant was harvested and subsequently used for the first viral amplification 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 at28°C The supernatant was harvested and the expression of the constructs in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 ml of Ni ^2*-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 amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate 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 centπfugation 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 ^2*-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, the 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% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C lmmunoadhesin (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, 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 ml of 1 M Tπs 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

Alternatively, a modified baculovirus procedure mav be used incoφorating hιgh-5 cells In this procedure the DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIE 1 - 1 (Novagen) The pIE 1 - 1 and pIE 1 -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus lei promoter in stably-transformed insect cells The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lei -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element pIEl- 1 and pIEl-2 include the translation initiation site and can be used to produce fusion proteins Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5 and 3' regions The 5' primer may incoφorate flanking (selected) restriction enzyme sites The product was then digested with those selected restriction enzymes and subcloned into the expression vector For example, derivatives of pIE 1 - 1 can include the Fc region of human IgG (pb PH IgG) or an 8 histidine (pb PH His) tag downstream (3'-of) the desired sequence Preferably, the vector construct is sequenced for confirmation

Hιgh-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no C02, NO pen/strep For each 150 mm plate, 30 μg of pIE based vector containing the sequence was 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 μl of CellFectin (CellFECTIN (GibcoBRL # 10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium The two solutions were combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex-Cell media was added to the 2 ml of DNA/CellFECTIN mix and this is layered on hιgh-5 cells that have been washed once with Ex-Cell media The plate is then incubated in darkness for 1 hour at room temperature The DNA/CellFECTIN mix is then aspirated and the cells are washed once with Ex-Cell to remove excess CellFECTIN 30 ml of fresh Ex-Cell media was added and the cells are incubated for 3 davs at 28 °C The supernatant was harvested and the expression of the sequence in the baculovirus 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 conditioned media from the transfected cells (0 5 to 3 L) was harvested by centπfugation to remove the cells and filtered through 0 22 micron filters For the poly-His tagged constructs, the protein comprising the sequence is purified using a Ni ²~-NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media was pumped onto a 6 ml Ni ⁷ -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/mm at 48°C After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein was then 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 lmmunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows The conditioned media 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 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 sequence ^~~ was assessed by SDS polyacrylamide gels and by N-terminal ammo acid sequencing by Edman degradation and other analytical procedures as desired or necessary PR0216 and PRO302 were successfully expressed by the above modified baculovirusprocedureincoφorating hιgh-5 cells

EXAMPLE 13 Preparation of Antibodies that Bind PRO230 PRQ216 or PRO302 This Example illustrates preparation of monoclonal antibodies that can specifically bind PRO230, PR0216 or PRO302

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 PRO230, PR0216 or PRO302 fusion proteins containing PRO230, PR0216 or PRO302, and cells expressing the gene encoding PRO230, PR0216 or PRO302 on the cell surface Selection of the immunogen can be made by the skilled artisan without undue experimentation

Mice, such as Balb/c, are immunized with the PRO230, PR0216 or PRO302 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1 to 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 with additional immunization injections Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect antι-PRO230, antι-PR0216 or antι-PRO302 antibodies

After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO230, PR0216 or PRO302 Three to four days later, the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU 1, available from ATCC, No CRL 1597 The fusions generate hybridoma cells that can then be plated in 96-well tissue culture plates containing HAT 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 PRO230, PR0216 or PRO302

Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies agaιnstPRO230, PR0216 or PRO302 is withm the skill in the art The positive hybridoma cells can be injected lntrapeπtoneally into syngeneic Balb/c mice to produce ascites containing the antι-PRO230, antι-PR0216 or antι-PRO302 monoclonal antibodies Alternatively, the hybridoma cells can be grown m tissue-culture flasks or roller bottles Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium-sulfate precipitation, followed by gel-exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed

Deposit of Material

The following mateπal(s) has/have been deposited with the American Type Culture Collection, 10801 University Blvd , Manassas, VA 201 10-2209, USA (ATCC) Material ATCC Pep No Deposit Date

DNA33223-1136 209264 September 16, 1997

DNA33087 209381 October 16, 1997

DNA40370-1217 209485 November 21, 1997

This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Puφose of Patent Procedure and the Regulations thereunder (Budapest Treaty)

This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit The deposit 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 public upon issuance of the pertinent U S patent or upon laying open to the public of any U S or foreign patent application whichever comes first, and assures availability of the progeny to one determined by the U S Commissioner of Patents and Trademarks to be entitled thereto 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 that if a culture of the mateπal(s) on deposit should die or be lost or destroyed when cultivated under suitable conditions, the mateπal(s) will be promptly replaced on notification with another of the same Availability of the deposited mateπal(s) is not to be construed 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 construct(s) deposited, since the deposited embodiment s) is/are intended as single ιllustratιon(s) of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention The deposit of mateπal(s) herein does not constitute an admission that the 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 construed as limiting the scope of the claims to the specific illustrations that it represents Indeed, various modifications of the invention m addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims

Claims

WHAT IS CLAIMED IS:

1 A composition comprising (a) a PRO230, PR0216 or PRO302 polypeptide, (b) an agonist of a PRO230, PR0216 or PRO302 polypeptide, or (c) an antagonist of a PRO230, PR0216 or PRO302 polypeptide, in admixture with a pharmaceutically acceptable carrier

2 The composition of Claim 1 comprising a therapeutically effective amount of(a) a PRO230, PR0216 or PRO302 polypeptide, (b) an agonist of a PRO230, PR0216 or PRO302 polypeptide, or (c) an antagonist of a PRO230, PR0216 or PRO302 polypeptide, thereof

3 The composition of Claim 1 , wherein the agonist is an antι-PRO230, antι-PR0216 or antι-PRO302 antibody

4 The composition of Claim 1, wherein the antagonist is an antι-PRO230, antι-PR0216 or antι-PRO302 antibody

5 The composition of Claim 1 further comprising a cardiovascular, endothelial, angiogenic or angiostatic agent

6 A method of preparing the composition of Claim 1 comprising admixing (a) a PRO230, PR0216 or PRO302 polypeptide, (b) an agonist of a PRO230, PR0216 or PRO302 polypeptide, or (c) an antagonist of a PRO230, PR0216 or PRO302 polypeptide, with a pharmaceutically acceptable carrier

7 An article of manufacture comprising

(a) a composition comprising (a) a PRO230, PR0216 or PRO302 polypeptide (b) an agonist of a PRO230, PR0216 or PRO302 polypeptide, or (c) an antagonist of a PRO230, PR0216 or PRO302 polypeptide, in admixture with a pharmaceutically acceptable carrier.

(b) a container containing said composition, and

(c) a label affixed to said container, or a package insert included in said pharmaceutical product referring to the use of said (a) PRO230, PR0216 or PRO302 polypeptide, (b) agonist of a PRO230, PR0216 or PRO302 polypeptide, or (c) antagonist of a PRO230, PR0216 or PRO302 polypeptide, in the treatment of a cardiovascular, endothelial, and angiogenic disorder

8 The article of manufacture of Claim 7, wherein said agonist is an antι-PRO230, antι-PR0216 or anti- PRO302 antibody

9 The article of manufacture of Claim 7, wherein said antagonist is an antι-PRO230, antι-PR0216 or anti- PRO302 antibody

10 The article of manufacture of Claim 7, wherein said composition comprises a therapeutically effective amount of said (a) PRO230, PR0216 or PRO302 polypeptide, (b) agonist of a PRO230, PR0216 or PRO302 polypeptide or (c) antagonist of a PRO230 PR0216 or PRO302 polypeptide, in admixture with said pharmaceutically acceptable carrier

11 A method for identifying an agonist of a PRO230, PR0216 or PRO302 polypeptide comprising

12 The method of Claim 1 1 , wherein the cellular response normally induced by said PRO230, PR0216 or PRO302 polypeptide is stimulation of cell proliferation

13 A method for identifying a compound that inhibits an activity of a PRO230, PR0216 or PRO302 polypeptide comprising contacting a test compound with a PRO230, PR0216 or PRO302 polypeptide under conditions and for a time sufficient to allow the test compound and polypeptide to interact and determining whether the activity of said PRO230, PR0216 or PRO302 polypeptide is inhibited

14 A method for identifying a compound the inhibits an activity of a PRO230, PR0216 or PRO302 polypeptide comprising the steps of

(a) contacting cells and a test compound to be screened in the presence of PRO230, PR0216 or PRO302 polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO230 PRO216 or PRO302 polypeptide, and

(b) determining the induction of said cellular response to determine if the test compound is an effective antagonist

15 The method of Claim 14, wherein the cellular response normally induced by said PRO230, PR0216 or PRO302 polypeptide is stimulation of cell proliferation

16 A method for identifying a compound that inhibits the expression of a PRO230, PR0216 or PRO302 polypeptide in cells that normally expresses the polypeptide, wherein the method comprises contacting the cells with a test compound under conditions suitable for allowing expression of said PRO230, PR0216 or PRO302 polypeptide and determining whether the expression of the PRO230, PR0216 or PRO302 polypeptide is inhibited 17 An agonist of a PRO230 PR0216 or PRO302 polypeptide

18 An antagonist of a PRO230 PR0216 or PR0302 polypeptide

19 A compound that inhibits the expression of a PRO230, PR0216 or PRO302 polypeptide in a mammalian cell

20 The compound of Claim 19 wherein said compound is an antisense oligonucleotide

21 An antibody that binds to a PRO230, PR0216 or PRO302 polypeptide

22 The antibody of Claim 21 which is a monoclonal antibody

23 The antibody of Claim 21 which is an antibody fragment

24 The antibody of Claim 21 which is a single-chain antibody

25 A method for diagnosing a disease or susceptibility to a disease which is related to a mutation in a PRO230, PR0216 or PRO302 polypeptide-encoding nucleic acid sequence comprising

(a) isolating a nucleic acid sequence encoding a PRO230, PR0216 or PRO302 polypeptide from a sample derived from a host, and

26 A method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises analyzing the level of expression of a gene encoding a PRO230, PR0216 or PRO302 polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in said mammal

27 A method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises detecting the presence or absence of a PRO230, PR0216 or PRO302 polypeptide in a test sample of tissue cells obtained from said mammal wherein the presence or absence of said PRO230, PR0216 or PRO302 polypeptide in said test sample is indicative of the presence of a cardiovascular endothelial or angiogenic disorder in said mammal 28 A method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal comprising

(a) contacting an antι-PRO230, antι-PR0216 or antι-PRO302 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antι-PRO230, antι-PR0216 or anti- PRO302 antibody and a PRO230, PR0216 or PRO302 polypeptide the test sample, wherein the formation of said complex is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in the mammal

29 A method for determining the presence of a PRO230, PR0216 or PRO302 polypeptide in a sample ^~~ comprising contacting a sample suspected of containing a PRO230, PR0216 or PRO302 polypeptide with an anti- PRO230, antι-PR0216 or antι-PRO302 antibody and determining binding of said antibody to a component of said sample

30 A cardiovascular, endothelial or angiogenic disorder diagnostic kit comprising an antι-PRO230, anti- PR0216 or antι-PRO302 antibody and a carrier in suitable packaging

31 The kit of Claim 30 further comprising instructions for using said antibody to detect the presence of the PRO230, PR0216 or PRO302 polypeptide

32 A method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal a therapeutically effective amount of (a) a PRO230, PR0216 or PRO302 polypeptide,

(b) an agonist of a PRO230, PR0216 or PRO302 polypeptide. or (c) an antagonist of a PRO230, PR0216 or PRO302 polypeptide

33 The method of Claim 32, wherein the disorder is cardiac hypertrophy, trauma, or cancer

34 The method according to Claim 32, wherein the mammal is human

35 The method of Claim 34, wherein the human has suffered myocardial infarction

36 The method of Claim 34, wherein the human has cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration

37 The method of Claim 36, wherein the cardiac hypertrophy is characterized by the presence of an elevated level of PGF_2(I

38 The method of Claim 32, wherein the PRO230, PR0216 or PRO302 polypeptide is administered together with a cardiovascular, endothelial or angiogenic agent 39 The method of Claim 36, wherein the PRO230, PR0216 or PRO302 polypeptide is administered following primary angioplasty

40 The method of Claim 32, wherein the cardiovascular, endothelial or angiogenic disorder is cancer

41 The method of Claim 40, wherein the PRO230, PR0216 or PRO302 polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent or a cytotoxic agent

42 The method of Claim 32 wherein said agonist is an antι-PRO230, antι-PR0216 or antι-PRO302 antibody

43 The method of Claim 32 wherein said antagonist is an antι-PRO230, antι-PR0216 or antι-PRO302 antibody

44 A method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal a nucleic acid molecule that encodes (a) a PRO230, PR0216 or PRO302 polypeptide, (b) an agonist of a PRO230, PR0216 or PRO302 polypeptide, or (c) an antagonist of a PRO230, PR0216 or PRO302 polypeptide

45 The method of Claim 44 wherein said agonist is an antι-PRO230, antι-PR0216 or antι-PRO302 antibody

46 The method of Claim 44 wherein said antagonist is an antι-PRO230, antι-PR0216 or antι-PRO302 antibody

47 The method of Claim 44, wherein the mammal is human

48 The method of Claim 44, wherein the nucleic acid molecule is administered via ex vivo gene therapy

49 A recombinant retroviral particle comprising a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO230, PR0216 or PRO302 polypeptide, (b) an agonist polypeptide of a PRO230, PR0216 or PRO302 polypeptide, or (c) an antagonist polypeptide of a PRO230, PR0216 or PRO302 polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein the retroviral vector is in association with retroviral structural proteins

50 An ex vivo producer cell comprising a nucleic acid construct that expresses retroviral structural proteins and also comprises a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO230, PR0216 or PRO302 polypeptide, (b) an agonist polypeptide of a PRO230, PR0216 or PRO302 polypeptide, or (c) an antagonist polypeptide of a PRO230, PR0216 or PRO302 polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein said producer cell packages the retroviral vector in association with the structural proteins to produce recombinant retroviral particles

51 A method for inhibiting endothelial cell growth in a mammal comprising administering to the mammal (a) a PR0216 polypeptide, (b) an agonist of a PR0216 polypeptide, (c) an antagonist of a PRO230, PR0216 or ^~~ PRO302 polypeptide, or (d) an antι-PR0216 antibody, wherein endothelial cell growth in said mammal is inhibited

52 A method for stimulating endothelial cell growth in a mammal comprising administering to the mammal (a) a PRO230 polypeptide, (b) an agonist of a PRO230 polypeptide, (c) an antagonist of a PRO230 polypeptide, or (d) an antι-PRO230 antibody, wherein endothelial cell growth in said mammal is stimulated

53 A method for inhibiting angiogenesis induced by a PRO302 polypeptide in a mammal comprising administering a therapeutically effective amount of an antι-PRO302 antibody to the mammal, wherein said angiogenesis is inhibited