JP2003531811A - Promotion or inhibition of angiogenesis and cardiovascularization - Google Patents

Abstract

(57) SUMMARY Disclosed are compositions and methods for stimulating or inhibiting angiogenesis and / or cardiovascular formation in mammals, including humans. Pharmaceutical compositions are based on the polypeptides or antagonists identified for one or more of these uses. Diseases diagnosed, prevented or treated by the compositions herein include trauma such as wounds, various cancers, and vascular diseases including atherosclerosis and cardiac hypertrophy. Furthermore, the present invention relates to novel polypeptides and nucleic acid molecules encoding these polypeptides. Also herein, vectors and host cells containing these nucleic acid sequences, chimeric polypeptide molecules containing the polypeptide of the present invention fused to a heterologous polypeptide, antibodies that bind to the polypeptide of the present invention, and polypeptides of the present invention A manufacturing method is also provided.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to compositions useful for promoting or inhibiting angiogenesis and / or cardiovascularization in mammals in need of such biological effects. And method.
This includes diagnosis and treatment of cardiovascular disorders as well as carcinogenic diseases.

(Description of Background) A. Heart Disease and Factors About 5 million Americans have heart failure, and about 400,000 new heart failure cases each year. It is the single most frequent cause of hospitalization for people over the age of 65 in the United States. Recent advances in the management of acute heart failure, including acute myocardial infarction, have resulted in an increasing number of patients with chronic heart failure. From 1979 to 1995, congestive heart failure (CHF
) Patients increased from 377,000 to 872,000 (up 130 percent), with 1 CHF death
16% increase. CHF is a syndrome characterized by left ventricular dysfunction, reduced exercise tolerance, poor living standards, and markedly reduced life expectancy. A prerequisite for heart failure is the inability of the heart to pump blood at a rate sufficient to meet the metabolic requirements of body tissues (in other words, lack of cardiac output). At least four major compensatory mechanisms are activated in the case of heart failure, including peripheral vasoconstriction, increased heart rate, increased cardiac contractility, and increased plasma volume. These effects are mediated primarily by the sympathetic nervous system and the renin-angiotensin system. Eichhor
n, American Journal of Medicine, 104: 163-169 (1998). Increased output from the sympathetic nervous system increases pulse rate, heart rate, and contractility. Angiotensin II 1) directly stimulates vascular smooth muscle contraction, 2) stimulates aldosterone and antidiuretic hormone secretion to enhance plasma volume expansion, 3) stimulates sympathetic nerve-mediated vascular tone, and 4) It raises blood pressure by catalyzing the denaturation of bradykinin, which has vasodilation and natriuretic activity. Brown and Vaughan, Circulation, 97: 1411
-1420 (1998). As described below, Angiotensin II also has a direct detrimental effect by promoting myocyte necrosis (exacerbated systolic function) and cardiac fibrosis (diastole and, in some cases, impaired systolic function). Have. Web
er, Circulation, 96: 4065-4082 (1998).

A common feature of congestive heart failure (CHF) is cardiac hypertrophy, the expansion of the heart is activated by both mechanical and hormonal stimuli, allowing the heart to meet the demand for increased cardiac output. To Morgan and Baker, Circulation, 83: 13-25 (1991). This hypertrophic response is often associated with various characteristic pathologies such as hypertension, aortic stenosis, myocardial infarction, cardiomyopathy, valvular regurgitation, and intracardiac shunts, all of which are associated with chronic hemodynamic hyperactivity. It becomes a load. Hypertrophy is usually defined as an increase in the size of an organ or structure that does not involve natural growth that does not involve tumor formation. Hypertrophy of the heart occurs due to either or both an increase in the mass of individual cells (myocytes) or an increase in the number of cells forming the tissue (hyperplasia). The expansion of the fetal heart depends mainly on the increase in the number of myocytes (which lasts until just after birth),
Postnatal cardiac myocytes lose their proliferative capacity. Further development occurs through hypertrophy of individual cells. Adult myocyte expansion is initially beneficial as a short-term response to impaired cardiac function by allowing the individual muscle fibers to be less stressed initially. However, under severe and prolonged overload, the hypertrophied cells begin to deteriorate,
To die. Katz, "Heart Failure", in: Katz AM ed., Physiology of the He
art (New York: Raven Press, 1992) pp. 638-668. Cardiac hypertrophy is a significant risk factor for mortality and morbidity in the clinical course of heart failure. Katz, Trends Cardiov
asc. Med., 5: 37-44 (1995). For further details of causes and symptoms of cardiac hypertrophy, see, for example, Heart Disease, A Textbook of Cardiovascular Medicine, Braunwald,
E. ed. (WB Saunders Co., 1988), Chapter 14, "Pathophysiology of Heart
Failure ". At the cellular level, the heart is composed of myocytes and surrounding supporting cells, collectively called non-myocytes. Non-myocytes are first fibroblasts / mesenchymal cells, and they are endothelial and smooth. Including myocytes, in fact myocytes form the majority of adult myocardial mass, but represent only about 30% of the total number of cells present in the heart: hormonal, physiological, hemodynamic, and In response to pathological stimuli, adult ventricular myocytes can adapt to an increased burden through activation of the hypertrophic process, which responds to cell division and atrial natriuretic peptide (ANP) Characterized by an increase in contractile protein content of individual cardiomyocytes and an increase in cell size of myocytes, which is accompanied by activation of fetal genes, including genes.
n et al., FASEB J., 5: 3037-3046 (1991); Chien et al., Annu. Rev. Physiol., 55: 77-
95 (1993). Increased myocardial mass resulting from increased myocyte size associated with the accumulation of interstitial collagen in the extracellular matrix and around the intramyocardial coronary artery is associated with left ventricular hypertrophy following pressure overload in humans. It has been described. Caspari et al., Cardiovasc. Res. , 11: 554-558 (1977); Schwarz et al., Am. J. c.
ardiol., 42: 895-903 (1978); Hess et al., Circulation, 63: 360-371 (1981); Pear
Lman et al., Lab. Invest., 46 158-164 (1982).

It has also been suggested that the parasecretory factor produced by non-myocyte supporting cells may be further involved in the development of cardiac hypertrophy, and further various non-myocyte-derived hypertrophic factors such as leukocyte inhibitory factor (LIF) and endothelin. Has been identified. Metcalf, Grow
th Factors, 7: 169-173 (1992); Kurzrock et al., Endocrinne Reviews, 12: 208-21.
7 (1991); Inoue et al., Proc. Natl. Acad. Sci. USA, 86: 2863-2867 (1989); Yanagi.
sawa and Masaki, Treds Pharm. Sci., 10: 374-378 (1989); U.S. Pat.
(Published November 12, 1996). A further exemplary factor that has been identified as a potential mediator of cardiac hypertrophy is cardiotrophin-1 (CT-1) (Pennica et al., Proc. Nat. Acad. Sci. USA,
92: 1142-1146 (1995)), catecholamines, corticosteroids, angiotensin, and prostaglandins. Catecholamines, corticosteroids, angiotensin, prostaglandins, LIF, endothelin, (including endothelin-1, -2, and -3 and big endothelin) and CT-1 are potentially hypertrophic. There are factors that are supposed to be intervening substances. For example, β-adrenergic receptor blockers (β-blockers such as propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, carvedilol, etc.) and verapamil. Is widely used in the treatment of hypertrophic cardiomyopathy. The beneficial effects of β-blockers on symptoms (e.g. chest pain) and exercise load are:
It mainly relies on reducing heart rate, which prolongs diastole and passively increases ventricular filling. Thompson et al., Br. Heart J., 44: 488-98 (1980); Harrison et al., Circ
ulation, 29: 84-98 (1964). Verapamil has been described to improve ventricular filling and possibly reduce myocardial ischemia. Bonow et al., Circulation, 72: 853
-64 (1985). Nifedipine and diltiazem are also sometimes used to treat 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 vasodilator properties, nifedipine can be harmful, especially to patients with outflow disorders. Disopyramide is used to relieve symptoms due to its negative muscle contractile properties. Pollic, N. Engl. J. Med., 307: 9
97-999 (1982). However, in many patients, the initial effect diminishes. Wigle et al., Circulation, 92: 1680-1692 (1995). Antihypertensive drug treatment has been reported to have a beneficial effect on cardiac hypertrophy associated with elevated blood pressure. Examples of drugs used alone or in combination for antihypertensive treatment include calcium antagonists such as nitrendipine; adrenergic receptor inhibitors such as those listed above; for example quinapril, captopril, enalapril, ramipril, Angiotensin converting enzyme (ACE) inhibitors such as benazepril, fosinopril, lisinopril; diuretics such as chlorothiazide, hydrochlorothiazide,
There are hydroflumethiazide, methylchlothiazide, benzthiazide, dichlorophenamide, acetazolamide, and indamide; and calcium channel inhibitors such as diltiazene, nifedipine, verapamil, and nicardipine. For example, treatment of hypertension with diltiazene and captopril showed a decrease in left ventricular muscle mass but did not normalize the Doppler index of diastolic function. Szlach
cic et al., Am. J. Cardiol., 63: 198-201 (1989); Shahi et al., Lancet, 336: 458-461 (
1990). These findings are interpreted to represent that excess amounts of interstitial collagen may remain after degeneration of left ventricular hypertrophy. Rossi et al., Am. Heart J., 124: 700-709 (
1992). Rossi et al., Supra, investigated the effect of captopril on the inhibition and degeneration of interstitial fibrosis and cardiomyocyte hypertrophy in pressure-bearing cardiac hypertrophy in experimental rats.

Agents that directly increase myocardial contractility (inotropic agents) were initially thought to be useful in patients with heart failure to improve cardiac output in the short term. However, all positive inotropic agents, except digoxigenin, increase mortality in the long term, even though they improve cardiac performance in the short term. Massie, Curr. Op. In Cardiology, 12:
209-217 (1997); Reddy et al., Curr. Opin. Cardiol., 12: 233-241 (1997). Recently,
β-adrenergic receptor inhibitors have been proposed for use in heart failure. The findings from clinical trials suggest that improved cardiac function can be achieved without increasing mortality, but improved survival of patients has not yet been demonstrated. Also, US Pat. Nos. 5,935,924, 5624806 for cardiotropin-1 or its antagonists for CHF treatment, or growth hormone and / or insulin-like growth factor-I.
See No. 5661122; and 5610134 and International Patent Application 95/28173. Another treatment modality is heart transplantation, which is limited by the availability of donor hearts. Endothelin is a vasoconstrictor peptide comprising 21 amino acids, isolated from porcine arterial endothelial culture supernatant and structurally determined. Yanagisawa et al., Nature, 332:
411-415 (1998). Endothelin was later discovered to have various effects, and endothelin antibodies such as endothelin antagonists have been shown to be effective in treating myocardial infarction, renal failure, and other diseases. Such endothelins are present in vivo and are shown to have vasoconstrictor activity, which is expected to be an endogenous factor in the regulation of the circulatory system, and also in hypertension, heart diseases such as myocardial infarction, acute renal failure, etc. Can be involved in various kidney diseases. Endothelin antagonists are described, for example, in US Pat. No. 5,773,414;
It is described in Japanese Patent Publication No. 3130299/1991, European Patent 457195, European Patent 460679; and European Patent 552489. A novel endothelin B receptor for identifying endothelin receptor antagonists is described in US Pat. No. 5,732,223. Current treatments for heart disease are primarily directed to the use of captopril and angiotensin converting enzyme (ACE) inhibitors such as diuretics. These agents improve hemodynamic profile, and exercise load, and reduce mortality and morbidity in patients with CHF. Kramer et al., Circulation, 67 (4): 807-816 (1983); Captopril Multicenter R
esearch Group, JACC, 2 (4): 755-763 (1983); The CONSENSUS Trial Study
Group, N. Engl. J. Med., 316 (23): 1429-1435 (1987); The SOLVD Investigato
rs, N. Engl. J. Med., 325 (5): 293-302 (1991). Furthermore, they are used in hypertension, left ventricular dysfunction, atherosclerotic vascular disease, and diabetic nephropathy. Brown and Vaughan, listed above. However, despite the proven benefits,
The response of ACE inhibitors is limited. For example, when prolonging life in the context of heart failure, ACE inhibitors appear to slow the progression of end-stage heart failure, and the majority of patients with ACE inhibitors have functional class III heart failure. Moreover, improvements in functional capacity and exercise time are negligible, and mortality rates continue to be high despite reduction. The CONSESUS 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.
Captopril-Digoxin Multicenter Research Group, JAMA, 259 (4): 539-544 (198
8). Thus, it is clear that ACE inhibitors can alleviate symptoms consistently in more than 60% of heart failure patients and reduce heart failure mortality by only about 15-20%.
For more side effects, see Broun and Vaughan, supra.

Alternatives to ACE inhibitors are represented by specific AT1 receptor antagonists. Clinical studies are planned to compare the effects of these two modalities in the treatment of cardiovascular and renal disease. However, animal model data suggest that the ACE / AngII pathway, although clearly associated with cardiac hypertrophy, is not the only one or the active major pathway in this role. Mouse gene "knockout" models have been created to test individual components of the pathway. In one such model,
ATsub1A, a major cardiac receptor II for AngII, was genetically deleted; these mice did not develop hypertrophy when AngII was given experimentally (secondary hypertrophy of AngII). Confirm basic success with model in removal). However, when the aorta is contracted in these animals (hypertensive heart stress model), the heart is still hypertrophic. This suggests that another signaling pathway unrelated to this receptor (ATsub1A) is activated in hypertension. ACE inhibitors will probably not be able to suppress these pathways. Harada et al., Circulat
ion, 97: 1952-1959 (1998). See also Homcy, Circulation, 97: 1890-1892 (1998) for mysteries related to the process and mechanism of cardiac hypertrophy. About 750,000 patients experience acute myocardial infarction (AMI) each year, and AMI accounts for approximately one-quarter of all deaths in the United States. Recently, thrombolytic agents such as streptokinase, urokinase, and especially tissue plasminogen activator (t-PA),
The survival rate of patients suffering from myocardial infarction was greatly improved. When administered as a continuous intravenous infusion for 1.5 to 4 hours, t-PA causes coronary vessel release in 90 minutes in 69% to 90% of treated patients. Topol et al., Am. J. Cardiol., 61: 723-728 (1998); Neu.
haus et al., J. Am. Coll. Cardiol., 12: 581-587 (1988); Neuhaus et al., J. Am. Coll.
Cardiol., 14: 1566-1569 (1989). The highest release rates have been reported in high dose or rapid dose therapy. Topol, J. Am. Coll. Cardiol., 15: 922-924 (
1990). Also, t-PA can be administered as a single bolus and, depending on its associated short half-life, is suitable for infusion therapy. Tebbe et al., Am. J. Cardiol.,
64: 448-453 (1989). The t-PA mutant has been engineered to have a particularly long half-life and high fibrin specificity, but TNKt-PA (aT103N, N117Q, KHRR (296-299) AAAA
The t-PA mutant, Keyt et al., Proc. Natl. Acad. Sci. USA, 91: 3670-3674 (1994)) is particularly suitable for bolus administration. However, despite all of these developments, the long-term prognosis of patient survival relies heavily on post-infusion monitoring and treatment of patients, including monitoring and treatment of cardiac hypertrophy.

B. Growth Factors Reportedly, various naturally occurring polypeptides induce proliferation of endothelial cells. These polypeptides are found in basic and acidic fibroblast growth factor (FGF) (Burgess
And Maciag, Annual Rev. Biochem., 58: 575 (1989)), platelet-derived endothelial cell growth factor (PD-ECGF) (Ishikawa et al., Nature, 338: 557 (1989)), and vascular endothelial growth factor (
VEGF). Leung et al., Science, 246: 1306 (1989); Ferrara and Henzel, Bioch.
em. Biophys. Res. Commun., 161: 851 (1989); Tischer et al., Biochem. Biophys. Re
s. Commun., 165: 1198 (1989); European Patent No. 4717 granted July 31, 1996.
No. 54B. Conditioned medium with cells transfected with human VEGF (hVEGF) cDNA promoted proliferation of capillary endothelial cells, whereas controls did not. Leung et al.,
Science 246: 1306 (1989). Some additional cDNAs are 121-, 189-, and 206-amino acid isoforms of hVEGF (also collectively referred to as hVEGF-related proteins)
Was identified in a human cDNA library encoding The 121-amino acid protein differs from hVEGF by virtue of the deletion of 44 amino acids between residues 116 and 159 of hVEGF. The 189-amino acid protein differs from hVEGF by the feature of inserting 24 amino acids at residue 116 of hVEGF and is clearly identical to human vascular permeability factor (hVPF). The 206-amino acid protein differs from hVEGF by the feature of inserting 41 amino acids at residue 116 of hVEGF. Houck et al., Mol. Endocrin., 5: 1806 (1991); Ferrara
Et al, Endocrine Reviews, 13:18 (1992); Keck et al, Science, 246: 1309 (1989); Co
nnolly et al., J. Biol. Chem., 264: 20017 (1989); European Patent No. 370989 published May 30, 1990. It is now well established that angiogenesis, including the formation of new blood vessels from pre-existing endothelium, is associated with the etiology of various diseases. These include solid tumors and metastases, atherosclerosis, post-lens fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular syndromes such as proliferative retinopathy (e.g. diabetic retinopathy), age-related macula. Includes degeneration (AMD), neovascular glaucoma, immune response of transplanted corneal cells and other cells, rheumatoid arthritis, and psoriasis. Folkman et al., J. Biol. Chem., 267: 1093
1-10934 (1992); Klagsbrun et al., Annu. Rev. Physiol., 53: 217-239 (1991); and
Garner A., "Vascular diseases", In: Pathobiology of Ocular Disease. A Dy
namic Approach, Garner A., Klintworth GK, eds., 2nd Edition (Marcel Dekke
r, NY, 1994), pp 1625-1710. In the case of tumor growth, angiogenesis appears to be of great importance in the transition from hyperplasia to tumorigenesis, providing nutrients for the growth of solid tumors. Folkman et al., Nature, 339:
58 (1989). Neovascularization favors the growth of tumor cells over normal cells and confers proliferative autonomy. Thus, there is a correlation between the microvessel density of tumor areas and patient survival in breast cancer as well as some other tumors. Weidner et al., N. Engl. J. Med
, 324: 1-6 (1991); Horak et al., Lancet, 340: 1120-1124 (1992); Macchiarini et al.,
Lancet, 340: 145-146 (1992). By exploring positive regulators of angiogenesis, aFGF, bFGF, TGF-α, TGF-β, HG
Many candidates were given, including F, TNF-α, angiotensin, IL-8, etc. Folkman et al., JBC, and Klagsbrun, et al., Supra. The negative regulator defined so far is thrombospondin (Good et al., Proc. Natl. Acad. Sci. USA., 87: 6624).
-6628 (1990)), a 16-kilodalton N-terminal fragment of prolactin (Clapp et al., Endocrino
logy, 133: 1292-1299 (1993)), angiostatin (O'Reilly et al., Cell, 79: 315-).
328 (1994)), and endostatin. O'Reilly et al., Cell, 88: 277-285 (1996).

Studies in the last few years have revealed an important role of VEGF in stimulating vascular endothelial cell proliferation as well as inducing vascular penetration and angiogenesis. Ferrara etc.,
Endocr. Rev., 18: 4-25 (1997). The finding that the loss of even one VEGF allele leads to fetal death indicates the irreplaceable role of this factor in vascular development and differentiation. Furthermore, VEGF has been shown to be an important mediator of neovascularization associated with tumor and intraocular disease. Ferrara et al., Endocr. Rev., listed above. VEGF mRNA is overexpressed in many human tumors examined. Berkman et al., J. Clin
.Invest., 91: 153-159 (1993); Brown et al., Human Pathol., 26: 89-91 (1995); B.
rown et al., Cancer Res., 53: 4727-4735 (1993); Mattern et al., Brit. J. Cancer, 73.
: 931-934 (1996); Dvorak et al., Am. J. Pathol., 146: 1029-1039 (1995). Also, the concentration level of VEGF in the ocular fluid is highly correlated with the presence of active proliferation of blood vessels in patients with diabetes and other ischemia-related retinopathy. Aiello et al., N.
Engl. J. Med., 331: 1480-1487 (1994). In addition, recent studies have shown that VEGF is localized in the choroidal neovascular membrane of patients affected by AMD. Lopez et al., Invest. Ophthalmol. Vis. Sci., 37: 855-868 (1996). Anti-VEGF neutralizing antibody suppresses the growth of various human tumor cell lines in nude mice (Kim et al., Nature, 362: 841-844 (1993); Warren et al., J. Clin. Invest.,
95: 1789-1797 (1995); Borgstrom et al., Cancer Res., 56: 4032-4039 (1996); Meln.
Yk et al., Cancer Res., 56: 921-924 (1996)) and also inhibit intraocular angiogenesis in ischemic retinal disease. Adamis et al., Arch. Ophthalmol., 114: 66-71 (1996). Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action have been suggested as candidates for the treatment of solid tumors and various intraocular neovascular diseases. Such antibodies are, for example, EP 817,648 published on January 14, 1998 and filed on April 3, 1998.
It is described in PCT / US98 / 06724.

Can rapidly induce a complex of genes expressed by certain cells,
There are several other growth and mitogens, including transforming oncogenes. Lau and Nathans. Molecular Aspects of Cellular Regulati
on, 6: 165-202 (1991). These genes, termed immediate early genes or early response genes, are transcriptionally activated within minutes after contact with growth or mitogens, independent of de novo protein synthesis. These groups of immediate-early genes encode secretory extracellular proteins that are required to coordinate 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 belonging to this group include cef10 (Simmons et al.
, Proc. Natl. Acad. Sci. USA, 86: 1178-1182 (1989)), cyr61 (O'Brien et al., Mol. Cel, which is rapidly activated by serum- or platelet-derived growth factor (PDGF)).
L. Biol., 10: 3569-3577 (1990)), transforming growth factor β (TGF-β).
), It is secreted by human vascular endothelial cells at high levels and exhibits PDGF-like biological and immunological activity, and PDGF is directed to specific cell surface receptors
Human connective tissue growth factor (CTGF), which competes with Bradham et al., J. Cell. Biol., 114.
: 1285-1294 (1991)), fisp-12 (Ryseck et al., Cell Growth Differ., 2: 235-
233 (1991)), human vascular IBP-like growth factor (VIGF) (WO96 / 17931), and normally quiescent in adult kidney cells and overexpressed in myeloblast-associated-virus type I-induced nephroblastoma. Nov which has been found to be included. Joloit et al., Mol. Cell. B
iol., 12: 10-21 (1992). Expression of these immediate-early genes acts as a "third messenger" in the cascade of growth factor-induced events. Also, they are believed to be necessary to integrate and coordinate wound healing, where complex biological processes such as differentiation and cell proliferation are normal events. Insulin-like growth factor binding protein (IG
FBPs) have been shown to stimulate in complex with insulin-like growth factor (IGF) to increase IGF binding to fibroblast and smooth muscle cell surface receptors. Clemmons et al., J. Clin. Invest., 77: 1548 (1986). The inhibitory effects of IGFBPs on various in vitro IGF actions include stimulation of glucose transport by adipocytes, sulphate uptake by chondrocytes, and thymidine uptake in fibroblasts. Zapf et al., J. Clin. Invest., 63: 1077 (1
979). Furthermore, IG in growth factor-mediated mitogen activity in normal cells
The inhibitory effect of FBP is shown.

C. The need for further treatment Given the role of vascular endothelial cell growth and angiogenesis in many diseases and disorders, it has the means to reduce or suppress one or more biological effects resulting from these processes. It is preferable. It is also desirable to have a means of testing for the presence of pathogenic polypeptides in normal and diseased conditions, especially cancer. Moreover, in certain aspects, the identification of factors that can prevent or reduce cardiac myocyte hypertrophy inhibit pathophysiological cardiac growth, as there is no generally applicable treatment for treating cardiac hypertrophy. It is of great importance in the development of new therapeutic strategies for Although there are several therapeutic modalities for various cardiovascular and oncogene disorders, further therapeutic approaches are still needed.

(Summary of Invention) A. Embodiments Accordingly, the present invention relates to compositions and methods for promoting or inhibiting angiogenesis and / or cardiovascularization in a mammal. The present invention is based on the identification of proteins that test positive in various cardiovascular assays that test the promotion or inhibition of certain biological activities. Thus, promoting or inhibiting angiogenesis, inhibiting or stimulating vascular endothelial cell growth, stimulating vascular endothelial cell growth or proliferation, inhibiting tumor growth, inhibiting angiogenesis-dependent tissue growth, stimulating angiogenesis-dependent tissue growth. , Where inhibition of cardiac hypertrophy and stimulation of cardiac hypertrophy, such as treatment of congestive heart failure, is desired, the protein is a drug useful for diagnosis and / or treatment (including prevention) of diseases. Conceivable. In one embodiment, the invention provides a composition comprising PRO polypeptide mixed with a pharmaceutically acceptable carrier. In one aspect, the composition comprises a therapeutically effective amount of the polypeptide. In another aspect, the composition comprises an additional active ingredient, namely a cardiovascular, endothelial or angiogenic or angiostatic agent,
Preferably it contains an angiogenic or angiostatic drug. Preferably the composition is sterile. PRO polypeptides can be administered in the form of liquid pharmaceutical preparations, which can be preserved for extended storage stability. Preserved liquid pharmaceutical preparations may contain multiple doses of the PRO polypeptide and are thus suitable for repeated use.

In a further embodiment, the present invention provides a mixture of a pharmaceutically acceptable carrier and a therapeutically effective amount of a PRO polypeptide, useful in the treatment of cardiovascular, endothelial or angiogenic diseases. Methods of preparing such compositions are provided. In another embodiment, the invention relates to P, admixed with a pharmaceutically acceptable carrier.
Compositions containing agonists or antagonists of RO polypeptides are provided. In one aspect, the composition contains a therapeutically effective amount of an agonist or antagonist. In another aspect, the composition contains an additional active ingredient, namely a cardiovascular, endothelial or angiogenic or angiostatic agent, preferably an angiogenic or angiostatic agent. Preferably the composition is sterile. Agonists or antagonists of PRO polypeptides may be administered in the form of liquid pharmaceutical preparations and may be stored such that extended stability on storage is achieved. Preserved liquid pharmaceutical preparations may contain multiple doses of PRO polypeptide agonists or antagonists and are thus suitable for repeated use. In a further embodiment, the invention provides a cardiovascular system comprising a mixture of a pharmaceutically acceptable carrier and a PRO polypeptide agonist or antagonist.
Methods of preparing such compositions useful in treating endothelial or angiogenic disorders are provided. In yet another embodiment, the invention relates to a composition containing anti-PRO antibody admixed with a pharmaceutically acceptable carrier. In one aspect, the composition comprises a therapeutically effective amount of the antibody. In another embodiment, the composition contains a further active ingredient, namely a cardiovascular, endothelial or angiogenic or angiostatic agent, preferably an angiogenic or angiostatic agent. Preferably the composition is sterile. The compositions may be administered in the form of liquid pharmaceutical preparations and may be stored such that extended stability on storage is achieved. Preserved liquid pharmaceutical preparations may contain multiple doses of anti-PRO antibody and are thus suitable for repeated use.
In a preferred embodiment, the antibody is a monoclonal antibody, antibody fragment, humanized antibody or single chain antibody. In a further embodiment, the invention provides such a composition useful in the treatment of cardiovascular, endothelial or angiogenic disorders, comprising a mixture of a pharmaceutically acceptable carrier and a therapeutically effective amount of anti-PRO antibody. A method for preparing a product is provided.

In yet a further aspect, the invention provides: (a) a composition of matter containing a therapeutically effective amount of a PRO polypeptide; (b) a container containing the composition; and (c) cardiovascular, endothelium. Or an article of manufacture comprising a label attached to the container or a packaging insert housed in the container, which describes the use of the PRO polypeptide in the treatment of an angiogenic disease, wherein the agonist or antagonist is PRO Poly It may be an antibody that binds to the peptide. The composition may include a therapeutically effective amount of the PRO polypeptide or an agonist or antagonist thereof. In another embodiment, the invention provides a method of identifying an agonist of a PRO polypeptide, which comprises: (a) bringing a cell and a test compound to be screened for a cellular response normally elicited by the PRO polypeptide. Contacting under conditions suitable for induction; and (b) measuring the induction of said cellular response to determine whether the test compound is an effective agonist, said Induction indicates that the test compound is an effective agonist. In another embodiment, the invention provides a method of identifying an agonist of a PRO polypeptide, which comprises: (a) bringing cells and a test compound to be screened under conditions suitable for stimulation of cell proliferation by the PRO polypeptide. Contacting under; and (b) measuring the proliferation of the cells to determine whether the test compound is an effective agonist, wherein stimulation of cell proliferation is effective for the test compound. Indicates an agonist.

In another embodiment, the invention provides a method of identifying a compound that inhibits the activity of a PRO polypeptide, which comprises allowing the test compound to interact with the PRO polypeptide and the test compound and the polypeptide. Contacting for sufficient conditions and time, and determining whether the activity of PRO polypeptide is inhibited. In a particularly preferred embodiment, either the test compound or the PRO polypeptide is immobilized on a solid support.
In another preferred embodiment, the non-immobilized component carries a detectable label. In a preferred embodiment, the method comprises: (a) contacting the cell with a test compound to be screened in the presence of the PRO polypeptide under conditions suitable for eliciting the cellular response normally elicited by the PRO polypeptide. And (b) measuring the induction of the cellular response to determine whether the test compound is an effective agonist. In another preferred embodiment, the method comprises: (a) contacting the cell with a test compound to be screened in the presence of the PRO polypeptide under conditions suitable for stimulation of cell proliferation by the PRO polypeptide;
And (b) measuring the proliferation of the cells to determine whether the test compound is an effective agonist. In another embodiment, the invention provides a method of identifying a compound that inhibits expression of a PRO polypeptide in a cell that normally expresses the PRO polypeptide, the method comprising contacting the cell with a test compound, the PRO poly Measuring whether the expression of the peptide is inhibited. In a preferred embodiment, the method comprises: (a) contacting the cell with a test compound to be screened under conditions suitable for expressing the PRO polypeptide; and (b) measuring inhibition of expression of said polypeptide. Including steps. In yet a further embodiment, the invention provides compounds that inhibit the expression of PRO polypeptides, such as the compounds identified by the methods above.

Another aspect of the invention is directed to PRO polypeptide agonists or antagonists that may be identified by the methods described above. One type of PRO polypeptide antagonist that inhibits one or more functions or activities of a PRO polypeptide is an antibody. Thus, in another aspect, the invention comprises a PR
An isolated antibody that binds to the O polypeptide is provided. In a preferred embodiment,
The antibody is a monoclonal antibody, preferably having non-human complementarity determining region (CDR) residues and human framework region (FR) residues. The antibody may be labeled or 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 invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal, which comprises (a) a test sample of tissue cells obtained from said mammal, and (b) the same. Analyzing the expression level of a gene encoding a PRO polypeptide in a control sample of known normal tissue cells of a cell type, wherein the level of expression level in a test sample when compared to a control sample is Indicates the presence of cardiovascular, endothelial or angiogenic disorders in animals. Expression of the gene encoding the PRO polypeptide may optionally be by measuring the level of mRNA or polypeptide in the test sample as compared to a control sample. In a still further aspect, the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal, which comprises detecting the presence or absence of a polypeptide in a test sample of tissue cells obtained from said mammal. The presence or absence of the polypeptide in the test sample indicates the presence of cardiovascular, endothelial or angiogenic disease in the mammal.

In yet a further aspect, the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal, which comprises (a) anti-testing a tissue cell test sample obtained from said mammal. Contacting with a PRO antibody, and (b) detecting the formation of a complex between said antibody and a PRO polypeptide in a test sample, the formation of said complex comprising cardiovascular, endothelium or Indicates the presence of angiogenic disease. Detection may be qualitative or quantitative and may be carried out in comparison to monitoring complex formation in a control sample of known normal tissue cells of the same cell type. The amount of complex formation in the test sample indicates the presence of cardiovascular, endothelium or hypovascularization 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, fluorometry, or other techniques known in the art. Test samples are usually obtained from individuals suspected of having cardiovascular, endothelial or angiogenic disorders. In another embodiment, the invention provides a method of determining the presence of PRO polypeptide in a sample, which comprises anti-assaying a sample suspected of containing PRO polypeptide.
Contacting with a PRO antibody and measuring the binding of said antibody to a component of said sample. In a particular embodiment, the sample comprises cells suspected of containing the PRO polypeptide and the antibody binds to the cells. The antibody is preferably detectably labeled and / or immobilized on a solid support. In a further aspect, the present invention provides a diagnostic kit for cardiovascular, endothelial or angiogenic diseases, which comprises an anti-PRO antibody and a carrier in a suitable package. Preferably, such a kit further comprises instructions for using said antibody to detect the presence of PRO polypeptide. Preferably the carrier is eg a buffer.
Preferably the cardiovascular, endothelial or angiogenic disease is cancer. In yet another embodiment, the invention provides a method of treating a cardiovascular, endothelial or angiogenic disorder in a mammal, which comprises administering to said mammal an effective amount of a PRO polypeptide. . Preferably, the disease is cardiac hypertrophy, trauma such as a wound or burn, or a type of cancer. In a further aspect, if the cardiovascular, endothelial or angiogenic disease is a type of cancer, the mammal is angioplasty or an agent for treating a cardiovascular, endothelial or angiogenic disease, such as an ACE inhibitor,
Or further exposure to chemotherapeutic agents. Preferably the mammal is a human, preferably a human at risk of developing cardiac hypertrophy, more preferably a human suffering from myocardial infarction. In another preferred embodiment, cardiac hypertrophy is characterized by the presence of elevated PGF _{2α ′} levels. Alternatively, cardiac hypertrophy is triggered by myocardial infarction,
Wherein, preferably the administration of PRO polypeptide is for 48 hours following myocardial infarction,
It is preferably started within 24 hours.

In another preferred embodiment, the cardiovascular, endothelial or angiogenic disease is cardiac hypertrophy and the PRO polypeptide is administered with a cardiovascular, endothelial or angiogenic drug. Preferred cardiovascular, endothelial or angiogenic agents for this purpose are selected from the group consisting of antihypertensive agents, ACE inhibitors, endothelin receptor antagonists and thrombolytic agents. When a thrombolytic agent is administered, preferably PRO
The polypeptide is administered after administration of such agents. More preferably, the thrombolytic agent is recombinant human tissue plasminogen activator. In another preferred embodiment, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy and the PRO polypeptide is administered following primary angioplasty for the treatment of acute myocardial infarction, wherein, preferably, the mammal Is further exposed to angioplasty or cardiovascular, endothelial or angiogenic drugs. In another preferred embodiment, the cardiovascular, endothelial or angiogenic disease is cancer and the PRO polypeptide is administered in combination with a chemotherapeutic agent, growth inhibitory agent or cytotoxic agent. In a further embodiment, the invention relates to a method of treating a cardiovascular, endothelial or angiogenic disorder in a mammal, which comprises administering to said mammal an effective amount of an agonist of PRO polypeptide. Preferably, the cardiovascular, endothelial or angiogenic disease is cardiac hypertrophy, trauma, cancer, or age-related macular degeneration. Also preferably the mammal is a human and an effective amount of an angiogenic or angiostatic drug is administered with an agonist. In a further embodiment, the invention relates to a method of treating a cardiovascular, endothelial or angiogenic disorder in a mammal, which comprises administering to the mammal an effective amount of an antagonist of PRO polypeptide. Preferably, the cardiovascular, endothelial or angiogenic disease is cardiac hypertrophy, trauma, cancer, or age-related macular degeneration. Also preferably the mammal is a human and an effective amount of an angiogenic or angiostatic drug is administered with the antagonist. In a further embodiment, the invention relates to a method of treating a cardiovascular, endothelial or angiogenic disorder in a mammal, which comprises administering to the mammal an effective amount of anti-PRO antibody. Preferably, the cardiovascular, endothelial or angiogenic disease is cardiac hypertrophy, trauma, cancer, or age-related macular degeneration. Also preferably the mammal is a human and an effective amount of an angiogenic or angiostatic drug is administered with the antibody.

In a further embodiment, the invention provides a method of treating a cardiovascular, endothelial or angiogenic disorder in a mammal suffering from a cardiovascular, endothelial or angiogenic disorder, which comprises (a) a PRO polypeptide. , (B) an agonist of the PRO polypeptide, or (c)
Comprising administering to a mammal a nucleic acid molecule encoding any of the antagonists of PRO polypeptides, wherein said agonist or antagonist is
-May be a PRO antibody. In a preferred embodiment the mammal is a human.
In another preferred embodiment, the gene is administered via ex vivo gene therapy. In a further preferred embodiment, the gene is contained within a vector, more preferably an adenovirus, adeno-associated virus, lentivirus or retroviral vector. In another aspect, the present invention provides a promoter, (a) PRO polypeptide,
It contains a retroviral vector consisting essentially of a nucleic acid molecule encoding (b) an agonist polypeptide of the PRO polypeptide, or (c) an antagonist polypeptide of the PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide. A recombinant retroviral particle is provided, wherein the retroviral vector is associated with a retroviral structural protein. Preferably, the signal sequence is from a mammalian, eg native PRO polypeptide. In yet a further embodiment, the present invention comprises a nucleic acid construct that expresses a retroviral structural protein, comprising a promoter, (a) PRO polypeptide, (b) P
Ex vivo production further comprising a retroviral vector consisting essentially of a nucleic acid molecule encoding an agonist polypeptide of the RO polypeptide, or (c) an antagonist polypeptide of the PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide. Producer cells are provided, wherein the producer cells include retroviral vectors associated with structural proteins that produce recombinant retroviral particles. In yet another embodiment, the invention provides a method of inhibiting the growth of mammalian endothelial cells, which comprises (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) a PRO polypeptide. May be administered to a mammal, the growth of endothelial cells of the mammal is inhibited, and the agonist or antagonist may be an anti-PRO antibody. Preferably, the mammal is a human and endothelial cell growth is associated with a tumor or retinal disease. In yet another embodiment, the invention provides a method of stimulating mammalian endothelial cell growth, which comprises (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) a PRO polypeptide. Is administered to a mammal, the endothelial cell growth of the mammal is stimulated, and the agonist or antagonist may be an anti-PRO antibody. Preferably the mammal is a human. In yet another embodiment, the invention provides a method of inhibiting cardiac hypertrophy in a mammal, which comprises (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide. To a mammal, wherein cardiac hypertrophy of the mammal is inhibited, and the agonist or antagonist is an anti-PRO antibody. Preferably, the mammal is a human and the cardiac hypertrophy is that induced by myocardial infarction.

In yet another embodiment, the invention provides a method of stimulating cardiac hypertrophy in a mammal, which comprises (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) a PRO polypeptide. The method may comprise administering an antagonist of the peptide to a mammal, wherein cardiac hypertrophy of the mammal is stimulated and the agonist or antagonist is an anti-PRO antibody. Preferably, the mammal is a human suffering from congestive heart failure. In yet another embodiment, the invention provides a method of inhibiting PRO polypeptide-induced angiogenesis in a mammal, comprising administering to said mammal a therapeutically effective amount of an anti-PRO antibody. Comprises. Preferably, the mammal is a human suffering from congestive heart failure. In yet another embodiment, the invention provides a method of stimulating PRO polypeptide-induced angiogenesis in a mammal, which comprises administering to said mammal a therapeutically effective amount of PRO polypeptide. Comprises. Preferably, the mammal is a human, more preferably angiogenesis promotes tissue regeneration or wound healing. In yet another embodiment, the invention provides a mammalian PRO333, PRO364.
, PRO877, PRO879, PRO882 or PRO885 polypeptide or an agonist thereof is provided for inhibiting endothelial cell growth in a mammal, whereby endothelial cell growth is inhibited in a mammal as previously described. In yet another embodiment, the invention provides a mammalian mammalian PRO179, PRO321.
, PRO840, PRO844, PRO846, PRO878 or PRO879.
Provided is a method of stimulating endothelial cell growth in a mammal comprising administering a polypeptide or agonist thereof, whereby endothelial cell growth is stimulated in a mammal as previously described. In yet another embodiment, the invention provides a mammalian mammalian PRO179, PRO321.
, PRO840, PRO844, PRO846, PRO878 or PRO879.
Provided is a method of inhibiting endothelial cell growth in a mammal, comprising administering an antagonist of the polypeptide, whereby endothelial cell growth is inhibited in a mammal as previously described. In yet another embodiment, the invention provides a mammalian PRO333, PRO364.
, PRO877, PRO879, PRO882 or PRO885 polypeptide comprising the administration of an antagonist of the polypeptide, thereby stimulating endothelial cell growth in a mammal as previously described. In yet another embodiment, the invention provides a mammalian PRO205, PRO882.
Alternatively, there is provided a method of inducing cardiac hypertrophy in a mammal comprising administration of a PRO887 polypeptide or agonist thereof, whereby cardiac hypertrophy is induced in a mammal as previously described. In yet another embodiment, the invention provides a mammalian mammalian PRO238, PRO878.
Or comprising administration of a PRO1760 polypeptide or agonist thereof,
A method of reducing cardiac hypertrophy in a mammal is provided, whereby cardiac hypertrophy is reduced in a mammal as previously described. In yet another embodiment, the invention provides a mammalian mammalian PRO238, PRO878.
Alternatively, there is provided a method of inducing cardiac hypertrophy in a mammal comprising administration of an antagonist of PRO1760 polypeptide, whereby cardiac hypertrophy is induced in a mammal as previously described. In yet another embodiment, the invention provides a mammalian PRO205, PRO882.
Alternatively, there is provided a method of reducing cardiac hypertrophy in a mammal comprising administration of an antagonist of PRO887 polypeptide, whereby cardiac hypertrophy is reduced in a mammal as previously described. In yet another embodiment, the invention provides a mammal with anti-PRO179, anti-PRO32.
1, PRO179, comprising administering a therapeutically effective amount of an anti-PRO840, anti-PRO844, anti-PRO846, anti-PRO878 or anti-PRO879 antibody,
A method of inhibiting angiogenesis induced by a PRO321, PRO840, PRO844, PRO846, PRO878 or PRO879 polypeptide,
This stimulates the angiogenesis already mentioned. In yet another embodiment, the invention comprises administering to a mammal a therapeutically effective amount of a polypeptide previously described, PRO179, PRO321, PR.
Provided is a method of stimulating angiogenesis induced by an O840, PRO844, PRO846, PRO878 or PRO879 polypeptide, which stimulates angiogenesis as previously described.

B. Further Embodiments In another aspect of the invention, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide. In one aspect, the isolated nucleic acid molecule comprises (a) the extracellular sequence of a full-length amino acid sequence disclosed herein, an amino acid sequence lacking a signal peptide disclosed herein, a transmembrane protein with or without a signal peptide disclosed herein. A DNA molecule encoding a PRO polypeptide having a domain, or other specifically identified fragment of the full-length amino acid sequence disclosed herein, or (b) at least about 8 relative to the complement of the DNA molecule of (a).
0% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, more preferably at least about 83% sequence identity, more preferably at least about 84%. Sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 86% sequence identity, more preferably at least about 87% sequence identity, more preferably at least about 88% sequence identity. Sex, more preferably at least about 89% sequence identity, more preferably at least about 90% sequence identity, more preferably at least about 91.
% Sequence identity, more preferably at least about 92% sequence identity, more preferably at least about 93% sequence identity, more preferably at least about 94% sequence identity, more preferably at least about 95%. Sequence identity, more preferably at least about 96% sequence identity, more preferably at least about 97% sequence identity, more preferably at least about 98% sequence identity, and more preferably at least about 99%. It includes nucleotide sequences that have sequence identity. In another aspect, the isolated nucleic acid molecule comprises (a) a PRO encoding the full-length PRO polypeptide cDNA disclosed herein, a PRO lacking a signal peptide disclosed herein.
A coding sequence for a polypeptide, a coding sequence for the extracellular domain of a transmembrane PRO polypeptide with or without a signal peptide disclosed herein, or a specially identified other fragment of the full-length amino acid sequence disclosed herein. A DNA molecule having a sequence, or (b) at least about 80% sequence identity to the complement of the DNA molecule of (a),
Preferably at least about 81% sequence identity, more preferably at least about 82.
% Sequence identity, more preferably at least about 83% sequence identity, more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 86%. Sequence identity, more preferably at least about 87% sequence identity, more preferably at least about 88% sequence identity, more preferably at least about 89% sequence identity, more preferably at least about 90% sequence identity. Sex, more preferably at least about 91% sequence identity, more preferably at least about 92% sequence identity, more preferably at least about 9%.
3% sequence identity, more preferably at least about 94% sequence identity, more preferably at least about 95% sequence identity, more preferably at least about 96% sequence identity, more preferably at least about 97%. More preferably at least about 98% sequence identity, and more preferably at least about 99% sequence identity.
A nucleotide sequence having the sequence identity of

In a further aspect, the invention provides (a) a DNA molecule encoding the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC disclosed herein, or (b) ( At least about 80% sequence identity to the complement of the DNA molecule of a), preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, more preferably at least about 83%. Sequence identity, more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 86% sequence identity, more preferably at least about 87% sequence identity. Identity, more preferably at least about 88% sequence identity, more preferably at least about 89% sequence identity,
More preferably at least about 90% sequence identity, more preferably at least about 91% sequence identity, more preferably at least about 92% sequence identity, more preferably at least about 93% sequence identity, more preferably. Is at least about 94%
Of sequence identity, more preferably at least about 95% sequence identity, more preferably at least about 96% sequence identity, more preferably at least about 97% sequence identity, more preferably at least about 98% sequence identity. It relates to an isolated nucleic acid molecule comprising a nucleotide sequence having identity, and more preferably at least about 99% sequence identity. Another aspect of the invention is a nucleotide sequence encoding a PRO polypeptide having a transmembrane domain deleted or transmembrane domain inactivated, or a nucleotide complementary to such a encoded nucleotide sequence. Provided is an isolated nucleic acid molecule comprising a sequence, the transmembrane domain of such a polypeptide being disclosed herein. Therefore, the soluble extracellular domain of the PRO polypeptides described herein is considered. Other embodiments are directed to fragments of the PRO polypeptide coding sequence, or complements thereof, which encode, eg, a PRO polypeptide which optionally encodes a polypeptide comprising a binding site for an anti-PRO antibody. It finds use as a fragment hybridization probe or as an antisense oligonucleotide probe. Such nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, more preferably at least about 50 nucleotides in length, more preferably at least about 60 nucleotides in length, More preferably at least about 70
Nucleotide length, more preferably at least about 80 nucleotides, more preferably at least about 90 nucleotides, more preferably at least about 100 nucleotides, more preferably at least about 110 nucleotides, more preferably at least about 120 nucleotides, more preferably Is at least about 130 nucleotides long, more preferably at least about 140 nucleotides long, more preferably at least about 150 nucleotides long, more preferably at least about 160 nucleotides long, more preferably at least about 170 nucleotides long, more preferably at least about 180 nucleotides long. Long, more preferably at least about 190 nucleotides long, more preferably at least about 200 nucleotides long, more preferably at least about 25. Nucleotide length, more preferably at least about 300 nucleotides length, more preferably at least about 350 nucleotides length, more preferably at least about 400 nucleotides length, more preferably at least about 450 nucleotides length, more preferably at least about 500 nucleotides length, more preferably. Is at least about 600 nucleotides in length, more preferably at least about 700 nucleotides in length, more preferably at least about 800 nucleotides in length, more preferably at least about 900 nucleotides in length, more preferably at least about 1000 nucleotides in length, where "about". The content of the term is meant to refer to a nucleotide sequence length that is plus or minus 10% of the referenced length. Novel fragments of the PRO polypeptide-encoding nucleotide sequence can be prepared by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs to Routine procedures may be identified by determining whether the PRO polypeptide-encoding nucleotide sequence fragment is novel. All such PRO polypeptide-encoding nucleotide sequences are considered herein. Also contemplated are PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably PRO polypeptide fragments that contain a binding site for an anti-PRO antibody.

In another embodiment, the invention provides an isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences identified above. In one aspect, the invention features a full-length amino acid sequence disclosed herein, an amino acid sequence lacking a signal peptide disclosed herein, an extracellular domain of a transmembrane protein with or without a signal peptide disclosed herein, or disclosed herein. At least about 80 relative to the PRO polypeptide with other specifically identified fragments of the full-length amino acid sequence
% Sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, more preferably at least about 83% sequence identity, more preferably at least about 84% sequence. Identity, more preferably at least about 85% sequence identity, more preferably at least about 86% sequence identity,
More preferably at least about 87% sequence identity, more preferably at least about 88% sequence identity, more preferably at least about 89% sequence identity, more preferably at least about 90% sequence identity, more preferably Is at least about 91%
Of sequence identity, more preferably at least about 92% sequence identity, more preferably at least about 93% sequence identity, more preferably at least about 94% sequence identity, more preferably at least about 95% sequence identity. Identity, more preferably at least about 96% sequence identity, more preferably at least about 97% sequence identity, more preferably at least about 98% sequence identity, and more preferably at least about 99% sequence identity. It relates to an isolated PRO polypeptide comprising amino acid sequences having identity. In a further aspect, the invention features at least about 80% sequence identity, preferably at least about 81% sequence identity to the amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC disclosed herein. Identity, more preferably at least about 82% sequence identity, more preferably at least about 83% sequence identity, more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity. , More preferably at least about 86% sequence identity, more preferably at least about 87% sequence identity, more preferably at least about 88% sequence identity, more preferably at least about 89% sequence identity, Preferably at least about 90% sequence identity, more preferably at least about 9
1% sequence identity, more preferably at least about 92% sequence identity, more preferably at least about 93% sequence identity, more preferably at least about 94% sequence identity, more preferably at least about 95%. Of sequence identity, more preferably at least about 96% sequence identity, more preferably at least about 97% sequence identity, more preferably at least about 98% sequence identity, and more preferably at least about 99%. Isolated PR containing amino acid sequences having the sequence identity of
O polypeptide.

In a further aspect, the invention provides a full-length amino acid sequence disclosed herein, an amino acid sequence lacking a signal peptide disclosed herein, an extracellular domain of a transmembrane protein with or without a signal peptide disclosed herein, or herein. At least about 80% positive, preferably at least about 81% positive, more preferably at least about 82% positive, and more preferably when compared to a PRO polypeptide having other specifically identified fragments of the full-length amino acid sequence disclosed in. Is at least about 83%
Positive, more preferably at least about 84% positive, more preferably at least about 85% positive, more preferably at least about 86% positive,
More preferably at least about 87% positive, more preferably at least about 8
8% positive, more preferably at least about 89% positive, more preferably at least about 90% positive, more preferably at least about 91% positive, more preferably at least about 92% positive, more preferably at least about 93% positive, and more. Preferably at least about 94% positive, more preferably at least about 95% positive, more preferably at least about 96% positive, more preferably at least about 97% positive, more preferably at least about 98% positive, and more preferably at least It relates to an isolated PRO polypeptide that comprises an amino acid sequence that scores about 99% positive. In a particular embodiment, the invention provides an isolated PRO polypeptide encoded by a nucleotide sequence encoding an amino acid sequence as described above, which has no N-terminal signal sequence and / or initiation methionine. Methods for producing them are also described herein, which methods involve culturing host cells containing a vector containing an appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide, and removing the PRO polypeptide from the culture medium. Including collecting. Another aspect of the invention provides an isolated PRO polypeptide having a transmembrane domain deleted or transmembrane domain inactivated. Also described herein are methods of producing them, which method comprises culturing host cells containing a vector containing an appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide, and removing the PRO polypeptide from the cell culture medium. Including collecting. In yet another embodiment, the invention relates to agonists and antagonists of the native PRO polypeptides identified herein. In a particular embodiment, the agonist or antagonist is an anti-PRO antibody or small molecule. In a further embodiment, the invention relates to a method of identifying an agonist or antagonist of a PRO polypeptide, which comprises contacting the PRO polypeptide with a candidate molecule and monitoring the biological activity mediated by said PRO polypeptide. including. Preferably, the PRO polypeptide is a native PRO polypeptide.

In a yet further embodiment, the invention relates to a composition of matter which comprises a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide described herein, or an anti-PRO antibody, in combination with a carrier. In some cases, the carrier is a pharmaceutically acceptable carrier. Another embodiment of the invention is the treatment of a PRO polypeptide, or an agonist or antagonist thereof, or an anti-PRO antibody as described above, of a condition resulting from a PRO polypeptide, an agonist or antagonist thereof or an anti-PRO antibody. It is intended for use in the preparation of useful medicaments. In a further embodiment of the invention, the invention provides a vector comprising DNA encoding any of the polypeptides described herein. Host cells containing any of such vectors are also provided. As an example, the host cell may be a CHO cell, E. coli, or a baculovirus infected insect cell. Further provided is a method of producing any of the polypeptides described herein, which comprises culturing a host cell under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture medium. In another embodiment, the invention provides a chimeric molecule comprising any of the polypeptides described herein fused to a heterologous polypeptide or amino acid sequence. Examples of such chimeric molecules include any of the polypeptides described herein fused to an epitope tag sequence or the Fc region of an immunoglobulin. In another embodiment, the invention provides an antibody that specifically binds to any of the above or below described polypeptides. In some cases, the antibody is a monoclonal antibody,
It is a humanized antibody, an antibody fragment or a single chain antibody. In yet another embodiment, the invention provides oligonucleotide probes useful for the isolation of genomic and cDNA nucleotide sequences or antisense probes,
The probes can be derived from any of the above or below nucleotide sequences.

Detailed Description of the Invention I. Definitions The terms "cardiovascular, endothelial and angiogenic disorders", "cardiovascular, endothelial and angiogenic dysfunction", "cardiovascular, endothelial or angiogenic disorders" and "cardiovascular, endothelial or angiogenic dysfunction" are mutually exclusive. Used interchangeably, and refers to some of the disease of blood vessels, such as arteries, capillaries, veins, and / or lymph vessels themselves. This includes signs of stimulating angiogenesis and / or cardiovascularization, as well as signs of inhibiting angiogenesis and / or cardiovascularization. Such diseases include, for example, arterial diseases such as atherosclerosis, hypertension, inflammatory vasculitis, Raynaud's disease and Raynaud's phenomenon, aneurysms, and arterial restenosis; venous and lymphatic vessel diseases such as thrombotic veins. Inflammation, lymphangiitis, and lymphedema; and other vascular diseases such as peripheral vascular disease, cancer, such as hemangiomas, such as hemangiomas (capillary and cavernous), glomus tumor, telangiectasia, bacterial hemangiomatosis, Hemangioendothelioma, hemangiosarcoma, hemangiopericytoma, Kaposi's sarcoma, lymphangioma, and lymphangiosarcoma, tumor angiogenesis, trauma such as wounds, burns, and other injured tissue, graft fixation, scarring, Includes ischemia-reperfusion injury, rheumatoid arthritis, cerebrovascular disease, kidney disease such as acute renal failure, and osteoporosis. Also included are angina, myocardial infarction, such as acute myocardial infarction, cardiac hypertrophy, and heart failure, such as CHF. "Hypertrophy" as used herein is defined as an increase in the size of a tissue or structure independent of normal growth that is not involved in tumorigenesis. Hypertrophy of an organ or tissue is due to either an increase in the size of individual cells (true hypertrophy), or an increase in the number of cells forming the tissue (hyperplasia), or both. Certain organs, such as the heart, lose their ability to divide shortly after birth. Thus, "cardiac hypertrophy" is defined as an increase in the size of the heart, and in adults, without concomitant cell division,
It is characterized by a contractile protein content and an increase in myocyte cell size. The characteristics of stress that cause hypertrophy (e.g., increased preload, increased afterload, loss of myocytes similar to those in myocardial infarction, primary contractility decline) are important in determining the nature of the response. It is clear that it plays a role. The early stages of cardiac hypertrophy are usually morphologically characterized by an increase in myofibril and mitochondrial size, and mitochondrial and nuclear expansion. At this stage, the myocytes are larger than normal and the tissue is well preserved. As the phase of cardiac hypertrophy progresses further, the number and size of certain organelles, such as mitochondria, increase preferentially and novel contractile elements are added to localized regions of cells in an irregular manner. Cells that have undergone hypertrophy for many years are in the cell body containing a fairly enlarged nucleus with a highly lobed membrane that replaces adjacent myofibrils and causes disruption of normal Z-membrane registration. Shows a more pronounced division. The term “cardiac hypertrophy” is used to include all stages in the progression of a medical condition, characterized by varying degrees of structural damage to the heart muscle, regardless of the underlying heart disease. Thus, the term also includes physiological conditions in the development of cardiac hypertrophy, such as elevated blood pressure, aortic stenosis, or myocardial infarction.

“Heart failure” refers to an abnormality of cardiac function in which the heart does not pump blood as fast as the demands of metabolizing tissues require. Heart failure can be caused by a variety of factors, including ischemia, congenital, rheumatic or idiopathic forms. "Congestive heart failure" (CHF) is the progression of the heart that is unable to provide sufficient cardiac output (the volume of blood pumped by the heart over time) to deliver oxygenated blood to peripheral tissues. It is a medical condition. As CHF progresses, structural and hemodynamic damage occurs. These damages appear in various ways, one characteristic sign of which is ventricular hypertrophy. CHF is a common end result of many heart diseases. "Myocardial infarction" is commonly regarded as the result of atherosclerosis of the coronary arteries, often with multiple coronary artery thrombosis. It is an intramural infarction with myocardial necrosis throughout the thickness of the ventricular wall, and the necrosis is in the subendothelial layer, within the myocardial wall, or both without extending through the ventricular wall and all the way to the epicardium. Accessory endocardium (non-mural)
Infarction can be divided into two types. Myocardial infarction is known to be due to both altered hemodynamic effects and altered structures in the damaged and healthy areas of the heart. Thus, for example, myocardial infarction reduces the maximum outflow of the heart and the heart beat volume. In addition, in association with myocardial infarction, interstitial DNA synthesis is stimulated and collagen formation is increased in the unaffected heart region. During long-term hypertension, increased cardiac stress and pressure result in cardiac hypertrophy associated with long-term "hypertension," for example, increased total peripheral tolerance. A characteristic of hypertrophied ventricles as a result of chronic pressure is impaired diastolic performance. Fouad et al., J. Am. Coll. Cardiol., 4: 1500-1506 (1984); Smith
Et al., J. Am. Coll. Cardiol., 5: 869-874 (1985). Long-term left ventricular relaxation was predominantly instinctively hypertensive despite normal or abnormal systolic function. Hartford et al., Hypertension, 6: 329-338 (1984). However, there is no parallel close proximity between blood pressure levels and cardiac hypertrophy. In humans, left ventricular function is repeatedly improved in response to antihypertensive treatment, but diuretics (hydrochlorothiazide), β
-Patients treated with blockers (prepanolol) or calcium channel blockers (diltiazem) have a relapse to left ventricular hypertrophy without improved diastolic function. Inouye et al., Am. J. Cardiol., 53: 1583-7 (1984).

Another compound heart disease associated with cardiac hypertrophy is “hypertrophic cardiomyopathy”. The condition is highly diverse in morphological, functional and clinical characteristics (Maron et al., N. Engl. J.
Med., 316: 780-789 (1987); Spirito et al., N. Engl. J. Med., 320: 749-755 (1
989); Louie and Edwards, Prog. Cardiovasc. Dis., 36: 275-308 (1994); Wigle
Et al., Circulation, 92: 1680-1692 (1995)), characterized by heterogeneity highlighted by facts that plague patients of all ages. Spirito et al., N. Engl. J. Med., 336: 77
5-785 (1997). Also, the factors that cause hypertrophic cardiomyopathy are diverse and poorly understood. Generally, mutations in the gene encoding the sarcomeric protein are involved in hypertrophic cardiomyopathy. Recent data suggests that β-myosin heavy chain mutations are estimated to account for approximately 30 to 40 percent of the causes of familial hypertrophic cardiomyopathy. Watkins et al., N. Engl. J. Med., 326: 1108-1114 (1992).
Schwartz et al., Circulation, 91: 532-540 (1995); Marian and Roberts, Circul.
ation, 92: 1336-1347 (1995); Thierfelder et al., Cell, 77: 701-712 (1994); Watk.
ins et al., Nat. Gen., 11: 434-437 (1995). In addition to β-myosin heavy chain, gene mutations at other positions 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). The superior valve "aortic stenosis" is a hereditary vascular disease characterized by narrowing of the ascending aorta, but other arteries, including the pulmonary artery, may also be affected. Untreated aortic stenosis results in increased intracardiac pressure, resulting in cardiac hypertrophy, which in fact leads to heart failure and death. Although the etiology of this disease is not fully understood, hyperplasia and hypertrophy of intermediate smooth muscle are hallmarks of this disease. A molecular variant of the elastin gene has been implicated in the pathogenesis of aortic stenosis, July 2, 1997.
It is reported in U.S. Pat. No. 5,650,282 published on the 2nd. "Heart valve regurgitation" results from heart disease as a result of heart valve disease. Various conditions, such as rheumatic fever, cause contraction or pulling of the valve orifice, while other conditions include endocarditis, inflammation of the endocardium or the membrane inside the atrial orifice, and cardiac surgery. become. Defects, such as valve stenosis or proximity of a valve defect, result in the accumulation of blood in the heart chamber or regurgitation of blood through the valve. Failure to heal the valve stenosis or insufficiency for a long period of time will result in cardiac hypertrophy and myocardial related damage, eventually requiring replacement of the valve. Included in the invention is all of these, and other cardiovascular, endothelial, and angiogenic disorders that may or may not be associated with cardiac hypertrophy.

The terms “cancer”, “cancerous” and “malignant” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, Hodgkin and non-Hodgkin lymphoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, Liver cancer, such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, colorectal cancer, colorectal cancer, endometrial cancer, salivary gland cancer, renal cell carcinoma and Wilms tumor, basal cell carcinoma , Melanoma, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer. The preferred cancers for treatment herein are breast cancer, colon cancer, lung cancer, melanoma, ovarian cancer and the above-mentioned vascular tumors. 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 refers to radioisotopes (eg, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y and ¹⁸⁶ Re), chemotherapeutic agents, and bacteria, fungi,
It is intended to include toxins such as enzyme-activated toxins of plant or animal origin, or fragments thereof. A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents are alkylating agents, folate antagonists, metabolites of nucleic acid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil, cisplatin, purine nucleosides, amines, amino acids, triazole nucleosides, or corticosteroids. Is included. Specific examples include adriamycin, doxorubicin, 5-fluorouracil, cytosine arabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin, taxol, toxotea, methotrexate,
Cisplatin, melphalan, vinblastine, bleomycin, etoposide,
Ifosfamide, mitomycin C, mitoxanthone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin,
Includes aminopterin, dactinomycin, mitomycin, esperamycin (see US Pat. No. 4,675,187), melphalan and related nitrogen mustards. Also included in this definition are hormonal agents that function to regulate or inhibit hormonal action on tumors such as tamoxifen and onapristone.

“Growth inhibitor” as used herein refers to a compound or composition that inhibits the growth of cells, eg, Wnt-overexpressing cancer cells, in vitro or in vivo.
That is, the growth inhibitor significantly reduces the percentage of malignant cells in the S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a position other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxol,
And topo II inhibitors such as doxorubicin, daunorubicin, etoposide, and bleomycin. These G1 arresting agents also affect S phase arrest and are, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and Ara-C. For more information, see The Molecular Basis of Cancer,
Mendelsohn and Israel, edited, Chapter 1, Murakami et al. Entitled "Cell cycle r
egulation, oncogenes, and antineoplastic drugs '' (WB Saunders: Philadelp
hia, 1995), especially on page 13. Further examples include tumor necrosis factor (TNF)
, An antibody capable of inhibiting or neutralizing the angiogenic activity of acidic or basic FGF or hepatocyte growth factor (HGF), an antibody capable of inhibiting or neutralizing the coagulation activity of tissue factor, protein C or protein S (1991) (See WO91 / 01753 published February 21)
, Or an antibody capable of binding to the HER2 receptor (WO89 / 06692), for example 4D5 antibody (
And their functional equivalents) (eg WO92 / 22653). "Treatment" is intended to prevent the progression or modification of pathological conditions of cardiovascular, endothelial and angiogenic diseases. The concept of treatment is used in its broadest sense and includes in particular the prevention (prevention), alleviation, reduction and cure of cardiovascular, endothelial and angiogenic diseases of any stage. Thus, "treatment" refers to both curative treatment and prophylactic or preventative measures,
Patients are prevented or delayed in cardiovascular, endothelial and angiogenic disorders. 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 has been prevented. Diseases are idiopathic, cardiotrophic
, Or myotrophic causes, or as a result of any cause, including ischemia or ischemic stroke, eg, myocardial infarction. “Chronic” administration refers to the administration of the drug in a continuous manner, as opposed to an acute manner, to maintain an initial therapeutic effect, eg, an antihypertrophic effect, over an extended period of time. A "mammal" for purposes of treatment is any animal classified as a mammal, including humans, domestic and farm animals, zoos, sports, or pet animals, such as dogs, horses, cats, cows, sheep, pigs, and the like. Refers to an animal. Preferably the mammal is a human. Administration "in combination" with one or more additional therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

The term “cardiovascular, endothelial and angiogenic agents” generally refers to any agent that acts in the treatment of cardiovascular, endothelial and angiogenic disorders. Examples of cardiovascular agents are those that promote blood pressure, heart rate, cardiac contraction, and vascular homeostasis that regulates endothelial and smooth muscle biology, all factors having a role in cardiovascular disease. Specific examples of these include angiotensin-II receptor antagonists: endothelin receptor antagonists such as BOSENTAN ^™ and MOXONODIN ^™ , interferon-gamma (IFN-γ); des-asparatate-angiotensin I: thrombolytic agents such as streptokinase, Urokinase, t-PA, and a t-PA variant with a longer half-life and designed for very high fibrin specificity, TNK-t-PA (T103N, N117Q, KHRR (296-299) AAAAt- P
A mutant, Keyt et al., Proc. Natl. Acad. Sci. USA, 91: 3670-3674 (1994)): cardiotonic or pressor agents such as digoxigenin, and β-adrenergic receptor blockers such as propranolol, timolol, Tartarolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, and carvedilol: angiotensin converting enzyme (
ACE) inhibitors such as quinapril, captopril, enalapril, ramipril, benazepril, fosinopril and lisinopril; diuretics,
Examples include chlorothiazide, hydrochlorothiazide, hydroflumetazide, methylclothiazide, benzthiazide, dichlorophenamide, acetazolamide, and indapamide; and calcium channel blockers such as diltiazem, nifedipine, verapamil and nicardipine. One preferred category of this kind is therapeutic agents used for the treatment of cardiac hypertrophy, or physiological conditions associated with the progression of cardiac 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 where appropriate, angiogenesis. This includes factors that promote wound healing, such as growth hormone, insulin-like growth factor-I (IGF-I), VE.
GF, VIGF, PDGF, epidermal growth factor (EGF), CTGF and its family members, FGF, and TGF-α and TGF-β. An "angiostatic drug" is an active agent that inhibits angiogenesis or tube formation, or inhibits or prevents the growth of cancer cells. Examples include antibodies to the angiogenic agents described above or other antagonists such as antibodies to VEGF. In addition, cell therapies,
For example, cytotoxic agents, chemotherapeutic agents, growth inhibitors, apoptotic agents, and other agents that treat cancer, such as anti-HER-2, anti-CD20, and bioactive and organic chemical agents. A "therapeutically effective amount" of an active agent, such as a PRO polypeptide, or an agonist or antagonist thereof, or an anti-PRO antibody, in the pharmacological sense of the present invention.
The term refers to an amount effective for treating cardiovascular, endothelial and angiogenic diseases in mammals and can be empirically determined. As used herein, an "effective amount" of an active agent, eg, PRO polypeptide, or agonist or antagonist thereof, or anti-PRO antibody, refers to an amount effective to carry out the stated purpose. Such an amount can be empirically determined depending on the desired effect. As used herein, the terms “PRO polypeptide” and “PRO” refer to various polypeptides when immediately followed by a numerical code, with the full code (eg PRO / number) referring to the particular polypeptide described herein. Means peptide sequence. As used herein, the terms “PRO / number polypeptide” and “PRO / number”, where “number” is given as the actual numerical symbol used herein, refer to native sequence polypeptides and variants (here Will be defined in more detail in). The PRO polypeptides described herein may be isolated from a variety of sources, such as human tissue types or other sources, and may be prepared by recombinant or synthetic methods.

A “native sequence PRO polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide of natural origin. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term “native sequence PRO polypeptide” specifically refers to naturally occurring truncated or secreted forms of a particular PRO polypeptide (eg,
Extracellular domain sequences), naturally-occurring mutant forms (eg, alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In various embodiments of the invention, a native sequence PRO polypeptide disclosed herein is a mature or full length native sequence polypeptide comprising the full length amino acid sequence shown in the accompanying figures. Start and stop codons are shown in bold and underlined in the figure.
However, the PRO polypeptide disclosed in the accompanying figures is labeled with amino acid 1
Although it is shown to begin with a methionine residue designated herein as, it is contemplated that other methionine residues located upstream or downstream of amino acid position 1 in the figure could be used as the starting amino acid residue of the PRO polypeptide. It is possible and possible. The PRO polypeptide "extracellular domain" or "ECD" means a form of the PRO polypeptide that is substantially free of transmembrane and cytoplasmic domains. Usually PR
O polypeptides ECD have less than 1% of their transmembrane and / or cytoplasmic domains, preferably less than 0.5% of such domains. PRO of the present invention
It will be appreciated that any transmembrane domain identified for a polypeptide will be identified according to the criteria routinely used in the art to identify that type of hydrophobic domain. The exact boundaries of the transmembrane domain may vary, but are likely not to exceed about 5 amino acids from either end of the first identified domain. Thus, a PRO polypeptide extracellular domain optionally comprises no more than about 5 amino acids from either side of the transmembrane domain and / or extracellular domain boundaries as identified in the Examples or specification. However, those polypeptides and their encoding nucleic acids, with or without a signal peptide, are contemplated by the present invention. The approximate location of the "signal peptides" of the various PRO polypeptides disclosed herein are shown in the accompanying figures. However, as noted, the C-terminal boundary of the signal peptide may vary, but is most likely to be less than about 5 amino acids on either side of the signal peptide C-terminal boundary as originally defined herein. High, the C-terminal boundary of the signal peptide can be identified according to the criteria routinely used to identify such types of amino acid sequence components (eg, Nielsen et al., Prot. Eng. 10: 1-
6 (1997) and von Heinje et al., Nucl. Acids. Res. 14: 4683-4690 (1986)). Furthermore, it is also recognized that in some cases cleavage of the signal peptide from the secreted polypeptide is not completely homogeneous, resulting in one or more secreted species. These mature polypeptides, and the polynucleotides encoding them, in which the signal peptide is cleaved within less than about 5 amino acids on either side of the C-terminal border of the signal peptide as defined herein, are contemplated by the present invention. It

A “PRO polypeptide variant” is a full-length native sequence PRO polypeptide disclosed herein, a full-length native sequence PRO polypeptide lacking the signal peptide disclosed herein, as defined above or below. An active PR having at least about 80% amino acid sequence identity with a sequence, the extracellular domain of a PRO disclosed herein with or without a signal peptide, or other fragments of the full-length PRO polypeptide disclosed herein.
O polypeptide. Such PRO polypeptide variants include, for example, PRO polypeptides in which one or more amino acid residues have been added or deleted at the N- or C-terminus of the full-length native amino acid sequence. Generally, a PRO polypeptide variant is a full length native amino acid sequence disclosed herein, a full length native sequence PRO polypeptide sequence lacking the signal peptide disclosed herein, the extracellular domain of a PRO disclosed herein with or without a signal peptide. Or the full length PR disclosed herein
At least about 80% amino acid sequence identity with the other fragment of the O polypeptide, 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% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably Is at least about 91% amino acid sequence identity, more preferred Or 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 more preferably at least about 99%. Have amino acid sequence identity. Generally, a PRO variant polypeptide is at least about 10 amino acids in length, more at least about 20 amino acids in length, more at least about 30 amino acids in length, more at least about 40 amino acids in length, more at least about 50 amino acids in length. Long, more at least about 60 amino acids in length, more at least about 70 amino acids in length, more at least about 80 amino acids in length,
More than at least about 90 amino acids long, more than at least about 100 amino acids long, more at least about 150 amino acids long, and more at least about 20 amino acids long.
It is 0 amino acids long, more than at least about 300 amino acids long, or more. As shown below, Table 1 provides the secure source code for the ALIGN-2 sequence comparison computer program. This source code is routinely compiled for use with the UNIX operating system to give the ALIGN-2 sequence comparison computer program. In addition, Tables 2-5 show the following methods for determining% amino acid sequence identity (Table 2-3) and% nucleic acid sequence identity (Table 4-5) using the ALIGN-2 sequence comparison computer program. Shows a hypothetical example to use, where "PRO" is the hypothetical PRO of interest
The amino acid sequence of the polypeptide is shown, and "comparative protein" is the target "PRO".
"Shows the amino acid sequences of the polypeptides with which the polypeptides are compared," PRO-D
"NA" indicates a hypothetical PRO-encoding nucleic acid sequence of interest, "comparative DNA" indicates the nucleotide sequence of the nucleic acid molecule to which the "PRO-DNA" nucleic acid molecule of interest is compared, and "X", "Y" And “Z” represent different hypothetical amino acid residues,
"N", "L" and "V" each represent different hypothetical nucleotides.

[0033]

[0034]

[0035]

[0036]

[0037]

“Percent (%) amino acid sequence identity” to a PRO polypeptide sequence, as defined herein, aligns the sequences and introduces gaps, if necessary, to obtain the maximum percent sequence identity, regardless of conservation. Substitutions are defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the PRO sequence, without also considering them as part of sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be performed by a variety of methods within the skill of the art, including BLAST, BLAST.
-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software can be achieved by using publicly available computer software. One of ordinary skill in the art can determine the appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes herein, the% amino acid sequence identity values are set forth in the sequence comparison program ALIGN-2, the complete source code for which is given in Table 1, as described below. Is obtained by using. ALI
The GN-2 sequence comparison computer program was created by Genentech, Inc.
The source code shown in is submitted to the US Copyright Office, Washington DC, 20559 with user documentation and is registered under US Copyright Registration Number TXU510087. A
LIGN-2 is publicly available through Genentech, Inc., South San Francisco, California and may be compiled from the source code given in Table 1. ALIG
N-2 program is a UNIX operating system, preferably Digital UNIX V4
Compiled for use with .0D. All sequence comparison parameters are ALIGN-2
It is set by the program and does not change. For purposes herein, the% amino acid sequence identity of a given amino acid sequence A, to or to a given amino acid sequence B (or to or to a given amino acid sequence B or A given amino acid sequence A with or including some degree of% amino acid sequence identity may also be calculated as: 100 times the fraction X / Y, where X is the sequence alignment program ALIGN. -2 is the number of amino acid residues with a score that is identical by alignment of A and B, and Y is the total number of amino acid residues in B. It will be appreciated that when the length of amino acid sequence A differs from the length of amino acid sequence B, the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A. As an example of calculating the% amino acid sequence identity using this method, Table 2-3 shows the calculation of the% amino acid sequence identity of the amino acid sequence referred to as "comparative protein" to the amino acid sequence referred to as "PRO". Show the method.

Unless otherwise indicated, all% amino acid sequence identity values herein are as above.
Obtained using the ALIGN-2 sequence comparison computer program. However,%
Amino acid sequence identity is determined by the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic
Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http://www.ncbi.nlm.nih.gov. And is otherwise available from the National Institute of Health, Bethesda, MD. NC
BI-BLAST2 uses several search parameters, all of which are set to initial values, eg unmask = OK, chain = all, expected occurrence = 10, minimum low compound length = 15/5 , Multipath e-value = 0.01, multipath constant = 25
, Final gap alignment drop-off = 25, and scoring matrix = BLOSUM62. In the situation where NCBI-BLAST2 is used for amino acid sequence comparisons, the% amino acid sequence identity of a given amino acid sequence A with or to a given amino acid sequence B (or with a given amino acid sequence B, or On the other hand, a certain percentage
A given amino acid sequence A with or containing amino acid sequence identity may also be calculated) as follows: 100 times the fraction X / Y, where X is A in the sequence alignment program NCBI-BLAST2. And the number of amino acid residues in the score that were matched by the alignment of B to be identical, where Y is B
Is the total number of amino acid residues. It will be appreciated that when the length of amino acid sequence A differs from the length of amino acid sequence B, the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A. In addition,% amino acid sequence identity values are calculated using the WU-BLAST-2 computer program (Al
tschul et al., Methods in Enzymology 266: 460-480 (1996)). Most WU-BLAST-2 search parameters are set to default values. Not set to initial values, ie adjustable parameters set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11,
And scoring matrix = BLOSUM62. For purposes herein, a% amino acid sequence identity value is (a) an amino acid sequence of a PRO polypeptide of interest having a sequence derived from a native PRO polypeptide and a comparative amino acid sequence of interest (ie, The number of matching identical amino acid residues as determined by WU-BLAST-2 between (a sequence which may be a PRO polypeptide variant with which the PRO polypeptide of interest is compared) and (b) Is determined by the quotient divided by the total number of residues of the PRO polypeptide. For example, "a polypeptide comprising an amino acid sequence A having or having at least 80% amino acid sequence identity with the amino acid sequence B".
In this expression, the amino acid sequence A is the target comparative amino acid sequence, and the amino acid sequence B is the target PRO polypeptide amino acid sequence.

By “PRO variant polynucleotide” or “PRO variant nucleic acid sequence” is meant a nucleic acid molecule that encodes an active PRO polypeptide, as defined below, the full length native sequence PRO polypeptide disclosed herein. Sequence, full length native sequence PRO polypeptide sequence lacking the signal peptide disclosed herein, PRO polypeptide extracellular domain with or without the signal peptide disclosed herein, or full length P disclosed herein.
At least about 8 relative to the nucleic acid sequence encoding the other fragment of the RO polypeptide sequence
It has 0% nucleic acid sequence identity. Generally, a PRO variant polynucleotide is a full length native sequence PRO polypeptide sequence disclosed herein, a full length native sequence PRO polypeptide sequence lacking the signal peptide disclosed herein, a PRO polypeptide with or without the signal peptide disclosed herein. At least about 80% with the nucleic acid sequence encoding the peptide extracellular domain, or other fragment of the full-length PRO polypeptide sequences disclosed herein.
Nucleic acid sequence identity, 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 Is 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 9
6% 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 more preferably at least about 99% nucleic acid sequence identity. Variants do not include the native nucleotide sequence. Generally, a PRO variant polynucleotide is at least about 30 nucleotides long, more at least about 60 nucleotides long, more at least about 90 nucleotides long, more at least about 120 nucleotides long, more at least about 150 nucleotides long. Long, more often at least about 180 nucleotides, more at least about 210 nucleotides, and more at least about 24
0 nucleotides, more at least about 270 nucleotides, more at least about 300 nucleotides, more at least about 450 nucleotides, more at least about 600 nucleotides, more at least about 900 nucleotides, or More than that.

The "percent (%) nucleic acid sequence identity" identified for a PRO polypeptide-encoding nucleic acid sequence as defined herein is that required to align the sequences and obtain the maximum percent sequence identity. It is then defined as the percentage of nucleotides in the candidate sequence that are identical to the nucleotides in the PRO polypeptide-encoding sequence, without introducing a gap and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent nucleic acid sequence identity can be performed by a variety of methods within the skill of the art, including BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNA
This can be achieved by using publicly available computer software such as STAR software. One of ordinary skill in the art can determine the appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein, the% nucleic acid sequence identity values are calculated as described below for AL
The complete source code for the IGN-2 program is obtained using the sequence comparison program ALIGN-2 given in Table 1. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code shown in Table 1 is the US Copyright Office.
, Washington DC, 20559, filed with user documentation and registered under US Copyright Registration Number TXU510087. ALIGN-2 is Genentech, South San Fran
It is publicly available through cisco, California and may be compiled from the source code given to Figure2AP. The ALIGN-2 program is compiled for use with a UNIX operating system, preferably Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. For the purposes herein, the% nucleic acid sequence identity of a given nucleic acid sequence C with or to a given nucleic acid sequence D (or with a given nucleic acid sequence D,
Or a given amino acid sequence C which has or contains some% nucleic acid sequence identity thereto) is calculated as follows: 100 times the fraction W / Z where W Is the number of nucleotides in the score that were matched by the C and D alignments of the sequence alignment program ALIGN-2, and Z is the total number of nucleotides in D. When the length of the nucleic acid sequence C is different from the length of the nucleic acid sequence D, C
It will be appreciated that the% nucleic acid sequence identity of D to C is different from the% nucleic acid sequence identity of D to C. As an example of calculating% nucleic acid sequence identity using this method, Tables 4-5 show% nucleic acid sequence identity of a nucleic acid sequence referred to as "comparative DNA" to a nucleic acid sequence referred to as "PRO-DNA." The calculation method of is shown. Unless otherwise noted, all% nucleic acid sequence identity values herein are ALIG as described above.
Obtained using the N-2 sequence comparison computer program. However,% nucleic acid sequence identity is determined by the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids R
es. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http://ww.ncbi.nlm.nih.gov. NCBI-BLAST
2 uses some search parameters, all of which are set to initial values, eg unmask = ok, chains = all, expected occurrence = 10, minimum low compound length = 15/5, multi Path e-value = 0.01, multipath constant = 25, final gap alignment dropoff = 25, and scoring matrix = BL
Including OSUM62.

In the situation where NCBI-BLAST2 is used for sequence comparisons, the% nucleic acid sequence identity of a given nucleic acid sequence C with or to a given nucleic acid sequence D (or with a given nucleic acid sequence D , Or a given nucleic acid sequence C which has or contains some% nucleic acid sequence identity thereto) is calculated as follows: 100 times the fraction W / Z where: W is the number of scored nucleotides that were matched by the C and D alignments of the sequence alignment program NCBI-BLAST2, and Z is D
Is the total number of nucleotides in. It will be appreciated that when the length of nucleic acid sequence C differs from the length of nucleic acid sequence D, the% nucleic acid sequence identity of C to D is different from the% nucleic acid sequence identity of D to C. In addition,% nucleic acid sequence identity values can be found in the WU-BLAST-2 computer program (Altsch
ul et al., Methods in Enzymology 266: 460-480 (1996)). Most WU-BLAST-2 search parameters are set to default values. Not set to default values, ie adjustable parameters set to the following values: overlap span =
1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. For purposes herein,% nucleic acid sequence identity values are (a) the nucleic acid sequence of the PRO polypeptide encoding sequence of interest having a sequence derived from the native sequence PRO polypeptide encoding nucleic acid, and To a comparative nucleic acid molecule (ie, a sequence which may be a variant polynucleotide with which the sequence of the PRO polypeptide-encoding nucleic acid molecule of interest is compared), the matching identity determined by WU-BLAST-2. It is determined by the quotient of the number of nucleotides divided by the total number of nucleotides of the (b) PRO polypeptide-encoding nucleic acid of interest. For example, in the expression "an isolated nucleic acid molecule comprising a nucleic acid sequence A having or having at least 80% amino acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the target nucleic acid of comparison. Sequence Nucleic Acid Sequence B is the nucleic acid sequence of the subject PRO polypeptide-encoding nucleic acid molecule. In another embodiment, the PRO variant polynucleotide is a nucleic acid molecule that encodes an active PRO polypeptide, preferably under stringent hybridization and wash conditions, respectively Figure2 (SEQ ID NO: 2), Figure4 (SEQ ID NO: 2). Number: 4), Fig
6 (SEQ ID NO: 6), Fig8 (SEQ ID NO: 8), Fig10 (SEQ ID NO: 10)
), FIG12 (SEQ ID NO: 12), FIG14 (SEQ ID NO: 14), FIG1
6 (SEQ ID NO: 16), FIG18 (SEQ ID NO: 18), FIG20 (SEQ ID NO: 20), FIG22 (SEQ ID NO: 22), FIG24 (SEQ ID NO: 24), F
ig26 (SEQ ID NO: 26), Fig28 (SEQ ID NO: 28), Fig30 (SEQ ID NO: 30), and FIG32 (SEQ ID NO: 32), which can hybridize to the nucleotide sequence encoding the full-length PRO polypeptide. The PRO variant polypeptide may be one encoded by the PRO variant polynucleotide.

The term “positive” in the context of amino acid sequence identity comparisons performed as described above includes amino acid residues which are identical as well as those which are identical in the compared sequences. Amino acid residues that score a positive value for the target amino acid residue are the same as the target amino acid residue, or of the target amino acid residue (as specified in Table 6 below). ) Are preferred substitutions. For the purposes herein, the% positive value of a given amino acid sequence A, with or with respect to a given amino acid sequence B (or with respect to a given amino acid sequence B, or to a certain extent thereto). A given amino acid sequence with or including% positives (also referred to as A) can be calculated as follows: 100 times the fraction X / Y, where X is the sequence alignment program ALIGN-2 A and B. Is the number of amino acid residues that are scored as positive by the alignment, and Y is the total number of amino acid residues in B. It will be appreciated that if the length of the amino acid sequence A differs from the length of the amino acid sequence B, then the% positive of A to B is different from the% positive of B to A. "Isolated," when used to describe the various polypeptides disclosed herein, means the polypeptides that have been identified, separated and / or recovered from the components of their natural environment. Preferably, the isolated polypeptide is free of association with all components that naturally bind. Contaminant components of its natural environment are materials that typically interfere with diagnostic or therapeutic uses for the polypeptide, and include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide is (1) sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a spinning cup sequenator, or (2) Coomassie blue or It is preferably purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using silver staining. Isolated polypeptide includes protein in situ within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step. An "isolated" nucleic acid molecule that encodes a PRO polypeptide, or an "isolated" nucleic acid molecule that encodes an anti-PRO antibody, has been identified and is a natural source of PRO-encoding nucleic acid, or anti-PRO- At least one normally associated with the natural source of the encoded nucleic acid
A nucleic acid molecule separated from one contaminant nucleic acid molecule. Preferably, the isolated nucleic acid molecule is free of all naturally bound contaminants. Isolated PRO-
The encoding nucleic acid molecule or isolated anti-PRO-encoding nucleic acid molecule is in a form or setting other than those found in nature. Thus, isolated nucleic acid molecule is distinguished from the PRO-encoding nucleic acid molecule or anti-PRO-encoding nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule that encodes a PRO polypeptide, or an isolated nucleic acid molecule that encodes an anti-PRO antibody, is, for example, a PRO polypeptide that has a chromosomal location different from that of the native cell. Peptide or anti-PR
It includes a PRO-nucleic acid molecule contained in cells that normally express O antibodies, or an anti-PRO-nucleic acid molecule.

The term "control sequences" refers to DNA sequences necessary for the expression of operably linked coding sequences in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA for a presequence or secretory leader is operably linked to the DNA for the PRO polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; the promoter or enhancer is the transcription of the sequence. Is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is in a position that facilitates translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in the reading phase. However, enhancers do not necessarily have to be in close proximity. Binding is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adapters or linkers are used in accord with conventional practice. The "stringency" of a hybridization reaction is readily determined by one of ordinary skill in the art and is an empirical calculation that generally depends on probe length, wash temperature, and salt concentration. In general, longer probes have higher temperatures for proper annealing and shorter probes have lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting point. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures make the reaction conditions more stringent, while lower temperatures reduce stringency. Further details and explanations of the stringency of the hybridization reaction can be found in Ausubel et al., Current Proto.
See cols in Molecular Biology, Wiley Interscience Publishers, (1995). As defined herein, "stringent conditions" or "high stringency conditions" means (1) low ionic strength and high temperature for washing, eg, 0.015M sodium chloride / 0.0015M at 50 ° C. Using sodium citrate / 0.1% sodium dodecyl sulphate; (2) Denaturants such as formamide during hybridization, eg 50% (v / v) formamide and 0.1% bovine serum albumin at 42 ° C. /0.1% Ficoll / 0.1% Polyvinylpyrrolidone / 50 mM pH 6
． 5 sodium phosphate buffer, and 750 mM sodium chloride, 75 m
With M sodium citrate; (3) 50% formamide at 42 ° C., 5 × SSC (0.75M NaCl, 0.075M sodium citrate),
50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhard's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1%
High stringency consisting of SDS and 0.1% SSC with 10% dextran sulfate, 0.2x SSC (sodium chloride / sodium citrate) wash at 42 ° C and 50% formamide at 55 ° C, then EDTA at 55 ° C. Identified by those using sexual wash. “Moderate stringency conditions” are described in Sambrook et al., Molecular Cloning: A Laboratory.
Manual (New York: Cold Spring Harbor Laboratory Press, 1989), including use of less stringent wash solutions and hybridization conditions (eg, temperature, ionic strength and% SDS) as described above. . Moderate stringency conditions were 20% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate pH 7.6, 5x denhard solution, 10% dextran sulfate, and 20 mg / ml. Denatured sheared salmon sperm DNA
Incubation in a solution containing at 37 ° C. overnight followed by 37-in 1 × SSC.
The condition is that the filter is washed at 50 ° C. One of ordinary skill in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

The modifier “epitope tag”, as used herein, refers to a chimeric polypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.
The tag polypeptide has a sufficient number of residues to provide an epitope against which the antibody can be produced, but its length is short enough so as not to interfere with the activity of the fused polypeptide. Also, the tag polypeptide is preferably fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues, usually about 8 to 50 amino acid residues.
(Preferably about 10 to about 20 amino acid residues). “Active” and “activity” in a PRO variant refer to natural or naturally occurring PRO
Refers to forms of PRO proteins that retain the biological and / or immunological activity of the polypeptide. "Biological activity" in a molecule that antagonizes a PRO polypeptide identifiable by the screening assays disclosed herein (eg, small organic or inorganic molecules, peptides, etc.) refers to the PRO polypeptide identified herein. Binding or conjugation,
Or interferes with the interaction of the PRO polypeptide with other cellular proteins, or PRO
Used to refer to a molecule's ability to inhibit the transcription or translation of a polypeptide.
Particularly preferred biological activities include systemic diseases affecting blood vessels, such as diabetes mellitus, and diseases of the arteries, capillaries, veins and / or lymph vessels, and cardiac hypertrophic activity affecting cancer. The term “antagonist” is used in its broadest sense to block, inhibit one or more biological activity of the native PRO polypeptide disclosed herein, eg, its mitogenic or angiogenic activity, where appropriate, Alternatively, it includes any molecule that neutralizes. P
An antagonist of a RO polypeptide interferes with the binding of the PRO polypeptide to a cell receptor, neutralizes or kills cells activated by the PRO polypeptide, or vascular endothelial cells after the PRO polypeptide binds to the cell receptor. It acts by interfering with the activation of. All of these points of mediation by a PRO polypeptide antagonist are considered equivalent to the purposes of this invention. Antagonists inhibit mitogenesis, angiogenesis, or other biological activity of PRO polypeptides, and thus tumors, particularly solid malignancies, rheumatoid arthritis, psoriasis,
Atherosclerosis, diabetes, other retinopathy, post lens fibroplasia, age-related plaque degeneration, neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, neovascular glaucoma, hemangiomas , Thyroid hyperplasia (Graves' disease), transplantation of the cornea and other tissues, and chronic inflammation, are useful in the treatment of diseases or disorders characterized by excessive neovascularization. Antagonists also include diseases or disorders characterized by undesired excessive vascular permeability, such as edema associated with brain tumors, ascites associated with malignant tumors, Megs syndrome, pneumonia, nephrotic syndrome,
Useful for treating epicardial fluid (eg, associated with pericarditis), pleural effusion. Similarly, the term "agonist" is used in its broadest sense and refers to the native PRO disclosed herein.
It includes any molecule that mimics the biological activity of a polypeptide. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of the native PRO polypeptide, peptides, small organic molecules and the like. A "small molecule" is defined herein as having a molecular weight of about 500 Daltons or less. The term "PRO polypeptide receptor" as used herein refers to PR
It refers to the cellular receptors of O polypeptides, cell surface receptors normally found on vascular endothelial cells, as well as variants thereof which retain the ability to bind PRO polypeptides.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. Antibodies show binding specificity for specific antigens,
Immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter type of polypeptide is produced, for example, at low levels by the lymphatic system and at increased levels by myeloma. The term "antibody" is used in its broadest sense and includes, but is not limited to, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (
(Eg, bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. "Natural antibodies" and "natural immunoglobulins" are heterotetrameric glycoproteins of about 150,000 daltons, usually composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. In addition, each heavy chain and light chain have regularly spaced interchain disulfide bonds. Each heavy chain has at one end a variable domain ( _VH ) followed by a number of constant domains. Each light chain has a variable domain ( _VL ) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the variable domain of the light chain is the heavy chain. Aligned with the variable domain of. Particular amino acid residues are believed to form the interface between the light and heavy chain variable domains. The term "variable" refers to the fact that certain sites in the variable domain differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. . However, the variability is not evenly distributed over the variable domain of the antibody. It is concentrated in three segments called hypervariable regions or complementarity determining regions (CDRs) of both light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR). The variable domains of the natural heavy and light chains are predominantly in the β-sheet arrangement linked by three CDRs that bind the β-sheet structure and in some cases form a loop bond forming part of it 4. It contains two FR regions. CDR of each chain
Are bound closer to the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. Kabat et al., NIH Publ.No.91-3242, Vol.I, 647-66
See page 9 [1991]. The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the involvement of the antibody in antibody-dependent cellular cytotoxicity.

An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ and F.
v fragment; diabodies; linear antibody (Zapata et al., Protein Eng. 8 (1
0): 1057-1062 [1995]); single chain antibody molecules; and multispecific antibodies formed from antibody fragments. Upon papain digestion of the antibody, two identical antigen-binding fragments, each called a "Fab" fragment, each having a single antigen-binding site, and a residual "Fc" fragment, whose name represents the ability to crystallize readily, are obtained. Produced. Pepsin treatment yields an F (ab ′) ₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen. "Fv" is the minimum antibody 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. In this arrangement, the three CDRs of each variable domain interact to form an antigen binding site on the surface of the _VH - _VL dimer. Collectively, 6 CDs
R confers antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has a lower affinity than the total binding site, but still has the ability to recognize and bind antigen. Have The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) 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 CH1 region including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab 'in which the cysteine residue (s) of the constant domains carry a free thiol group. F (ab ′) ₂ antibody fragments were produced as pairs of Fab ′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The “light chain” of an antibody (immunoglobulin) from any vertebrate species is based on the amino acid sequence of its constant domain, in two distinct types called kappa (κ) and lambda (λ). One is assigned. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA,
There are IgD, IgE, IgG and IgM, some of which are further subclasses
(Isotype), eg, IgG1, IgG2, IgG3 and IgG4, IgA and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, σ, ε, γ and μ, respectively. The subunit and three-dimensional structures of different classes of immunoglobulins are well known.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies that make up the population naturally occur in small amounts. Identical except for possible mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, different determinants
Each monoclonal antibody is directed against a single determinant on the antigen, as compared to conventional (polyclonal) antibodies that typically include different antibodies directed against (epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized from hybridoma cultures uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and does not mean that the antibody must be produced in any particular fashion. For example, the monoclonal antibody used in the present invention is
It can be made by the hybridoma method disclosed by Kohler et al., Nature 256, 495 (1975) or can be made by the recombinant DNA method (eg,
See U.S. Pat. No. 4,816,567). Also, "monoclonal antibody" refers to, for example, Clackson.
Et al., Nature 352: 624-628 (1991), and Marks et al., J. Mol. Biol. 222: 581-597 (1
It can also be isolated from a phage antibody library using the technique described in 991). Here, the monoclonal antibody has a part of the heavy chain and / or the light chain which is identical or homologous to the corresponding sequence of an antibody derived from a specific species or an antibody belonging to a specific antibody class or subclass, and the rest of the chains. A "chimeric" antibody (immunoglobulin) whose portion is identical or homologous to the corresponding sequence of an antibody from another species or an antibody of another antibody class or subclass, as well as which has the desired biological activity In particular, fragments of these antibodies are specifically included (US Pat. No. 4,816,567; Morrison et al., Proc. Na.
tl. Acad. Sci. USA, 81: 6851-6855 [1984]). "Humanized" forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ', F (ab')).
₂ or other antigen-binding subsequence of an antibody), which contains a minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies will have residues in the CDRs of the recipient replaced by those in the CDRs of a non-human species (donor antibody) having the desired specificity, affinity and capacity, such as mouse, rat or rabbit. Human immunoglobulin (recipient antibody). In some cases, human immunoglobulin F
The vFR region residues are replaced by the corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody properties. Generally, a humanized antibody has at least one, and typically no, all or almost all CDR regions of a non-human immunoglobulin sequence and all or almost all FR regions of a human immunoglobulin sequence. It contains substantially all of the two variable domains. The humanized antibody optimally comprises 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); Re.
ichmann et al., Nature 332, 323-329 (1988); and Presta, Curr. Op. Struct. Biol.
. 2, 593-596 (1992). Humanized antibodies include primatized PRIMATIZED ^™ antibodies in which the antigen binding region of the antibody is derived from an antibody produced by immunizing a macaque with the antigen of interest.

“Single-chain Fv” or “sFv” antibody fragments contain the V _H and V _L domains of the antibody, and these domains are present in the polypeptide single chain. Preferably,
The Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains which enables the sFv to form the desired structure for antigen binding. sFv
Reviews include, for example, Pluckthun, The Pharmacology of Monoclonal Antibo
dies, vol.113, edited by Rosenburg and Moore (Springer-Verlag, New York, 1994) pp.2
See 69-315. The term “diabody” refers to a small antibody fragment having two antigen binding sites, the fragment comprising a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) of the same polypeptide chain. ( _VH - _VL ). By using a linker that is so short that no pairing between the two domains on the same chain is allowed, the domain is forced to pair with the complementary domain of the other chain, creating two antigen binding sites. Diabodies are described in more detail, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. 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 its diagnostic or therapeutic use for the antibody, including enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is (1) Lori method.
More than 95% antibody, most preferably 99% by weight when measured by (Lowry method)
By using (2) Spinning cup sequenator until
Enough to obtain an N-terminal or internal amino acid sequence of at least 15 residues,
Alternatively, (3) purification to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. Isolated antibody includes the antibody in 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 term "label" as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody to produce a "labeled" antibody. The label may itself be detectable (eg a radioactive or fluorescent label) or, in the case of an enzymatic label, may catalyze the chemical conversion of a detectable substrate compound or composition. Radionuclides that can be provided as the detectable label include, for example, I-131, I-123, and I-125.
, Y-90, Re-188, At-211, Cu-67, Bi-212, and Pd-1
Including 09. The label may also be an undetectable substance such as a toxin. By "solid phase" is meant a non-aqueous matrix to which the antibodies of the invention can adhere. Examples of solid phases included herein include those formed partly or wholly from glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol and silicone. In certain embodiments, depending on the context, the solid phase can comprise the wells of an assay plate; in others it can be a purification column (eg, an affinity chromatography column). The term also includes a discontinuous solid phase of discrete particles, such as those described in US Pat. No. 4,275,149.

“Liposomes” are small vesicles composed of various types of lipids, phospholipids, and / or surfactants, which are drugs for mammals (PRO polypeptides or their antibodies disclosed herein). Etc.) is useful for transportation. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. The term “immunoadhesin” as used herein refers to antibody-like molecules that combine the binding specificity of a heterologous protein (“adhesin”) with the effector functions of immunoglobulin constant domains. Structurally, an immunoadhesin contains a fusion of an immunoglobulin constant domain sequence with an amino acid sequence that has the desired binding specificity and is other than the antigen recognition and binding site of an antibody (ie, "heterologous"). . The adhesin portion of the immunoadhesin molecule is typically a contiguous amino acid sequence containing at least the binding site for the receptor or ligand. The immunoglobulin constant domain sequence of the immunoadhesin has IgG-1, IgG-2, IgG-3 or IgG-4 subtype, I
It can be obtained from any immunoglobulin such as gA (including IgA-1 and IgA-2), IgE, IgD or IgM.

II. Compositions and Methods of the Invention A. PRO179, PRO238, PRO364, PRO844, PRO84
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
And PRO887 variants, full-length native sequence PRO179, PRO238, PRO364, P described herein.
RO844, PRO846, PRO1760, PRO205, PRO321, P
RO333, PRO840, PRO877, PRO878, PRO879, PR
In addition to the O882, PRO885 and PRO887 polypeptides, PRO179
, PRO238, PRO364, PRO844, PRO846, PRO1760
, PRO205, PRO321, PRO333, PRO840, PRO877,
It is believed that the PRO878, PRO879, PRO882, PRO885 and PRO887 variants can also be prepared. PRO179, PRO238, PRO364,
PRO844, PRO846, PRO1760, PRO205, PRO321,
PRO333, PRO840, PRO877, PRO878, PRO879, P
The RO882, PRO885 and PRO887 mutants are the same as PRO179, PRO2.
38, PRO364, PRO844, PRO846, PRO1760, PRO2
05, PRO321, PRO333, PRO840, PRO877, PRO87
8, PRO879, PRO882, PRO885 or PRO887 by introducing appropriate nucleotide changes and / or the desired PRO179, P
RO238, PRO364, PRO844, PRO846, PRO1760, P
RO205, PRO321, PRO333, PRO840, PRO877, PR
It can be prepared by synthesizing an O878, PRO879, PRO882, PRO885 or PRO887 polypeptide. Those skilled in the art will recognize that amino acid changes, such as changes in the number or position of glycosylation sites or changes in membrane anchoring properties, may be associated with PRO179, PR.
O238, PRO364, PRO844, PRO846, PRO1760, PR
O205, PRO321, PRO333, PRO840, PRO877, PRO
It will be appreciated that the post-translational process of 878, PRO879, PRO882, PRO885 or PRO887 may be altered. Natural full-length sequences PRO179, PRO238, PRO364, PRO844, P
RO846, PRO1760, PRO205, PRO321, PRO333, P
RO840, PRO877, PRO878, PRO879, PRO882, PR
O885 or PRO887 or PRO179, PRO238, P described herein
RO364, PRO844, PRO846, PRO1760, PRO205, P
RO321, PRO333, PRO840, PRO877, PRO878, PR
Mutations in various domains of O879, PRO882, PRO885 or PRO887 can be made using any of the techniques and guidelines for conservative and non-conservative mutations described, for example, in US Pat. No. 5,364,934. The mutation results in the native sequence PRO179, PRO238, PRO364, PRO844,
PRO846, PRO1760, PRO205, PRO321, PRO333,
PRO840, PRO877, PRO878, PRO879, PRO882, P
PRO179, PRO238, PRO3 compared to RO885 or PRO887
64, PRO844, PRO846, PRO1760, PRO205, PRO3
21, PRO333, PRO840, PRO877, PRO878, PRO87
P in which the amino acid sequence of 9, PRO882, PRO885 or PRO887 is changed
RO179, PRO238, PRO364, PRO844, PRO846, PR
O1760, PRO205, PRO321, PRO333, PRO840, PR
O877, PRO878, PRO879, PRO882, PRO885 or PR
It may be a substitution, deletion or insertion of one or more codons encoding O887.
In some cases, the mutation is at least one amino acid of PRO179, PRO23.
8, PRO364, PRO844, PRO846, PRO1760, PRO20
5, PRO321, PRO333, PRO840, PRO877, PRO878
, PRO879, PRO882, PRO885 or PRO887 in any one or more domains by any other amino acid. A guideline for which amino acid residues are inserted, substituted or deleted without adversely affecting the desired activity is PRO.
179, PRO238, PRO364, PRO844, PRO846, PRO1
760, PRO205, PRO321, PRO333, PRO840, PRO8
77, PRO878, PRO879, PRO882, PRO885 or PRO8
It is found by comparing the 87 sequences with the sequences of known protein molecules of homology and minimizing the amino acid sequence changes made within the regions of high homology. Amino acid substitutions can be the result of substitutions of one amino acid for another amino acid with similar structural and / or chemical properties, eg, replacement of leucine with serine, a conservative amino acid substitution. Insertions and deletions can optionally be in the range of 1 to 5 amino acids. Permissible mutations are determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting variants for the activity exhibited by the full-length or mature native sequence.

In a particular embodiment, the conservative substitutions of interest are shown in Table 6, beginning with the preferred substitution. If such a substitution results in a change in biological activity, more substitutional changes are introduced and the product screened, as shown in Table 6 for exemplary substitutions or as further described below in the amino acid taxonomy. Table 6 Original residues Exemplary substitution Preferred substitution Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe Norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala ; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met phe; ala; norleucine leu PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
Substitutional modification of the function and immunological identity of a PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptide may be accomplished by (a) a structure of the polypeptide backbone of the substitution region, eg a sheet or helical arrangement, (b) a target site. This is accomplished by selecting substituents that are substantially different in their effect, while maintaining the charge or hydrophobicity of the molecules of, or (c) the bulk of the side chains. Naturally occurring residues can be divided into groups based on common side chain properties: (1) Hydrophobicity: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilicity: cys, ser. , thr; (3) Acidity: asp, glu; (4) Basicity: asn, gln, his, lys, arg; (5) Residues affecting chain orientation: gly, pro; and (6) aromatic: Non-conservative substitutions for trp, tyr, phe will require exchanging a member of one of these classes for another class. Residues so substituted may also be introduced at conservative substitution sites, preferably left-over (non-conserved) sites.

Mutations can be made using techniques known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Re
s., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34: 315 (1985)], restriction selective mutagenesis. [Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)] or other well-known techniques are described in PRO179, PRO238, PRO364, PRO844, P.
RO846, PRO1760, PRO205, PRO321, PRO333, P
RO840, PRO877, PRO878, PRO879, PRO882, PR
D cloned to produce O885 or PRO887 mutant DNA
Can be implemented in NA. Also, scanning amino acid analysis can be used to identify one or more amino acids along a flanking sequence. The preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine,
Contains serine and cysteine. Alanine is typically the preferred scanning amino acid in this group because it excludes side chains above the beta carbon and does not alter the backbone structure of the mutant [Cunningham and Wells, Science, 244: 1081-1085]. (198
9)]. Also, alanine is typically preferred because it is the most common amino acid. Moreover, it is often found in both buried and exposed locations [Crei
ghton, The Proteins, (WH Freeman & Co., NY); Chothia, J. Mol. Bi
ol., 150: 1 (1976)]. If alanine substitution does not yield a sufficient amount of variant, an isoteric amino acid can be used.

B. PRO179, PRO238, PRO364, PRO844, PRO84
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
And modifications of PRO887 PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
Covalent modifications of PRO877, PRO878, PRO879, PRO882, PRO885 and PRO887 are included within the scope of the invention. One type of covalent modification is PRO179, PRO238, PRO364, PRO844, PRO8.
46, PRO1760, PRO205, PRO321, PRO333, PRO8
40, PRO877, PRO878, PRO879, PRO882, PRO88
5 or the targeted amino acid residue of the PRO887 polypeptide is replaced with PRO179,
PRO238, PRO364, PRO844, PRO846, PRO1760,
PRO205, PRO321, PRO333, PRO840, PRO877, P
Reaction with an organic derivatizing reagent capable of reacting with selected side chains or N- or C-terminal residues of RO878, PRO879, PRO882, PRO885 or PRO887. Derivatization with bifunctional reagents has been described, for example, in PRO179, PRO238.
, PRO364, PRO844, PRO846, PRO1760, PRO205
, PRO321, PRO333, PRO840, PRO877, PRO878,
PRO879, PRO882, PRO885 or PRO887 is a water-insoluble support matrix or anti-PRO179, anti-PRO238, anti-PRO364, anti-
PRO844, anti-PRO846, anti-PRO1760, anti-PRO205, anti-P
RO321, anti-PRO333, anti-PRO840, anti-PRO877, anti-PRO
878, anti-PRO879, anti-PRO882, anti-PRO885 or anti-PRO8
87 antibodies are useful for cross-linking surfaces for use in purification methods and vice versa. Commonly used cross-linking agents are, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide ester such as 4-azidosalicylic acid, 3,3′-dithiobis (succinimidyl). Homobifunctional imide ester including disuccinimidyl ester such as propionate), bifunctional maleimide such as bis-N-maleimido-1,8-octane, and methyl-3-[(
Including reagents such as p-azidophenyl) -dithio] propioimidate. Other modifications include deamination of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, lysine, arginine, and histidine side chains. Methylation of α-amino groups of [TE Creighton, Proteins: Structur
e and Molecular Properties, WH Freeman & Co., San Francisco, pp.79-86
(1983)], acetylation of N-terminal amines, and amidation of any C-terminal carboxyl groups.

PRO179, PRO238, PRO364, PR included within the scope of the present invention
O844, PRO846, PRO1760, PRO205, PRO321, PR
O333, PRO840, PRO877, PRO878, PRO879, PRO
Another type of covalent modification of the 882, PRO885 or PRO887 polypeptides involves altering the native glycosylation pattern of the polypeptide. By "altering the native glycosylation pattern" is intended herein the native sequence PRO179, PR.
O238, PRO364, PRO844, PRO846, PRO1760, PR
O205, PRO321, PRO333, PRO840, PRO877, PRO
Deletion of one or more carbohydrate moieties found in 878, PRO879, PRO882, PRO885 or PRO887 (either by removal of existing glycosylation sites or deletion of glycosylation by chemical and / or enzymatic means), and /
Or native sequence PRO179, PRO238, PRO364, PRO844, PR
O846, PRO1760, PRO205, PRO321, PRO333, PR
O840, PRO877, PRO878, PRO879, PRO882, PRO
By addition of one or more glycosylation sites not present in 885 or PRO887. In addition, this clause includes qualitative changes in glycosylation of native proteins, including changes in the nature and proportion of the various carbohydrate moieties present. PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
Addition of glycosylation sites to the PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptides may be accompanied by amino acid sequence alterations. This modification may be made, for example, by the native sequence PRO179, PRO238, PRO364, PRO844, PRO8 of one or more serine or threonine residues.
46, PRO1760, PRO205, PRO321, PRO333, PRO8
40, PRO877, PRO878, PRO879, PRO882, PRO88
5 or PRO887 (O-linked glycosylation site), or by substitution. PRO179, PRO238, PRO364, PRO844,
PRO846, PRO1760, PRO205, PRO321, PRO333,
PRO840, PRO877, PRO878, PRO879, PRO882, P
The RO885 or PRO887 amino acid sequence is optionally altered at the DNA level, in particular PRO179, PRO238, PRO364, PRO844, P.
RO846, PRO1760, PRO205, PRO321, PRO333, P
RO840, PRO877, PRO878, PRO879, PRO882, PR
It may be altered through mutating the DNA encoding the O885 or PRO887 polypeptides at preselected bases to generate codons that are translated into the desired amino acid.

PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
Another means of increasing the number of carbohydrate moieties on the PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are known in the art, for example WO 87/05330, published September 11, 1987, and Ap.
lin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981). PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
Removal of the carbohydrate moiety present on the PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptides results in mutational replacement of the codons encoding the amino acid residues presented chemically or enzymatically or as targets for glycosylation. Can be done by Chemical deglycosylation technology
Known in the art, for example Hakimuddin et al., Arch. Biochem. Biophys., 2
59:52 (1987) and by Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides is described by Thotakura et al., M.
This is achieved by using various endo and exoglycosidases, as described in eth. Enzymol. 138: 350 (1987). PRO179, PRO238, PRO364, PRO844, PRO of the present invention
846, PRO1760, PRO205, PRO321, PRO333, PRO
840, PRO877, PRO878, PRO879, PRO882, PRO8
85 or other types of covalent modifications of PRO887 are PRO179, PRO23.
8, PRO364, PRO844, PRO846, PRO1760, PRO20
5, PRO321, PRO333, PRO840, PRO877, PRO878
, PRO879, PRO882, PRO885 or PRO887 polypeptides to one of a variety of non-proteinaceous polymers such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, US Pat. No. 4,640,835.
No. 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,17
Includes binding in the manner described in 9,337.

In addition, PRO179, PRO238, PRO364, PRO844 of the present invention,
PRO846, PRO1760, PRO205, PRO321, PRO333,
PRO840, PRO877, PRO878, PRO879, PRO882, P
RO885 or PRO887 is PRO179, PRO238, PRO364, PRO844, PRO846 fused to another heterologous polypeptide or amino acid sequence.
, PRO1760, PRO205, PRO321, PRO333, PRO840
, PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 may be modified by a method for forming a chimeric molecule. In one embodiment, such a chimeric molecule comprises PRO179, PRO238, PRO and a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind.
364, PRO844, PRO846, PRO1760, PRO205, PRO
321, PRO333, PRO840, PRO877, PRO878, PRO8
79, PRO882, PRO885 or PRO887. Epitope tags typically include PRO179, PRO238, PRO364, PRO84.
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, PRO885 or PRO887 at the amino- or carboxyl-terminus. Such PRO179, PRO238, PRO364, PRO844, PRO8
46, PRO1760, PRO205, PRO321, PRO333, PRO8
40, PRO877, PRO878, PRO879, PRO882, PRO88
The presence of the epitope-tagged form of 5 or PRO887 can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag may be accomplished by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag, PRO179, PRO238, PRO364, PRO844, P.
RO846, PRO1760, PRO205, PRO321, PRO333, P
RO840, PRO877, PRO878, PRO879, PRO882, PR
Allow easy purification of O885 or PRO887. Various tag polypeptides and their respective antibodies are well known in the art. For example, poly-
Histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tag; flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol
., 8: 2159-2165 (1988)]; c-myc tag and its corresponding 8F9, 3C7, 6E1
0, G4, B7 and 9E10 antibodies [Evan et al., Molecular and Cellular Biology,
5: 3610-3616 (1985)]; and herpes simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides are flag peptides [Hopp et al., BioTechnology, 6: 120.
4-1210 (1988)]; KT3 epitope peptide [Martin et al., Science, 255: 192-19].
4 (1992)]; α-tubulin epitope peptide [Skinner et al., J. Biol. Chem.
, 266: 15163-15166 (1991)]; and T7 gene 10 protein peptide tag [Lu
tz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397 (1990)]. In an alternative embodiment, the chimeric molecule is PRO179, PRO238, PR.
O364, PRO844, PRO846, PRO1760, PRO205, PR
O321, PRO333, PRO840, PRO877, PRO878, PRO
A fusion of 879, PRO882, PRO885 or PRO887 with an immunoglobulin or a particular region of an immunoglobulin may be included. The divalent form of the chimeric molecule ("
(Also referred to as "immunoadhesin"), such a fusion may be the Fc region of an IgG molecule. The Ig fusion preferably replaces at least one variable region within the Ig molecule with PRO179, PRO238, PRO364, PRO844.
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
Includes solubilized (transmembrane domain deleted or inactivated) forms of PRO885 or PRO887 polypeptides. In a particularly preferred embodiment, the immunoglobulin fusion is
IgG1 molecule hinge, CH2 and CH3, or hinge, CH1, CH2 and C
Includes H3 region. See US Pat. No. 5,428,130 issued Jun. 27, 1995 for the preparation of immunoglobulin fusions.

C. PRO179, PRO238, PRO364, PRO844, PRO84
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
And PRO887 Polypeptide Preparation The present invention relates to PRO179, PRO238, PRO364, PR in the present application.
O844, PRO846, PRO1760, PRO205, PRO321, PR
O333, PRO840, PRO877, PRO878, PRO879, PRO
There is provided a newly identified and isolated nucleotide sequence encoding polypeptide, designated 882, PRO885 or PRO887. Especially, PRO1
79, PRO238, PRO364, PRO844, PRO846, PRO17
60, PRO205, PRO321, PRO333, PRO840, PRO87
7, PRO878, PRO879, PRO882, PRO885 or PRO88
The cDNA encoding the 7 polypeptide is identified and isolated as disclosed in further detail in the Examples below. Different PRO numbers are assigned to the proteins produced in the separately expressed circular region, but UNQ numbers are assigned to any given DN.
It is unique for A and the encoded protein and will not change. However, for the sake of simplicity, in this application DNA 16451-1388, D
NA35600-1162, DNA47365-1206, DNA59838-1
462, DNA44196-1353, DNA76532-1702, DNA30
868, DNA34433, DNA41374, DNA53987, DNA58
120, DNA58121, DNA58122, DNA58125, DNA58
128 or a protein encoded by DNA58130, a further natural homolog,
And PRO179, PRO238, PRO364, PRO844, PRO846
, PRO1760, PRO205, PRO321, PRO333, PRO840
, PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887, regardless of their origin or method of preparation,
"PRO179", "PRO238", "PRO364", "PRO8"
44 "," PRO846 "," PRO1760 "," PRO205 "," PRO "
321 ”,“ PRO333 ”,“ PRO840 ”,“ PRO877 ”,“ PRO ”
878 ”,“ PRO879 ”,“ PRO882 ”,“ PRO885 ”or“ PR ”
"O887". The following description mainly deals with PRO179, PRO238, PRO364, PR.
O844, PRO846, PRO1760, PRO205, PRO321, PR
O333, PRO840, PRO877, PRO878, PRO879, PRO
PRO179 by culturing cells transformed or transfected with a vector containing a nucleic acid encoding a 882, PRO885 or PRO887 polypeptide,
PRO238, PRO364, PRO844, PRO846, PRO1760,
PRO205, PRO321, PRO333, PRO840, PRO877, P
A method for producing a RO878, PRO879, PRO882, PRO885 or PRO887 polypeptide. Of course, PRO179, PRO238, PRO364, PRO844 using other methods well known in the art.
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
It is believed that PRO885 or PRO887 can be prepared. For example,
PRO179, PRO238, PRO364, PRO844, PRO846, P
RO1760, PRO205, PRO321, PRO333, PRO840, P
RO877, PRO878, PRO879, PRO882, PRO885 or P
The RO887 polypeptide sequence, or a portion thereof, may be produced by direct peptide synthesis using solid phase technology. Stewart et al., Solid-Phase Peptide Synthesis, (W.
H. Freeman Co., San Francisco, CA 1969); Merrifield, J. Am. Chem. Soc.,
See 85: 2149-2154 (1963). In vitro protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions.
PRO179, PRO238, PRO364, PRO844, PRO846, P
RO1760, PRO205, PRO321, PRO333, PRO840, P
RO877, PRO878, PRO879, PRO882, PRO885 or P
Various portions of RO887 are separately chemically synthesized and combined using chemical or enzymatic methods to combine full length PRO179, PRO238, PRO364, PRO84.
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, PRO885 or PRO887 polypeptides may be produced.

I. PRO179, PRO238, PRO364, PRO844, PRO84
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
Alternatively, isolation of DNA encoding PRO887 PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
The DNA encoding the PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptide is PRO179, PRO238.
, PRO364, PRO844, PRO846, PRO1760, PRO205
, PRO321, PRO333, PRO840, PRO877, PRO878,
MR encoding PRO879, PRO882, PRO885 or PRO887
It can be obtained from a cDNA library prepared from tissues that possess NA and are suspected of expressing it at detectable levels. Therefore, human PRO179
, Human PRO238, human PRO364, human PRO844, human PRO846
, Human PRO1760, human PRO205, human PRO321, human PRO33
3, human PRO840, human PRO877, human PRO878, human PRO87
9, D encoding human PRO882, human PRO885 or human PRO887
NA can be conveniently obtained from a cDNA library prepared from human tissue, as described in the Examples. In addition, PRO179, PRO238, PRO3
64, PRO844, PRO846, PRO1760, PRO205, PRO3
21, PRO333, PRO840, PRO877, PRO878, PRO87
The gene encoding the 9, PRO882, PRO885 or PRO887 polypeptide can also be obtained from a genomic library or by oligonucleotide synthesis. The library includes probes (PRO179, PRO238, PRO3) designed to identify a gene of interest or a protein encoded by the gene.
64, PRO844, PRO846, PRO1760, PRO205, PRO3
21, PRO333, PRO840, PRO877, PRO878, PRO87
9, PRO882, PRO885 or PRO887 polypeptides or an oligonucleotide of at least about 20-80 bases). Screening of a cDNA or genomic library with selected probes can be performed using standard procedures described, for example, in Sambrook et al., Supra. Desired PRO179, PRO238, PRO364, PR
O844, PRO846, PRO1760, PRO205, PRO321, PR
O333, PRO840, PRO877, PRO878, PRO879, PRO
Another method of isolating the gene encoding 882, PRO885 or PRO887 is to use the PCR method. Sambrook et al., Supra; Dieffenbach et al., PCR Prim.
er: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1
995). The examples below describe techniques for screening a cDNA library. The oligonucleotide sequence selected as a probe should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably detectably labeled upon hybridisation with the DNA in the library to be screened. Methods of labeling are well known in the art and include the use of radiolabels such as ³² P labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate and high stringency, are given in Sambrook et al., Supra. Sequences identified in such library screening methods are Genbank
Can be compared and aligned with well-known sequences that have been deposited in public databases such as the above or individual sequence databases and are made available to the public. Sequence identity (either at the amino acid or nucleic acid level) within a determined region of a molecule or over the full length sequence is determined by a computer software program, such as ALIGN, which employs various algorithms to measure homology. It can be determined by DNAstar and INHERIT. Nucleic acids having protein-encoding sequences are used for the first time using the deduced amino acid sequences disclosed herein and, if necessary, to detect intermediates and precursors of mRNA that were not reverse transcribed into cDNA, Sambrook et al., Supra. It can be obtained by screening the selected cDNA or genomic library using conventional primer extension methods as described in.

Ii. Selection and Transformation of Host Cells Host cells were prepared according to PRO179, PRO238, PRO364, P described herein.
RO844, PRO846, PRO1760, PRO205, PRO321, P
RO333, PRO840, PRO877, PRO878, PRO879, PR
Transfected or transformed with an expression or cloning vector for the production of O882, PRO885 or PRO887, inducing a promoter, selecting transformants, or appropriately modified to amplify the gene encoding the desired sequence. Cultivated in conventional nutrient medium. Culture conditions such as medium, temperature, pH, etc. can be selected by those skilled in the art without undue experimentation. In general, the principles, protocols, and practical techniques for maximizing cell culture productivity are described in Mammalian Cell Biotechnology.
: A Practical Approach, edited by M. Butler (IRL Press, 1991) and Sambrook et al., Supra. Methods of transfection, such as CaPO ₄ treatment and electroporation, are known to those of skill in the art. Depending on the host cell used, transformation is done using standard methods appropriate to the cell. Calcium treatment with calcium chloride or electroporation as described in Sambrook et al., Supra, is commonly used for prokaryotes or other cells that contain substantial cell wall barriers. Infection by Agrobacterium tumefaciens was reported by Shaw et al., G.
ene, 23: 315 (1983) and in International Patent Application No. WO 89/05859 published June 29, 1989, for the transformation of certain plant cells. For mammalian cells lacking such cell walls, Graham and van der Eb, Virology, 52:45.
The calcium phosphate precipitation method of 6-457 (1978) is used. General aspects of mammalian cell host system transformations have been described in US Pat. No. 4,399,216. Transformation into yeast is typically performed by Van solingen et al., J. Bact., 130: 946 (1977) and Hsi.
Ao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979). However, other methods of introducing DNA into cells, such as nuclear microinjection, electroporation, intact cells, or bacterial protoplast fusion using polycations such as polybrene, polyornithine and the like can also be used. For various techniques for transforming mammalian cells, see Ke.
own et al., Methods in Enzymology, 185: 527-537 (1990) and Mansour et al., Nature,
336: 348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectors described herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, for example E. coli K12 strain MM294 (ATCC31,446); E. coli X1776.
(ATCC31,537); E. coli strains W3110 (ATCC27,325) and K5772 (ATCC53,6)
35). Other preferred prokaryotic host cells are E. coli, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcesans ( Serratia marcescans), and Shigella, and bacilli, such as B. subtilis and B. licheniformis.
) (Eg, Bacillus licheniformis 41P described in DD 266,710 issued Apr. 12, 1989), Pseudomonas, for example, Pseudomonas aeruginosa and Enterobacteriaceae such as Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host as it is a common host strain for recombinant DNA production and publishing. Preferably, the host cell secretes a minimal amount of proteolytic enzyme. For example, strain W3110 may be modified to make a genetic mutation in a gene encoding a protein that is foreign to the host, and examples of such hosts include E. coli W3110 strain 1A2 having the complete genotype tonA; Genotype ton
E. coli W3110 strain 9E4 carrying A ptr3; complete genotype tonA p
rt3 phoA E15 (argF-lac) 169 degP ompTk
an E. coli having ^r W3110 strain 27C7 (ATCC 55,244); the complete genotype tonA ptr3 phoA E15 (algF-lac ) 169 degP
E. coli W3110 strain 37D6 harboring ompT rbs7ilvGkan ^r ;
Escherichia coli W3110 which is a 37D6 strain having a non-kanamycin resistant degP deletion mutation
Strain 40B4; and the E. coli strain having a mutant periplasmic protease disclosed in US Pat. No. 4,946,783 issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, such as PCR or other nucleic acid polymerase reactions are preferred.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are PRO179,
PRO238, PRO364, PRO844, PRO846, PRO1760,
PRO205, PRO321, PRO333, PRO840, PRO877, P
Suitable cloning or expression host for RO878, PRO879, PRO882, PRO885 or PRO887 encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. In addition, Schizosaccharomyces prombe (Beach and Nurse, Natu
re, 290: 140 [1981]; EP 139,383, issued May 2, 1985; Kluveromyces hosts (US Pat. No. 4,943,529; Fleer et al., Bio / Technology,
9: 968-975 (1991)), for example K. lactis (MW98-8C, CBS683, CBS
4574; Louvencourt et al., J. Bacteriol. 737 [1983]), K. fragilis.
lis) (ATCC 12,424), cable gallix (K. bulgaricus) (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (A.
TCC 56,500), K. drosophilarum (ATCC 36,906; Van d
en Berg et al., Bio / Technology, 8: 135 (1990)), Katemoto Tolerance (K. themot)
olerans) and K. marxianus; Yarrowia (EP 4
02,226); Pichia pastoris (EP 183,070; Sheekrishna et al.
, J. Basic Microbiol, 28: 265-278 [1988]); Candida; Trichodel Malaysia (reesia) (EP 244,234); Panax mold (Case et al., Proc. Natl. Acad. Sci. U)
SA, 76: 5259-5263 [1979]); Schwanniomyces, such as Schwanniomyces occidentalis (EP 3 issued October 31, 1990).
94,538); and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium (WO 91/00357, issued January 10, 1991); and Koji fungus,
For example, P. falciparum (Ballance et al., Biochem. Biophys. Res. Commun., 112: 2
84-289 [1983]; Tilburn et al., Gene, 26: 205-221 [1983]; Yelton et al., Proc. Natl.
Acad. Sci. USA, 81: 1470-1474 [1984]) and black mold (Kelly and Hynes, EM).
BO J., 4: 475-479 [1985]). The preferred methylotropic (met
hylotropic yeasts include, but are not limited to, Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, and Torulopsis.
rulopsis), and a yeast capable of growing in methanol selected from the genus consisting of Rhodotorula. A list of specific species that is exemplary of this yeast classification is C.
Anthony, The Biochemistry of Methylotrophs, 269 (1982). Glycosylated PRO179, PRO238, PRO364, PRO844, P
RO846, PRO1760, PRO205, PRO321, PRO333, P
RO840, PRO877, PRO878, PRO879, PRO882, PR
Suitable host cells for the expression of nucleic acid encoding O885 or PRO887 are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodaspera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese Hamster Ovary (CHO) and COS cells. A more detailed example is the monkey kidney CV1 strain transformed with SV40 (COS-
7, ATCC CRL 1651); human embryonic kidney strain (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster. Ovarian cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad.
Sci. USA, 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Repr
od., 23: 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human hepatocytes (He
p G2, HB 8065); and mouse breast tumor cells (MMT 060562, ATCC CCL51).
Selection of the appropriate host cell is within the skill of the art.

Iii. Selection and use of replicable vectors PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
A nucleic acid (eg, cDNA or genomic DNA) encoding PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 is inserted into a replicable vector for cloning (amplification of DNA) or expression. Various vectors are publicly available. The vector can be in the form of, for example, a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. Generally, the DNA is inserted into the appropriate restriction endonuclease sites using techniques well known in the art. Vector components are generally, but not limited to, if the sequence is secreted, one or more signal sequences, an origin of replication, one or more marker genes,
It includes enhancer elements, promoters, and transcription termination sequences. Construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those of ordinary skill in the art. PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 are not only directly recombinantly produced, but are also signal sequences or mature proteins or other polypeptides having a specific cleavage site at the N-terminus of the polypeptide. It is also produced as a fusion peptide with a heterologous polypeptide. Generally, the signal sequence is a component of the vector or is inserted into the vector PRO179, PRO238, PRO364, PRO844, PRO84.
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
Alternatively, it is a part of DNA encoding PRO887. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II leaders. For yeast secretion, the signal sequences are yeast invertase leader, alpha factor leader (Saccharomyces and Kluyveromyces α).
Factor leaders, the latter of which are described in U.S. Pat.No. 5,010,182), or acid phosphatase leaders, C. albicans glucoamylase leaders (1990 4
It may be the signal described in EP 362179) issued on Apr. 4, 2004, or WO 90/13646 published on Nov. 15, 1990. In mammalian cell expression, mammalian signal sequences may be used for the direct secretion of proteins 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 common to many bacteria,
It is well known for yeasts and viruses. The origin of replication derived from 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 mammalian. It is useful as a cloning vector in animal cells. Expression and cloning vectors typically contain a selection gene, also termed a selectable marker. Typical selection genes include (a) resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) complementing auxotrophic defects, or (c) the genetic code for, for example, basili. It encodes a protein, such as D-alanine racemase, that supplies important nutrients not available from complex media. Examples of selectable markers suitable for mammalian cells are PRO179, PRO238, PRO364, PRO844, such as DHFR or thymidine kinase.
PRO846, PRO1760, PRO205, PRO321, PRO333,
PRO840, PRO877, PRO878, PRO879, PRO882, P
It is possible to identify a cellular component capable of incorporating a nucleic acid encoding RO885 or PRO887. Suitable host cells using wild-type DHFR are those that have been prepared and propagated as described in Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). Defective CHO cell line. A preferred selection gene for use in yeast is the yeast plasmid YRp7.
Is the trp1 gene. Stinchcomb et al., Nature, 282: 39 (1979); Kin
gman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980). The trp1 gene provides a selectable marker for mutant strains of yeast that lack the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). Expression and cloning vectors are usually PRO179, PRO238, PR
O364, PRO844, PRO846, PRO1760, PRO205, PR
O321, PRO333, PRO840, PRO877, PRO878, PRO
It includes a promoter operably linked to the nucleic acid sequence encoding 879, PRO882, PRO885 or PRO887 and controlling mRNA synthesis. Suitable promoters recognized by a variety of possible host cells are known. Suitable promoters for use in prokaryotic hosts are the β-lactamase and lactose promoter systems [Cahng et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)].
, Alkaline phosphatase, tryptophan (trp) promoter system [Goeddel, Nu
cleic Acids Res., 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:
21-25 (1983)]. Promoters used in bacterial systems are also PRO179, PR
O238, PRO364, PRO844, PRO846, PRO1760, PR
O205, PRO321, PRO333, PRO840, PRO877, PRO
It has a Shine-Dalgarno (SD) sequence operably linked to DNA encoding 878, PRO879, PRO882, PRO885 or PRO887. Examples of suitable promoter sequences for use with yeast hosts include 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemist
ry, 17: 4900 (1987)), for example, enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, Pyruvate kinase, trioselate isomerase, phosphoglucose isomerase, and glucokinase are included. Other yeast promoters are inducible promoters that have the additional effect of transcription being regulated by growth conditions, such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glycerin. There are promoter regions for aldehyde-3-phosphate dehydrogenase and enzymes that control the utilization of maltose and galactose. Vectors and promoters suitable for expression in yeast are further described in EP 73,657.

From vectors in mammalian host cells to PRO179, PRO238, PR
O364, PRO844, PRO846, PRO1760, PRO205, PR
O321, PRO333, PRO840, PRO877, PRO878, PRO
Transcription of a 879, PRO882, PRO885 or PRO887 nucleic acid can be performed, for example, by
Polyoma virus, infectious epithelioma virus (UK 2,211,504 published July 5, 1989)
, A promoter derived from the genome of viruses such as adenovirus (eg adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40), heterologous mammals Promoters, such as the actin or immunoglobulin promoters, and promoters derived from heat shock promoters are controlled as long as such promoters are compatible with the host cell line. Desirable PRO179, PRO238, PRO364 by higher eukaryotes
, PRO844, PRO846, PRO1760, PRO205, PRO321
, PRO333, PRO840, PRO877, PRO878, PRO879,
Transcription of the DNA encoding PRO882, PRO885 or PRO887 can be enhanced by inserting an enhancer sequence into the vector. Enhancers, usually about 10 to 300 base pairs, are cis-acting elements of DNA that act on promoters to enhance their transcription. Many enhancer sequences from mammalian genes are currently known (globin, elastase, albumin, α-fetoprotein and insulin). However, typically enhancers from eukaryotic viruses will be used. As an example, SV40 on the late side of the replication origin
Includes enhancers (100-270 base pairs), cytomegalovirus early promoter enhancer, polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers are PRO179, PRO238, PR
O364, PRO844, PRO846, PRO1760, PRO205, PR
O321, PRO333, PRO840, PRO877, PRO878, PRO
5'of the sequence encoding 879, PRO882, PRO885 or PRO887.
Alternatively, it may be spliced into the vector at the 3'position, but is preferably located at the 5'position from the promoter. Expression vectors for use in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also include termination of transcription and mRNA.
It also contains the sequences required for the stabilization of Such sequences are used in eukaryotic or viral DNs.
It can be obtained from the A or cDNA, usually 5'and sometimes 3'untranslated regions. These areas are PRO179, PRO238, PRO364, PRO844, PRO.
846, PRO1760, PRO205, PRO321, PRO333, PRO
840, PRO877, PRO878, PRO879, PRO882, PRO8
The untranslated portion of the mRNA encoding 85 or PRO887 contains a nucleotide segment transcribed as a polyadenylated fragment. PRO179, PRO238, PRO364, P in recombinant vertebrate cell culture
RO844, PRO846, PRO1760, PRO205, PRO321, P
RO333, PRO840, PRO877, PRO878, PRO879, PR
Other methods, vectors and host cells suitable for adapting the synthesis of O882, PRO885 or PRO887 are described by Gething et al., Nature, 293: 620-625 (1981); Mante.
i et al., Nature, 281: 40-46 (1979); EP 117,060; and EP 117,058.

Iv. Detection of Gene Amplification / Expression Amplification and / or expression of a gene can be performed by appropriately labeled probes based on the sequence provided herein, for example, conventional Southern blotting, Northern quantifying mRNA transcription. Blotting (Thomas, Proc. Natl. Acad. Sci. USA, 77: 520
1-5205 (1980)), the dot blot method (DNA analysis), or the in situ hybridization method, and can be directly measured in a sample. Alternatively, an antibody capable of recognizing a specific double strand including a DNA double strand, an RNA double strand and a DNA-RNA hybrid double strand or a DNA-protein double strand can be used. The antibody can then be labeled and the assay performed, wherein the duplex is surface bound, such that the antibody bound to the duplex at the time of surface duplex formation. Presence can be detected. Alternatively, gene expression can be measured by immunological methods that directly quantitate expression of the gene product, such as immunohistochemical staining of cells or tissue sections and cell culture or body fluid assays. Antibodies useful for immunohistochemical staining and / or assay of sample fluids may be monoclonal or polyclonal and can be prepared in any mammal. Conveniently, the antibody is the native sequence PRO179,
PRO238, PRO364, PRO844, PRO846, PRO1760,
PRO205, PRO321, PRO333, PRO840, PRO877, P
To a RO878, PRO879, PRO882, PRO885 or PRO887 polypeptide, or to a synthetic peptide based on the DNA sequences provided herein, or PRO179, PRO238, PRO364, PRO844,
PRO846, PRO1760, PRO205, PRO321, PRO333,
PRO840, PRO877, PRO878, PRO879, PRO882, P
It can be prepared against an exogenous sequence which is fused to the DNA encoding RO885 or PRO887 and which encodes a specific antibody epitope.

V. Purification of polypeptide PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
Forms of the PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptides can be recovered from the culture medium or lysates of host cells. If membrane-bound, a suitable wash solution (eg Triton-X ^™ 10
0) or can be detached from the membrane using enzymatic cleavage. PRO179, PR
O238, PRO364, PRO844, PRO846, PRO1760, PR
O205, PRO321, PRO333, PRO840, PRO877, PRO
The cells used to express the 878, PRO879, PRO882, PRO885 or PRO887 polypeptides can be freeze-thaw cycled, sonicated, mechanical disrupted,
Alternatively, it can be disrupted by various chemical or physical means such as cytolytic agents. PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
It may be desirable to purify PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptides from recombinant cell proteins or polypeptides. Purified by the following procedure, which is an example of a suitable purification procedure: Fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or cation exchange resins such as DEAE; chromatofocusing; SDS-. PAGE; ammonium sulfate precipitation; eg Sephadex G-
Gel filtration using 75; Protein A Sepharose column to remove contaminants such as IgG; and PRO179, PRO238, PRO364, PRO844, PR
O846, PRO1760, PRO205, PRO321, PRO333, PR
O840, PRO877, PRO878, PRO879, PRO882, PRO
Metal chelation columns that bind epitope-tagged forms of 885 or PRO887 polypeptides. Known in the art, for example Deutcher, Methodes in En
zymology, 182 (1990); Scopes, Protein Purification: Principles and Pract
Many protein purification methods described in ice, (Springer-Verlag, New York 1982) can be used. The purification process chosen may depend, for example, on the production method used and the particular PRO179, PRO238, PRO364, PRO8 produced.
44, PRO846, PRO1760, PRO205, PRO321, PRO3
33, PRO840, PRO877, PRO878, PRO879, PRO88
2, depending on the nature of PRO885 or PRO887.

D. PRO179, PRO238, PRO364, PRO844, PRO84
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
Or uses of the PRO887 polypeptide i. Cardiovascular, Endothelial and Angiogenic Activity Assays Various assays can be used to test the cardiovascular, endothelial and angiogenic activity of the polypeptides described herein. Assays for testing endothelin antagonist activity disclosed in US Pat. No. 5,773,414 include rat ventricular binding assay, which tests the ability of a polypeptide in a receptor assay to inhibit iodinated endothelin-I binding, rabbit renal artery smooth muscle cells. Endothelin-receptor binding assay to test the intact cell binding of radiolabeled endothelin-I using functionally, by measuring intracellular levels of second messengers with functional activity in Rat-I cells In a measured inositol phosphate accumulation assay, in vivo (isolated tube) studies using endothelium of male New Zealand rabbits, and in vitro studies using SD rats, added compounds were released into cultured vascular smooth muscle. Ability to reduce endothelin-stimulated arachidonic acid Include arachidonic acid release assay that measures. Assays for tissue-generating activity are described in, but not limited to, WO95 / 16035 (bone, cartilage, tendon); WO95 / 05846 (nerve, neuron), and WO91 / 07491 (skin, endothelium). Things are included. Wound healing activity assays are described, for example, by Eaglelstein and Mertz, J. Invest. Derm.
atol., 71: 382-384 (1978), modified by Winter, Epidermal Wound.
Healing, Maibach, HI and Rovee, DT, edited (Year Book Medical Publishers. Inc.
., Chicago), pp71-112 are included. Endothelin B ₁ (ETB ₁₎ binds to the receptor polypeptide, a screening assay of a test molecule relating to a PRO polypeptide that modulates signal transduction activity, as described in U.S. Patent No. 5,773,223, endothelin B ₁
Providing a host cell transformed with a DNA encoding a receptor polypeptide,
The cells are exposed to the test candidate drug and the endothelin B ₁ receptor signal transduction activity is measured. There are several cardiac hypertrophy assays. In vitro assays include induction of adult rat heart myocyte diffusion. In this assay, ventricular myocytes were identified by Piper et al., “Adult ventricular rat heart muscle cells”, Cell Culture Te
chniques in Heart and Vessel Research.HM Piper.ed (Berlin: Springer-V
erlag, 1990), pp. 36-60 and isolated essentially from single (male SD) rats according to a modified version of the procedure. This procedure allows for isolation of growing ventricular myocytes and long-term culture of rod phenotype cells. Phenylephrine and prostaglandin F _2α (PGF _2α ) have been shown to induce a metastatic response in these growing cells. The inhibition of myocyte metastasis induced by PGF _2α or PGF _2α analogs (eg fluprostenol) and phenylephrine by various potential inhibitors of cardiac hypertrophy is then tested.

One example of an in vivo assay is to test the inhibition of fluprostenol-induced cardiac hypertrophy in vivo. This pharmacological model is based on the subcutaneous injection of fluprostenol (an agonist analog of PGF _2α ) in rats (eg,
Tests the ability of PRO polypeptides to inhibit male Wistar or SD-induced cardiac hypertrophy. It is known that PGF _2α in the myocardium is chronically elevated to a detectable level in rats with pathological cardiac hypertrophy induced by myocardial infarction. Lai et al., Am. J. Physiol. (Heart Circ. Physiol.), 271: H2197
-H2208 (1996). Therefore, factors capable of inhibiting the effects of fluprostenol on myocardial growth in vivo may be useful in the treatment of cardiac hypertrophy. Efficacy of PRO polypeptides in cardiac hypertrophy is determined by weighing the heart, ventricle and left ventricle (normalized by body weight) to fluprostenol treated rats that do not receive PRO polypeptide. Another example of an in vivo assay is the pressure overload cardiac hypertrophy assay. In in vivo tests, it is common to induce pressure overload cardiac hypertrophy by contraction of the abdominal aorta of test animals. In a typical protocol, rats (e.g. male Wistar
Or SD) is anesthetized and the abdominal aorta of each rat is stenotic to just below the diaphragm. Beznak M., Can. J. Biochem. Physiol., 33: 985-94 (1955). The aorta is exposed by surgical incision and a short thick needle is placed next to the tube. The aorta is ligated with silk thread around the needle, contracted and immediately removed, reducing 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, PSEMB, 200: 95-100 (1992). In another in vivo assay, the effect on cardiac hypertrophy, followed by experimentally induced myocardial infarction (MI), is measured. In rats, acute MI is induced by left coronary artery ligation, which is further confirmed by electrocardiography. Also, a sham-operated group of animals is prepared as a control animal. Early data showed that cardiac hypertrophy was present in the MI group, with an 18% increase in heart weight relative to body weight. Lai and others listed above. Treatment of these animals with candidate blockers of cardiac hypertrophy, such as PRO polypeptides, provides valuable information on the therapeutic potential of the candidate drugs tested. Further assay tests in the induction of cardiac hypertrophy using SD rats are disclosed in US Pat. No. 5,773,415.

Further understanding the role of the genes identified herein in the pathogenesis and progression of tumors in cancer and testing the efficacy of therapeutic candidates including antibodies to the native PRO polypeptide and other antagonists, such as small molecule antagonists. For this purpose, various well-known animal models can be used. The in vivo nature of such models makes human patients particularly prognostic in their response. Animal models of tumors and cancers (breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodents, such as murine models. Such models use standard techniques, such as subcutaneous injection, tail vein injection, spleen transplantation, peritoneal transplantation, subrenal capsule or orthopin transplantation, such as colon cancer cells transplanted into colon tissue, It can be prepared by transferring tumor cells into syngeneic mice. See, for example, PCT Publication No. WO97 / 33551 published September 18, 1997. Probably the most frequently used animal species for oncogene studies are immunodeficient mice, especially nude mice. The finding that nude mice with thymus stimulation / dysplasia successfully act as hosts for human tumor xenografts has led to their widespread use for this purpose. Autosomal recessive nu genes include, for example, ASW, A / He, BALB / c, B10.LP, C17, CH
3, C57BL, C57, CBA, DBA, DDD, I / st, NC, NFR,
It has been introduced into a large number of clearly cognate nude mice, including NFS, NFS / N, NZB, NZC, NZW, P, RIII and SJL. In addition to nude mice, a wide variety of other animals that have inherited immunological deficiencies are raised and used as recipients of tumor xenografts. For more details, see The Nude Mouse in Oncology Res
See earch, E. Boven and B. Winograd. eds. (CRC Press, Inc., 1991). Cells introduced into such animals are well known tumor / cancer cells such as any of the tumor cell lines listed above, eg, the B104-1 cell line (stable NIH-3T3 cells transfected with the neu protooncogene. Caco-2 (ATCC HTB-37); or a moderately differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC H).
TB-38), or from tumors and cancer. Tumor or cancer cell samples are used in standard conditions, including freezing and storage in liquid nitrogen, and can be obtained from patients by surgery. Karmali et al., Br. J. Cancer. 48: 689-696 (19
83).

Tumor cells can be introduced into animals such as nude mice by various procedures.
The subcutaneous (sc) space of mice is very suitable for tumor implantation. Tumors can also be implanted as solid blocks with needle biopsies, eg by use of trocars, or cell suspensions. Tumor tissue fragments of suitable size for solid block or trocar implantation are introduced into the subcutaneous space. Cell suspensions are prepared fresh from primary tumor or stable tumor cell lines and injected subcutaneously. Tumor cells can also be injected as a subcutaneous implant. In this position, the implant is applied between the bottom of the dermal connective tissue and the subcutaneous tissue. Animal models of breast cancer are described by Drebin et al., Proc. Nat. Acad. Sci. USA, 83: 9129-913.
3 (1986), e.g., rat neuroblast cells (ne
u oncogene) or neu-transformed NIH-3T3 cells into nude mice. Similarly, animal models of colon cancer can be made by passing colon cancer cells into animals, eg, nude mice, and allowing these animals to develop tumors.
An orthotopic model of human colon cancer in nude mice is described, for example, in Wang et al., Cancer R
esearch, 54: 4726-4728 (1994) and Too et al., Cancer Research, 55: 681-684 (1995).
). This model is based on AntiCancer, Inc., (San Diego, Californi
It is based on the so-called "METAMOUSE ^™ " sold by a). Tumors generated in animals are removed and can be cultured in vitro. The cells from the in vitro culture are then passed to the animal. Such tumors may serve the purpose of further testing or drug screening. Alternatively, the tumors obtained by passage can be isolated and RNA of pre-passage cells and cells isolated after one or more passage steps analyzed for differential expression of the gene of interest. To be done. Such passaging techniques can be performed on any well known tumor or cancer cell line. For example, Meth A, CMS4, CMS5, CMS21 and WEHI-164.
Is a highly controlled model to chemically induce fibrosarcoma in BALB / c female mice (DeLeo et al., J. Exp. Med., 146: 720 (1977)) and to study the antitumor activity of various drugs. Provide the system. Palladino et al., J. Immunol., 138: 4023-4032 (1987). Briefly, tumor cells are grown in vitro in cell culture. The cell lines are washed and suspended in buffer at a cell density of 10x10 ⁶ to 10x10 ⁷ cells / ml before injecting the animals. The animals are then injected subcutaneously with 10 to 100 μl of cell suspension, with tumors appearing in 1 to 3 weeks. In addition, Lewis lung in mice, one of the most thoroughly studied trial tumors
The (3LL) carcinoma can be used as a tumor model for research. The efficacy of this tumor model correlates with favorable effects in human patients diagnosed with small cell carcinoma of the lung (SCCL). This tumor can be introduced into normal mice by injecting tumor fragments from diseased mice or cells in culture. Zupi et al., Br. J. Cancer, 41: suppl. 4. 30 (1980). There was evidence that the tumors started with single cell injections and that a fairly high proportion of infected cells survived. For more information on this tumor model, see Zacharski, Haemostasis, 16
: 300-320 (1986).

One method of assessing the efficacy of test compounds in animal models with transplanted tumors is to measure tumor size before or after treatment. Traditionally, the size of transplanted tumors is measured with two or three dimensional slide calipers. Measurements limited to two dimensions do not accurately reflect tumor size; thus, mathematical formulas are usually used and converted to the corresponding amount. However, tumor size measurements are very inaccurate. The therapeutic effect of a candidate drug can be described as better if the treatment-induced growth is delayed and the specific growth is delayed. Another important variable in the description of tumor growth is the time to double tumor burden. Also, computer programs for calculating and describing tumor growth, such as Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop on Immune-
The program reported by Deficient Animals. Wu and Sheng. Eds. (Basel. 1989), p. 301 is available. However, it should be noted that the post-treatment necrosis and inflammatory response may actually result, at least initially, in an increase in tumor size. Therefore, these changes need to be carefully monitored by a combination of morphometry and flow cytometric analysis. In addition, the recombinant (transgenic) animal model is based on the PRO identified here.
The coding site for the gene can be introduced into the genome of the animal of interest and engineered using standard techniques for making transgenic animals. Animals that can be provided as targets for transgenic manipulation include, but are not limited to,
Includes mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates such as baboons, chimpanzees and monkeys. Techniques well known in the art for introducing transgenes into such animals include pronuclear microinjection (US Pat. No. 4,873,191); retrovirus-mediated gene transfer into the embryo (eg, Van der
Putten et al., Proc. Nat. Acad. Sci. USA, 82: 6148-615 (1985)); targeting genes in embryonic stem cells (Thompson et al., Cell, 56: 312-321 (1989)); embryo electroporation. (Lo, Mol. Cell. Biol., 3: 1803-1814 (1983)); and sperm-mediated gene transfer. Lavitrano et al., Cell, 57: 717-73 (1989). The review includes
See, for example, U.S. Pat. No. 4,736,866. For purposes of this invention, transgenic animals include those carrying the transgene in only some of their cells ("mosaic animals"). The transgenes can be integrated as a single transgene or in chains, eg, head-to-head or head-to-tail tandems. Also, selective introduction of the transgene into a particular cell line is described, for example, in Lasko et al., Proc. Nat. Acad. Sci. USA,
89: 6232-636 (1992). Expression of the transgene in transgenic animals can be monitored by conventional techniques. For example, Southern blot analysis or PCR amplification can be used to verify transgene integration. The expression level of mRNA can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR or immunocytochemistry. Animals are further tested for signs of tumor or cancer progression.

PRO identified herein by homologous recombination between the modified genomic DNA encoding the PRO polypeptide and the endogenous gene encoding the PRO polypeptide introduced into the embryonic cell of an animal. "Knockout" animals can be constructed that have defective or altered genes that encode the polypeptides. For example, a specific P
The cDNA encoding the RO polypeptide can be used to clone genomic DNA encoding the polypeptide according to established techniques. A portion of the genomic DNA encoding the particular PRO polypeptide can be deleted or replaced with another gene, such as the gene encoding a selectable marker used to monitor integration. Typically, the vector will contain unchanged flanking DNA (5 ′ and 3
Includes'both ends' for several kilobases. For example, T for homologous recombination vectors
See homas and Capecchi, Cell, 51: 503 (1987). The vector is introduced into embryonic stem cells (for example, by electroporation), and cells in which the introduced DNA is homologously recombined with the endogenous DNA are selected. For example, Li et al., Cell, 69: 915.
See (1992). The selected cells are then injected into the blastocyst of an animal (eg mouse or rat) to form an assembled chimera. For example, Bradley, Teratocarcinomas and Em
bryonic Stem Cells: A Practical Approach, EJ Robertson, ed. (IRL, Oxf
Ord, 1987), pp. 113-152. The chimeric embryos are then transplanted into a suitable pseudopregnant female nanny and at intervals, "knockout" animals are created. Offspring with DNA that has been homologously recombined into germ cells are identified by standard techniques and can be used to breed animals in which all cells of the animal contain homologously recombined DNA. . Knockout animals are characterized by the ability to protect against certain pathological conditions and the progression of that pathological condition due to the absence of PRO polypeptide. The effects of antibodies that specifically bind the PRO polypeptides identified herein, and other drug candidates, can be further tested in the treatment of spontaneous animal tumors. A suitable target for this study is feline oral squamous cell carcinoma (SCC). The feline oral SCC is highly invasive, and this malignant tumor is considered to be the most common oral malignant tumor in cats, and it is measured that 60% or more of oral tumors are repeated in this species. Although it rarely metastasizes to distant sites, the low incidence of metastases is reflected in the short survival of cats bearing this tumor. These tumors cannot be usually surgically treated due primarily to the anatomy of the feline oral cavity. Currently, there is no effective treatment for this tumor. Prior to entering this study, each cat is biopsied for a complete clinical trial and scanned by computed tomography (CT). Cats diagnosed with squamous cell tumors of the sublingual mouth are excluded from this study. The tongue is paralyzed as a result of such tumors, and the animals are unable to feed themselves when the tumor dies from treatment. Each cat is treated repeatedly over a long period of time. Tumors will be photographed daily and then rechecked during the treatment period. After treatment, each cat is subjected to another CT scan. CT scans and chest radiographs are evaluated every 8 weeks thereafter. The data were assessed as different in viability, reactivity and toxicity relative to the control group. Positive reactions require evidence of tumor regression, preferably with improved quality of life and / or increased lifespan. In addition, spontaneous animal tumors of dogs, cats and baboons such as fibrosarcoma, adenocarcinoma, lymphoma, chondroma, or leiomyosarcoma can also be tested. Of course, canine and feline mammary adenocarcinomas are a preferred model, and their appearance and properties are very similar to those of humans. However, the use of this model is limited by the incidence of this type of tumor in animals. Also, other in vitro and in vivo cardiovascular, endothelial and angiogenic tests well known in the art are suitable here.

Ii. Tissue distribution The results of cardiovascular, endothelial and angiogenic assays can now be demonstrated by further study, eg by measuring mRNA expression in various human tissues. As described above, gene amplification and / or expression in various tissues can be performed by appropriately labeled probes based on the sequences provided herein, for example, conventional Southern blotting, quantifying mRNA transcription. Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)), dot blot method (DN
A analysis) or an in situ hybridization method.
Alternatively, an antibody capable of recognizing a specific double strand including a DNA double strand, an RNA double strand and a DNA-RNA hybrid double strand or a DNA-protein double strand can be used. Alternatively, gene expression in various tissues can be measured by immunological methods that directly quantitate the expression of the gene product, such as immunohistochemical staining of tissue sections and cell culture or body fluid assays. Antibodies useful for immunohistochemical staining and / or assay of sample fluids may be monoclonal or polyclonal and can be prepared in any mammal. Conveniently, the antibody is a native sequence PR
It can be prepared against O polypeptides, or against synthetic peptides based on the DNA sequences provided herein, or against exogenous sequences fused to PRODNA and encoding specific antibody epitopes. General techniques for antibody production, and specific protocols for in situ-hybridization are provided below.

Iii. Antibody-Binding Studies of Cardiovascular, Endothelial and Angiogenic Studies, which test the ability of anti-antibodies to inhibit the effects of PRO polypeptides on endothelial cells or other cells used in cardiovascular, endothelial and angiogenic assays. Results can be substantiated by studying antibody binding. Examples of antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies, the preparation of which is described below. Antibody binding studies can be performed in any of the well known assay methods, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola
, Monoclonal Antibodies: A Manual of Techniques, (CRC Press, Inc. 1987) p
p. 147-158. Competitive binding assays rely on the ability of a labeled standard to compete for a test sample in binding a finite amount of antibody. The amount of target protein in the test sample is inversely proportional to the amount of standard bound by the antibody. To facilitate measurement of the amount of bound standard, the antibody is preferably insolubilized before and after competition, and analyte and standard that bind to the antibody are conveniently separated from unbound residual standard and analyte. To do. The sandwich assay uses two antibodies that are capable of binding different immunogenic moieties, or epitopes, or proteins to be detected. In the sandwich assay, the test sample to be analyzed is a first support immobilized on a solid support.
Of the antibody, followed by binding of the second antibody to the analyte, thus forming an insoluble three-site complex. See, eg, US Pat. No. 4,376,100. The second antibody is a detectable moiety, which may itself be labeled (direct sandwich assay) or may be measured using an anti-immunoglobulin antibody which is labeled with a detectable moiety (indirect sandwich assay). Assay). For example, one type of sandwich assay is an ELISA assay, where the detectable moiety is an enzyme. In immunohistochemistry, tissue samples may be fresh or frozen, may be embedded in paraffin, and may be fixed with preservatives such as formalin.

Iv. Cell-Based Tumor Assays Cell-based assays and animal models for cardiovascular, endothelial and angiogenic diseases, eg tumors, here demonstrate the discovery of cardiovascular, endothelial and angiogenic assays and are further identified herein. Genes can be used to understand the relationship between unwanted genes and the progression and pathogenesis of unwanted cardiovascular, endothelial and angiogenic cell growth. The role of the gene products identified herein in unwanted cardiovascular, endothelial and angiogenic cell growth, eg, tumor progression and pathogenesis, may be achieved using cells or cell lines identified as stimulated or inhibited by the PRO polypeptide. Can be tested. Such cells include, for example, those shown in the following examples. In a different approach, the ability of cells of well-known cell types involved in certain cardiovascular, endothelial and angiogenic diseases to be transfected with cDNA, which cDNA induces excessive growth or inhibits growth. To analyze. Where the cardiovascular, endothelial and angiogenic disease is cancer, suitable tumor cells include, for example, stable tumor cell lines, such as the B104-1-1-1 cell line (stable NIH-3T transfected with the neu protooncogene.
3 cell lines) and ras-transfected NIH-3T3 cells, which are capable of transfecting the desired gene and capable of monitoring tumorigenic growth. Then, using such transfected cell lines, the poly- or monoclonal antibody or antibody composition exerts cytostatic or cytotoxic activity in the growth of the transfected cells, or antibody-dependent cellular cells. The ability to inhibit tumorigenic cell growth by mediating toxicity (ADCC) is tested. In addition, cells transfected with the coding sequences of the genes identified herein can be used to identify candidate drugs for the treatment of cardiovascular, endothelial and angiogenic disorders such as cancer. In addition, primary media obtained from tumors of transgenic animals (described above) can be used in cell-based assays, although stable cell lines are preferred. Techniques for deriving certain cell lines from transgenic animals are well known in the art. See, eg, Small et al., Mol. Cell. Biol., 5: 642-648 (1985).

V. Gene therapy where PRO179, PRO238, PRO364, PRO844, PRO
846, PRO1760, PRO205, PRO321, PRO333, PRO
840, PRO877, PRO878, PRO879, PRO882, PRO8
85 or PRO887 polypeptides, and polypeptidyl agonists and antagonists may be used in accordance with the present invention by in vivo expression of those peptides, often referred to as gene therapy. There are two main ways to get the nucleic acid (sometimes contained within a vector) into the cells of the patient: in vivo and ex vivo. For in vivo delivery, the nucleic acids are typically PRO179, PRO238, PRO364, PRO844,
PRO846, PRO1760, PRO205, PRO321, PRO333,
PRO840, PRO877, PRO878, PRO879, PRO882, P
The site at which the RO885 or PRO887 polypeptide is required, ie P
RO179, PRO238, PRO364, PRO844, PRO846, PR
O1760, PRO205, PRO321, PRO333, PRO840, PR
O877, PRO878, PRO879, PRO882, PRO885 or PR
Synthetic site of O887 polypeptide, PRO179, PR if known
O238, PRO364, PRO844, PRO846, PRO1760, PR
O205, PRO321, PRO333, PRO840, PRO877, PRO
The 878, PRO879, PRO882, PRO885 or PRO887 polypeptides are directly injected into the patient at the site (eg, wound) where the biological activity is required.
In ex vivo treatment, the patient's cells are removed, nucleic acids are introduced into these isolated cells, and the modified cells are administered to the patient either directly or encapsulated in, for example, a porous membrane implanted in the patient (US (See Patent Nos. 4,892,538 and 5,283,187)
. There are various techniques available for introducing nucleic acids into living cells. These techniques depend on whether the nucleic acid is transferred in vitro to cultured cells or in vivo to a host of interest. Suitable techniques for transferring nucleic acids to mammalian cells in vitro include liposomes, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation and the like. Transduction involves replication defects, the binding of recombinant viral (preferably retroviral) particles to cellular receptors, and then the introduction of the nucleic acid contained in the particles into cells. A commonly used vector for ex vivo delivery of genes is the retrovirus.

Currently preferred in vivo nucleic acid transfer techniques are viral or non-viral vectors (adenovirus, lentivirus, herpes simplex I virus, or adeno-associated virus (AAV)), and lipid-based systems (lipid-mediated transfer of genes). Useful lipids for example are DOTMA, DOPE, and DC-Chol; for example Ton
Kinson et al., Cancer Investigation, 14 (1): 54-65 (1996)). The most preferred vector for use in gene therapy is a virus, most preferably adenovirus, AAV, lentivirus, or retrovirus.
Viral vectors, such as retroviral vectors, include at least one transcription promoter / enhancer or locator, or alternative splicing, nuclear R
Other factors that control gene expression by NA export, or other means such as post-translational modification of messengers are included. Furthermore, viral vectors such as retrovirus vectors are PRO179, PRO238, PRO364, PRO844, PRO84.
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
Or when transcribed in the presence of a gene encoding a PRO887 polypeptide,
It includes a nucleic acid molecule that is operably linked to it and that functions as a translation initiation sequence. Such vector constructs also include packaging signals suitable for the virus used,
It contains terminal repeats (LTRs) or parts thereof, and positive and negative strand primer binding sites, if they are not already present in the viral vector. In addition, these vectors typically represent PRO179, PRO238, PRO364, PRO844, PRO846, from the host cell in which they are located.
PRO1760, PRO205, PRO321, PRO333, PRO840,
It includes a signal sequence that secretes a PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptide. Preferably, the signal sequence for this purpose is a mammalian signal sequence, most preferably PRO17.
9, PRO238, PRO364, PRO844, PRO846, PRO176
0, PRO205, PRO321, PRO333, PRO840, PRO877
, PRO878, PRO879, PRO882, PRO885 or PRO887.
The native signal sequence for the polypeptide. In some cases, the vector construct also contains polyadenylation and signal and translation termination sequences that direct one or more restriction sites. By way of example, such vectors are typically 5'LTR, tR
NA binding site, packaging signal, origin of second-strand DNA synthesis, and 3 '
Includes an LTR or part thereof. Other non-viral vectors such as cationic lipids, polylysines, and dendrimers can also be used. In some situations, it may be desirable to provide the nucleic acid source with a reagent that targets the target cell, such as an antibody or target cell specific for a cell surface membrane protein, a ligand for a receptor on the target cell, and the like. When liposomes are used, proteins that bind to cell surface membrane proteins with endocytosis are used to promote targeting and / or uptake, such as capsid proteins of specific cell types or fragments thereof, Antibodies to proteins that undergo internalization during cycling and proteins that target intracellular localization and improve intracellular half-life. Techniques for receptor-mediated endocytosis are described, for example, in Wu et al., J. Biol. Chem., 262: 4429-4432 (1987) and Wagner et al., Proc.
Natl. Acad. Sci. USA, 87: 3410-3414 (1990). For a review of currently known gene labeling and gene therapy protocols, see Anderson et al.
, Science, 256: 808-813 (1992). See also WO 93/25673 and references cited therein. Suitable gene therapy and methods for making retroviral particles and structural proteins include
Found in US Pat. No. 5,681,746.

Vi. Use of Gene as Diagnostic Method The present invention also relates to PRO179, PRO238, PRO364 as a diagnostic method,
PRO844, PRO846, PRO1760, PRO205, PRO321,
PRO333, PRO840, PRO877, PRO878, PRO879, P
It relates to the use of genes encoding RO882, PRO885 or PRO887 polypeptides. PRO179, PRO238, PRO364, PRO844, P
RO846, PRO1760, PRO205, PRO321, PRO333, P
RO840, PRO877, PRO878, PRO879, PRO882, PR
Mutants in the O885 or PRO887 polypeptides are responsible for tumors, so mutated forms of PRO179, PRO238, PRO364, PRO844, PR
O846, PRO1760, PRO205, PRO321, PRO333, PR
O840, PRO877, PRO878, PRO879, PRO882, PRO
Detection of 885 or PRO887 polypeptides allows for the diagnosis of cardiovascular, endothelium and angiogenic diseases, or cardiovascular, endothelium and angiogenic diseases such as tumor infectivity. Human PRO179, human PRO238, human PRO364, human PRO844
, Human PRO846, human PRO1760, human PRO205, human PRO32
1, human PRO333, human PRO840, human PRO877, human PRO87
8, human PRO879, human PRO882, human PRO885 or human PRO8
Individuals carrying variants in the gene encoding the 87 polypeptide can have DNA levels detected by a variety of techniques. Diagnostic nucleic acids can be obtained from patient cells, such as blood, urine, saliva, tissue biopsies, and autopsy materials. Genomic DNA is enzymatically amplified using PCR prior to analysis (Saiki et al., Nature, 324: 163-166.
(1986)) or can be used for direct detection. RNA or cDNA can be used for the same purpose. As an example, PRO179, PRO238,
PRO364, PRO844, PRO846, PRO1760, PRO205,
PRO321, PRO333, PRO840, PRO877, PRO878, P
PCR primers complementary to the nucleic acids encoding the RO879, PRO882, PRO885 or PRO887 polypeptides were added to PRO179, PRO238, PR.
O364, PRO844, PRO846, PRO1760, PRO205, PR
O321, PRO333, PRO840, PRO877, PRO878, PRO
It can be used to identify and analyze 879, PRO882, PRO885 or PRO887 polypeptide variants. For example, deletions and insertions can be detected by changes in the size of the amplification product compared to the normal genotype. Point mutation (point m
utations) is PRO179, PRO238, PRO364, PRO844, P
RO846, PRO1760, PRO205, PRO321, PRO333, P
RO840, PRO877, PRO878, PRO879, PRO882, PR
Radiolabeled RNA encoding O885 or PRO887 polypeptide,
Or PRO179, PRO238, PRO364, PRO844, PRO846
, PRO1760, PRO205, PRO321, PRO333, PRO840
, PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptide-labeled antisense DNA
It can be identified by hybridizing amplified DNA to the sequence. Preferred compatible sequences can be distinguished from non-compatible duplexes by RNase A digestion or different melting temperatures. Genetic testing based on DNA sequence differences is accomplished by detecting alterations 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 are distinguished in denaturing formamidine gradient gels, and migration of different DNA fragments is inhibited at different positions in the gel by the particular melting or partial melting temperature. For example, Myers et al., Science, 23.
0: 1242 (1985). Also, sequence changes at specific positions may be accompanied by nuclease protection assays, such as RNase and SI protection, or chemical cleavage methods, such as Cotton et al., Proc. Natl. Acad.
Sci. USA, 85: 4397-4401 (1985). Thus, the deletion of specific DNA sequences may result in hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes such as restriction fragment length polymorphism (RFLP), and Southern blots of genomic DNA. Can be achieved by a method.

Vii. Uses for PRO polypeptide level detection In addition to the more convenient gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis. Expression of the nucleic acid encoding the PRO polypeptide is linked to vascular disease associated with tumorigenesis, or neovascularization. When the PRO polypeptide has a signal sequence, mRNA is highly expressed in endothelial cells, whereas it is low in smooth muscle cells, which indicates that PRO polypeptide is present in serum. Is shown. Therefore, anti-PRO polypeptide antibodies may be used in the diagnosis of such disorders, since changes in the levels of this PRO polypeptide are indicative of vascular disease associated with tumorigenesis, or neovascularization. can do. An antibody specific for the PRO polypeptide is attached to a solid support and labeled P
Competitive assays in which the RO polypeptide and a sample derived from the host are passed over a solid support can also be used, where the amount of detected level attached to the solid support correlates with the amount of PRO polypeptide in the sample. There is.

Viii. Chromosome Mapping The sequences of the present invention are also important in chromosome identification. The sequence is specifically targeted to and can hybridize to a specific location on an individual's human chromosome.
In addition, there is currently a need for identifying specific sites on the chromosome. Several chromosomal marking reagents based on actual sequence data (repeated polymorphisms) are currently available for marking chromosomal locations. DNA mapping of the chromosomes of the present invention is an important first step in correlating sequences with disease-related genes. Briefly, the sequence varies from cDNA to PCR primer (preferably 15-25
It can be mapped to a chromosome by preparing base pairs). Using computer analysis of the 3'-untranslated region, primers that are not spanned by more than one exon of genomic DNA are quickly selected, thus further complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosome genes. Only hybrids containing the human gene corresponding to the primer will make an amplified fragment. PCR mapping of somatic cell hybrids is a rapid procedure for assigning specific DNA to specific chromosomes. Using similar oligonucleotide primers in the present invention, sublocalization can be achieved in a similar manner, with pools of large genomic clones or panels of fragments from specific chromosomes. Similarly, other mapping methods that can be used to map to that chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to assemble a chromosome-specific cDNA library. Is included. Fluorescent in situ hybridization of cDNA clones to metaphase chromosome expansion (F
ISH) can be used to provide the correct chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases;
Clones longer than 2000 base pairs are likely to bind to a unique chromosomal location with sufficient signal strength for simple detection. For FISH, PRO
179, PRO238, PRO364, PRO844, PRO846, PRO1
760, PRO205, PRO321, PRO333, PRO840, PRO8
77, PRO878, PRO879, PRO882, PRO885 or PRO8
The use of clones to induce the gene encoding the 87 polypeptide,
The longer the better, the better. For example, 2000 base pairs is good, 4000
More preferred in base pairs, above 4000, is probably not necessary to get good results for a reasonable percentage of time. For a review of this technology, see Verma et al., Human Chromosomes; a Manual of Basic Techniques (Pergamon P
ress, New York. 1988). Once the sequence is mapped to exact chromosomal locations, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data is, for example, V. M.
cKusick, Mendelian Inheritance in Man (Johns Hopkins University Welch Me
dical library). The relationship between genes and diseases that map to the same chromosomal region is then identified by linkage assay (co-inheritance of physically adjacent genes). Next, it is necessary to determine the differences in the cDNA or genomic sequence between diseased or non-diseased individuals. A mutation is considered to be the cause of a disease if the mutation is found in some or all of the individuals affected by the disease and not in normal individuals. According to the current resolution of physical mapping and gene mapping techniques, a cDNA located exactly in a chromosomal region associated with disease is one of between 50 and 500 potentially causative genes (which means 1 megabase). Mapping resolution of 20k
(It is assumed that there is one gene per b).

Ix. Candidate Drug Screening Assays The present invention provides PRO179, PRO238, PRO364, PRO844, PR.
O846, PRO1760, PRO205, PRO321, PRO333, PR
O840, PRO877, PRO878, PRO879, PRO882, PRO
Similar to 885 or PRO887 polypeptides (agonist) or PRO17
9, PRO238, PRO364, PRO844, PRO846, PRO176
0, PRO205, PRO321, PRO333, PRO840, PRO877
, PRO878, PRO879, PRO882, PRO885 or PRO887.
Also included are methods of screening compounds to identify those that inhibit the effects of the polypeptide (antagonists). A screening assay for antagonist candidate drugs is performed by using the genes identified herein, PRO179, PRO238, P.
RO364, PRO844, PRO846, PRO1760, PRO205, P
RO321, PRO333, PRO840, PRO877, PRO878, PR
It is designed to identify compounds that bind or complex with the O879, PRO882, PRO885 or PRO887 polypeptides, or otherwise inhibit the interaction of the encoded polypeptide with other cellular proteins. Such screening assays include those applicable to high throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. The assay is performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, including those known in the art. All assays for antagonists include PRO179, PRO238, PRO364, PRO8, in which they are encoded by the nucleic acids identified herein as drug candidates.
44, PRO846, PRO1760, PRO205, PRO321, PRO3
33, PRO840, PRO877, PRO878, PRO879, PRO88
2, PRO885 or PRO887 polypeptides are common in that they require contact under conditions and for a time sufficient for these two components to interact. In the binding assay, the interaction is binding and the complex formed is isolated or detected in the reaction mixture. In a particular embodiment, PRO179, PRO238, PRO364, PRO84 encoded by the genes identified herein.
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, PRO885 or PRO887 polypeptides or drug candidates are covalently or non-covalently immobilized to a solid phase, eg, a microtiter plate. Non-covalent bond is
Generally, solid surfaces are designated PRO179, PRO238, PRO364, PRO844.
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
This is accomplished by coating with a solution of PRO885 or PRO887 polypeptide and drying. Alternatively, the immobilized PRO179, PRO238, PRO3
64, PRO844, PRO846, PRO1760, PRO205, PRO3
21, PRO333, PRO840, PRO877, PRO878, PRO87
Immobilized antibodies specific for the 9, PRO882, PRO885 or PRO887 polypeptides, such as monoclonal antibodies, can be used to anchor it to a solid surface. The assay involves the immobilization component, for example a coated surface containing the fixation component,
It is carried out by adding a non-immobilized component which may be labeled with a detectable label. When the reaction is complete, unreacted components are removed, for example by washing, and the complex adhered to the solid surface is detected. When the first non-immobilized component carries a detectable label, detection of the label immobilized on the surface indicates that complex formation has occurred. If the first non-immobilized component has no label, complex formation can be detected, for example, by a labeled antibody that specifically binds to the immobilized complex.

Specific PRs with which the candidate compound interacts but is encoded by the gene identified here
O179, PRO238, PRO364, PRO844, PRO846, PRO
1760, PRO205, PRO321, PRO333, PRO840, PRO
877, PRO878, PRO879, PRO882, PRO885 or PRO
If it does not bind to the 887 polypeptide, its interaction with the polypeptide can be assayed by well-known methods for detecting protein-protein interactions. Such assays include traditional techniques such as cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. further,
Protein-protein interactions are described by Chevray and Nathans, Proc. Natl. Acad.
Sci. USA 89, 5789-5793 (1991), (Fields and coworkers [Fiels and Song, Nature (London) 340, 245-246 (1989); Chien et al.
It can be monitored by using the yeast-based gene system described in Proc. Natl. Acad. Sci. USA 88, 9578-9582 (1991)). Many transcriptional activators, such as yeast GAL4, consist of two physically distinct modular domains, one acting as a DNA binding domain and the other functioning as a transcriptional activation domain. The yeast expression system described in the previous literature (generally referred to as the "2-hybrid system") takes advantage of this property and uses two hybrid proteins,
On the one hand the target protein is fused to the DNA binding domain of GAL4, and on the other hand
The candidate activating protein is fused to the activation domain. GAL1-la
Expression of the cZ reporter gene under the control of the GAL4-activating promoter depends on reconstitution of GAL4 activity via protein-protein interactions. Colonies containing interacting polypeptides are detected with a chromogenic substance for β-galactosidase. Complete kit for identifying protein-protein interactions between two specific proteins using 2-hybrid technology (MATCHMAKER (
Trade name)) is commercially available from Clontech. This system can be extended to the mapping of protein domains involved in a particular protein interaction, as well as the identification of amino acid residues important for this interaction. PRO179, PRO238, PRO364, PRO844 identified here
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
A compound that inhibits the interaction of a gene encoding a PRO885 or PRO887 polypeptide with an intracellular or extracellular component can be tested as follows: Usually, the reaction mixture is a mixture of the gene product and the extracellular or intracellular component, The two products are prepared to include under conditions and for time of interaction and binding. To test the ability of a candidate compound to inhibit binding, the reaction is performed in the absence and presence of the test compound. In addition, placebo may be added to the third reaction mixture to provide a positive control. The binding (complex formation) of the test compound present in the mixture with intracellular or extracellular components is monitored as described above. The formation of the complex in the control reaction but not in the reaction mixture containing the test compound indicates that the test compound inhibits the interaction of the test compound with its binding partner.

If the PRO polypeptide has the ability to stimulate endothelial cell proliferation in the presence of the co-mitogen ConA, then one example of a screening method takes advantage of this ability. In particular, in a proliferation assay, human umbilical vein endothelial cells were obtained, cultured in 96-well flat bottom culture dishes (Costar, Cambridge, MA), supplemented with an appropriate reaction mixture to facilitate cell proliferation, and the mixture was Con-A (Calbiochem, La Jol
la, CA). Add Con-A and the compound to be screened,
After incubation at 37 ° C., cultures are labeled with ^3- H thymidine and collected on glass fiber filters (phD; Cambridge Technology, Watertown, MA). Three
The mean ^3- H thymidine incorporation (cpm) of the media is measured using a liquid scintillation counter (Beckman Instruments. Irvine. CA). Significant ^3- (H) thymidine contamination was shown upon stimulation of endothelial cells. To assay antagonists, the assay described above is performed, in which PRO polypeptide may be added with the compound to be screened, and compounds that inhibit ^3- (H) thymidine incorporation in the presence of PRO polypeptide. Ability indicates that the compound is an antagonist of the PRO polypeptide. Alternatively, the antagonist may be detected by binding the PRO polypeptide and a potential antagonist with the membrane-bound PRO polypeptide receptor or recombinant receptor under conditions suitable for competitive inhibition assays. PRO polypeptides can be labeled, such as radioactively, and the number of PRO polypeptide molecules bound to the receptor can be used to determine the efficacy of a potential antagonist. The gene encoding the receptor can be identified by many methods known to those of skill in the art, such as ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun.
, 1 (2): Chapter 5 (1991). Preferably, expression cloning is used and the polyadenylated RNA is prepared from cells responsive to the PRO polypeptide, which RNA is
The cDNA library generated from the
Used for transfection of other cells that are not polypeptide responsive. Transfected cells grown on glass slides are exposed to labeled PRO polypeptide. PRO polypeptides can be labeled by a variety of means including iodination or the inclusion of recognition sites for site-specific protein kinases. After fixation and incubation, slides are subjected to autoradiographic analysis. Positive pools are identified and subpools are prepared and retransfected using an interactive subpooling and rescreening step, resulting in the generation of a single clone encoding the putative receptor.

As an alternative approach to receptor identification, labeled PRO polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. The crosslinked material is dissolved in PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excited, separated into peptide fragments and subjected to protein microsequencing. The amino acid sequences obtained from the microsequences are used to design a set of degradable oligonucleotide probes that screen a cDNA library to identify genes encoding putative receptors. In another assay for antagonists, mammalian cells expressing the receptor or a membrane preparation are incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction is then measured. Compositions useful in the treatment of cardiovascular, endothelial and angiogenic disorders include, but are not limited to, antibodies, small organic and inorganic molecules, peptides, phosphopeptides that inhibit the activity and / or expression of target gene products, Includes antisense and ribozyme molecules, triple helix molecules. More particular examples of potential antagonists are oligonucleotides that bind to fusions of immunoglobulins and PRO polypeptides, particularly but not limited to poly- and monoclonal antibodies and antibody fragments, single chain antibodies, anti-idio. It includes type antibodies and chimeric or humanized forms of these antibodies or fragments, as well as antibodies, including human antibodies and antibody fragments. Alternatively, the potential antagonist may be a closely related protein, eg, a mutated form of the PRO polypeptide that recognizes but does not affect the receptor, thereby competitively inhibiting the action of the PRO polypeptide. Other potential PRO polypeptide antagonists are antisense RNA or DNA constructs prepared using antisense technology, eg, antisense RNA or DNA hybridizes to a target mRNA to interfere with protein translation. This acts to directly block the translation of mRNA. Antisense technology can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of polynucleotides to DNA or RNA. For example, the 5'coding portion of the polypeptide nucleotide sequence encoding the mature PRO polypeptide herein is used to design an antisense RNA oligonucleotide of about 10 to 40 base pairs in length.
DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription (Triple Helix-Lee et al., Nucl, Acid Res., 6: 3073 (1979);
Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 251: 1360 (1991).
), Thereby preventing transcription and production of PRO polypeptide. Antisense RNA oligonucleotides hybridize to mRNA in vivo to form mRNA
Block the translation of NA molecules into PRO polypeptides (Antisense-Okano, Neu
rochem., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors o
f Gene Expression (CRC Press: Boca Raton, FL, 1988)). The oligonucleotides described above can also be delivered to cells to express antisense RNA or DNA in vivo and inhibit the production of PRO polypeptides. Antisense DN
When A is used, the translation initiation site, eg, about − of the target gene nucleotide sequence.
Oligodeoxyribonucleotides derived from between the 10 and +10 positions are preferred.

Antisense RNA or DNA molecules generally will be at least about 5 bases long, about 1 base long.
0 base length, about 15 base length, about 20 base length, about 25 base length, about 30 base length, about 35 base length
Base length, about 40 base length, about 45 base length, about 50 base length, about 55 base length, about 60 base length, about 65 base length, about 70 base length, about 75 base length, about 80 base length, about 85 base length Base length, about 90 bases, about 95 bases, about 100 bases, or more. Potential antagonists include small molecules that bind to the active site, receptor binding site, or growth factor or other related binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptide organic or inorganic compounds. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within potential RNA targets can be identified by known techniques. Further details can be found in, for example, Rossi, Current B
See Biology 4: 469-471 (1994) and PCT publication, number WO 97/33551 (published September 18, 1997). Nucleic acid molecules in triple-helix formation used to inhibit transcription are single-stranded and composed of deoxynucleotides. The basic composition of these oligonucleotides is designed to promote triple helix formation via Hoogsteen base pairing rules,
It generally requires resizable extension of purines or pyrimidines on one strand of the duplex. Further details are given, for example, in the above-mentioned PCT publication, number WO 97/3355.
See 1. These small molecules can be identified by any one or more of the screening assays discussed above and / or by any other screening technique well known to those of skill in the art.

X. Types of Cardiovascular, Endothelial and Angiogenic Diseases Treated Having Activity in the Cardiovascular, Endothelial and Angiogenic Assays Described Here,
And / or their gene products have been found to be located in the cardiovascular system P
RO polypeptides or their agonists or antagonists are likely to have therapeutic use in systemic disorders affecting blood vessels, such as various cardiovascular, endothelial and angiogenic disorders including diabetes mellitus. Their therapeutic effectiveness may include diseases of the arteries, capillaries, veins, and / or lymphatic vessels. Therapeutic examples below include muscle wasting disease treatment, osteoporosis treatment, transplant fixation aids to stimulate cell growth around the transplant, thus facilitating binding to its intended site, IGF stability in tissue or serum. And, where appropriate, increased binding to the IGF receptor (as it has been shown in vitro that IGF enhances the growth of human bone marrow erythroid and granulocyte progenitor cells). The PRO polypeptide or agonist or antagonist thereof also stimulates erythropoiesis or granulopoiesis, stimulates wound healing and tissue regeneration, and promotes tissue, eg connective tissue, skin, bone, cartilage, muscle, lung or kidney. It can be used to stimulate or inhibit endothelial cell migration in connection with regrowth-related therapies. Increased angiogenesis mediated by PRO polypeptides or antagonists is beneficial for ischemic tissue, collateral coronary arteries of the heart, and subsequently coronary stenosis. Antagonists are used to inhibit the action of such polypeptides and limit excessive connective tissue production, for example during wound healing or embryonic fibrosis. This includes acute myocardial infarction and heart failure. Further, the present invention relates to treatment of cardiac hypertrophy, regardless of cause, by administering a therapeutically effective amount of a PRO polypeptide, or an agonist or antagonist thereof. When the purpose is treatment of human patients, the PRO polypeptide is preferably recombinant human PRO polypeptide (rhPRO polypeptide). Treatment of cardiac hypertrophy can be performed at any of its various stages resulting in a wide variety of medical conditions, including myocardial infarction, hypertension, hypertrophic cardiomyopathy, and heart valve regurgitation. Treatment can be extended to all stages of the progression of cardiac hypertrophy, with or without structural damage to the myocardium, regardless of the underlying heart disease. The determination of whether to use the molecule itself or an agonist thereof for a particular indication when counteracting an antagonist of the molecule is primarily driven by the molecule promoting neovascularization, endothelial cell development, or angiogenesis, Alternatively, it depends on whether or not these pathologies are inhibited. For example, if the molecule promotes angiogenesis, its antagonists would be useful in the treatment of diseases where it is desired to limit or prevent angiogenesis. Examples of such diseases relate to hemangiomas such as hemangiomas, tumor angiogenesis, retinal neovascularization, diabetic retinopathy or premature infantile retinopathy or macular degeneration and proliferative vitreoretinopathy. Choroids, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation, psoriasis, neovascularization-associated endometriosis, restenosis followed by balloon angioplasty, scar tissue overproduction, eg after surgery. Kind of keloids formed,
Includes fibrosis after myocardial infarction, or fibrotic disorders associated with embryonic fibrosis. However, if the molecule inhibits angiogenesis, it is expected to be used directly in the treatment of the above mentioned pathologies.

On the other hand, if the molecule stimulates angiogenesis, it may prompt itself for angiogenesis, such as peripheral vascular disease, hypertension, vasculitis, Raynaud's disease and phenomenon, aneurysms, and arterial restenosis. Used for thrombophlebitis, lymphangitis, lymphedema, wound healing and tissue repair, ischemia-reperfusion injury, angina, myocardial infarction such as acute myocardial infarction, chronic heart disease, heart failure such as congestive heart failure, and osteoporosis To be done. However, if the molecule inhibits angiogenesis, the antagonist will be used in the treatment of conditions where angiogenesis is desired. Certain types of diseases are described below, where the PRO polypeptide or an antagonist thereof is provided as a therapeutic target for the prevention or treatment of the disease, or as a useful vascular release agent to target. Atherosclerosis is a disease characterized by the accumulation of plaques in the thickened intima of the arteries due to fluid accumulation, smooth muscle cell proliferation, and the formation of fibrous tissue within the arterial wall. is there. The disease affects large, middle and small arteries in any organ. Altered endothelial and vascular smooth muscle cell function is known to play an important role in regulating the accumulation and relaxation of these plaques. Hypertension is characterized by elevated blood pressure in the systemic arteries, pulmonary arteries, or portal system. Elevated blood pressure results from or results in defective endothelial function and / or vascular disease. Vasculitis includes giant cell arteritis, Takayas arteritis, multiple arterial nodosa (including microangiopathy forms), Kawasaki disease, micropolyangiitis, Wegener's granulomatosis, and various infectious vascular diseases ( (Including Henoch-Schonlein prupura). Altered endothelial cell function has been shown to be important in these diseases. Raynaud's disease and Raynaud's phenomenon are characterized by intermittent abnormal defects in the circulation through limbs exposed to cold air. Altered endothelial cell function has been shown to be important in these diseases. An aneurysm is a saccular or fusiform dilatation of an arterial or venous dendritic tree associated with altered endothelial cells and / or vascular smooth muscle cells. Arterial restenosis (arterial wall restenosis) is the result of altered endothelial and vascular smooth muscle cell proliferation and function, followed by angiogenesis. Thrombophlebitis and lymphangitis are inflammatory diseases of the veins and lymphatic vessels that result and / or result in altered endothelial cell function, respectively. Similarly, lymphedema is a condition associated with defects in the lymphatic vessels from endothelial cell function. The family of benign and malignant vascular tumors is characterized by abnormal growth and growth of cellular elements of the vasculature. For example, lymphangioma is a congenital, often benign tumor of the lymphatic system of the bladder, a lymphoid malformation that usually occurs in newborns. Bladder tumors tend to grow in adjacent tissues. Bladder tumors usually occur in the cervix and axilla. They also occur in the soft tissues of the extremities. The main symptom is swelling, with occasionally reticulated structured lymphoid and lymphocysts surrounded by connective tissue. Lymphangioma has been hypothesized to result from improper binding to fetal lymphatic vessels or their deletions. The result is local damage to lymph drainage. Griener et al., Lymphology, 4: 140-144 (1971).

Another use of the PRO polypeptides or antagonists thereof herein is in tumor angiogenesis, which is involved in angiogenesis of tumors that can grow and / or metastasize. This process depends on the growth of new blood vessels. Examples of related pathologies involving neoplasia and tumor angiogenesis include breast cancer, lung carcinoma, gastric carcinoma, esophageal carcinoma, colorectal carcinoma, liver carcinoma, ovarian carcinoma, tecoma, male embryonal tumor, cervical carcinoma, uterus. Endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcoma, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinoma, blood vessels Tumor, marine hemangioma, hemangioblastoma, pancreatic carcinoma, retinal carcinoma, astrocytoma, glioblastoma, schwannoma, oligodendroma, medulloblastoma, neuroblastoma, rhabdomyosarcoma, bone Includes primary sarcoma, leiomyosarcoma, urosarcoma, thyroid carcinoma, Wilms tumor, renal cell carcinoma, prostate carcinoma, abnormal blood vessel growth associated with phakomatoses, edema (e.g. associated with brain tumors), and Meigs syndrome . Age-related macular degeneration (AMD) is the cause of severe visual loss in the elderly population. The exudative form of AMD is characterized by choroidal neovascularization and retinal pigment endothelial cell detachment. PR because choroidal neovascularization is associated with a dynamic decline in prognosis
The O polypeptides or their antagonists are expected to be useful in reducing severe AMD. Also, wound healing, such as wound healing and tissue repair, is an intended use for PRO polypeptides or their antagonists herein. The formation and relaxation of new blood vessels is essentially tissue healing and repair. This category includes bone or cartilage, tendon, ligament, and / or nerve tissue growth or regeneration, as well as wound healing and tissue regeneration and replacement, and treatment of burns, amputations, and ulcers. PRO polypeptides or antagonists thereof that induce cartilage and / or bone growth in environments where bone is not normally formed are indicated for the treatment of bone fracture and cartilage damage, or human and other animal defects. Such preparations using PRO polypeptides or antagonists thereof are prophylactically used in reducing fractures and improving fixation of artificial joints. The formation of new bone induced by osteogenic agents contributes to the repair of congenital, trauma-induced, or oncological, resection-induced cranial defects and is also useful in cosmetic plastic surgery. Moreover, PRO polypeptides or antagonists thereof better and faster occlude unhealed wounds including, but not limited to, pressure ulcers, ulcers associated with vascular insufficiency, surgical or traumatic wounds, etc. It is useful for promoting things. Still further, PRO polypeptides or antagonists thereof may be administered to other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, sequences or endothelium), muscle (smooth muscle,
It may exhibit skeletal muscle or myocardium) and vascular (including vascular endothelium) tissue generation or regeneration, or activity promoting cell growth involving such tissue. Part of the desired effect is to inhibit or regulate fibrotic scarring and regenerate normal tissue.

The PRO polypeptides or antagonists herein are also useful for pathology from systemic cytokine damage and for reperfusion of wounds in various tissues, treatment of lung or liver fibrosis, and regeneration or protection. The PRO polypeptides or antagonists thereof are also useful for promoting or inhibiting the differentiation of the above-mentioned tissues from precursor tissues or cells, or for inhibiting the growth of the above-mentioned tissues. In addition, PRO polypeptides or antagonists thereof can be used in the treatment of periodontal disease and other tooth repair processes. Such agents are provided in an environment that attracts osteogenic cells, stimulates the growth of osteogenic cells, or induces the differentiation of osteogenic cell progenitors. The PRO polypeptide or antagonists herein stimulates osteoporosis or osteoarthritis, eg, bone and / or cartilage repair, or inflammation because blood vessels play an important role in regulating bone rotation and growth. It is useful for treatment by blocking the process of process-mediated tissue destruction (collagenase activity, osteoclasts, etc.) or inflammation. Another category of tissue renewal activity referred to herein as PRO polypeptides or its antagonists is tendon / ligament formation. Proteins that induce the formation of tendon / ligament-like tissue or other tissues in environments where tissue is not normally formed are applied to the healing of tendon or ligament tears, malformations, and tendon or ligament defects in humans and other animals. It Such preparations are prophylactically used in the prevention of damage to tendon or ligament tissue, as well as the improved fixation of tendon or ligament to bone or other tissue, and the repair of tendon or ligament tissue defects. New tendon / ligament-like tissue formation induced by the compositions of PRO polypeptides or antagonists herein contributes to the repair of tendon or ligament deficiencies of congenital, traumatic or other origin, It is also useful in cosmetic plastic surgery for the attachment or repair of tendons or ligaments. The composition herein attracts tendon- or ligament-forming cells, stimulates growth of tendon- or ligament-forming cells, induces differentiation of tendon- or ligament-forming cell progenitors, or recovers ex vivo. It is provided in an environment that induces the growth of tendon / ligament cells or progenitors in a tissue repair effect. Also, the composition here is
It is also useful for tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. In addition, the composition comprises the same suitable matrix and / or sequestrant as carriers well known in the art.

PRO polypeptides or antagonists thereof are central and peripheral nervous system diseases and neuropathies involved in nerve cell proliferation and regeneration of nerve and brain tissue, ie degeneration of nerve cells or nerve tissue, death or trauma, and It is useful for treating mechanical or traumatic diseases. In particular, PRO polypeptides or antagonists thereof may be used for peripheral nervous system diseases such as peripheral nerve injury, peripheral neuropathy and focal neuropathy, and central nervous system diseases such as Alzheimer's disease, Parkinson's disease, Huntingdon's disease, muscle atrophy. Used in the treatment of lateral sclerosis and Shy-Drager syndrome. Additional medical conditions treated with the present invention include mechanical or traumatic disorders such as spinal cord disorders, head trauma and cerebrovascular disorders such as stroke. Peripheral neuropathy resulting from chemotherapy or other medical therapies can also be treated using PRO polypeptides or antagonists thereto. Ischemia-reperfusion injury is another sign. Endothelial cell dysfunction is important both in the initiation and regulation of the sequelae of the events that follow ischemia-reperfusion injury. Rheumatoid arthritis is a further sign. The target of inflammatory cells that cross the blood vessel growth and vasculature is an important factor in the pathogenesis of the rheumatoid and serum negative forms of arthritis. PRO polypeptides or antagonists thereof are also administered prophylactically to patients with cardiac hypertrophy to prevent the progression of the condition and to avoid sudden death, including death of asymptomatic patients. Such prophylactic treatment is for cases of large left ventricular cardiac hypertrophy (maximum wall thickness of 35 mm or more in adults, or comparable in children), or when the hemangioma load in the heart becomes particularly strong. Is particularly justified in the example of. In addition, PRO polypeptides or antagonists thereof are useful in the treatment of atrial fibrillation, which is expressed in a significant proportion of patients diagnosed with hypertrophic cardiomyopathy. Further signs include angina, myocardial infarction, eg acute myocardial infarction, and heart failure,
For example, congestive heart failure is included. Further, non-neoplastic conditions include psoriasis, diabetes, and premature retinopathy, posterior lens fibroplasia, other proliferative retinopathy including neovascular glaucoma, thyroid hyperplasia (including Graves' disease), cornea and others. Tissue transplantation, chronic inflammation, pneumonia, nephrotic syndrome, preeclampsia, pericardial effusion (eg, associated with pericarditis), and pleural effusion. In view of the above, the PRO polypeptides described herein, or agonists or antagonists thereof, which have been shown to alter or impact endothelial cell function, proliferation and / or morphology, may include many or all of the above. Plays an important role in the etiology and etiology of the diseases of, and could be provided as a vascular-related agent that targets these diseases, or as a therapeutic target to augment or inhibit these processes.

Xi. Administered Procotol, Schedule, Dose and Formulation The molecules described herein and their agonists and antagonists are pharmaceutically useful as prophylactic and therapeutic agents for the various diseases and conditions mentioned above. The therapeutic compositions of PRO polypeptides or agonists or antagonists may be prepared using any desired pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol) of the desired molecule with suitable purity. , A. Hen, (19
80)) in the form of a lyophilized preparation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are preferably non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrate, and other organic acids; ascorbine. Antioxidants containing acids and methionines; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol Cyclopentanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine; Amino acids such as lutamin, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates containing glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; Salt forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and / or TWE
Includes nonionic surfactants such as EN ^™ , PLURONICS ^™ or polyethylene glycol (PEG). Further examples of such carriers are ion exchangers, alumina, aluminum stearate, lecithin, serum albumins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids. Partial glyceride mixture, water, salt, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, and Propylene glycol. Topical carriers or gel-based forms include polysaccharides such as sodium carboxymethyl cellulose or methyl cellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene block copolymers, polyethylene glycols, and mokuro alcohol. For any administration, conventional depot forms are preferably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nasal sprays, sublingual tablets, and sustained release formulations. The PRO polypeptide or agonist or antagonist is typically formulated in such vehicles at a concentration of about 0.1 mg / ml to 100 mg / ml.

Other preparations are included with the PRO polypeptides or their antagonists incorporated into the formed product. Such products can be used to regulate endothelial cell growth and angiogenesis. In addition, tumor invasion and metastasis may be regulated with these products. The PRO polypeptide or antagonist used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filter membranes before or after lyophilization and reconstitution. PRO polypeptides are usually stored in lyophilized form or in solution when administered systemically. PRO in lyophilized form
The polypeptide or agonist is typically formulated in combination with other ingredients, including a suitable diluent at the time of use. An example of a liquid formulation of a PRO polypeptide or antagonist is a sterile, clear, colorless, fresh solution filled in single-dose vials for subcutaneous injection. Preservative pharmaceutical compositions suitable for repeated use depend, for example, largely on the type and indication of the polypeptide, a) the PRO polypeptides or their agonists or antagonists; b) the stability of the polypeptides or other molecules in solution. A buffer capable of maintaining a pH within a range of maximization, preferably a pH of about 4-8; c) a detergent / surfactant that primarily stabilizes the polypeptide or molecule against agitation-induced assembly; d) An isotonic agent; e) a preservative selected from the group of phenols, benzyl alcohol and benzethonium halides such as chlorides; and f) water. If the cleaning agent used is nonionic, it can be, for example, polysorbate.
(Eg, POLYSORBATE ^™ (TWEEN ^™ ) 20, 80, etc.), poloxamer (eg, P
OLOXAMER ^™ 188). By using a nonionic surfactant, the formulation can be exposed to shear of surface stress without causing protein denaturation. Furthermore, such surfactant-containing preparations are used in aerosol devices such as those used for intravenous administration and needleless jet injection guns,
Can be used (see eg EP 257,956). An isotonicity agent is present to ensure isotonicity of the liquid composition of the PRO polypeptide or agonist or antagonist thereof, and is a polyhydric sugar alcohol, preferably 3
Or higher sugar alcohols such as glycerin, erythritol, arabitol,
Includes xylitol, sorbitol and mannitol. These sugar alcohols can be used alone or in combination. Also, sodium chloride or other suitable salt may be used to equalize the solution. The buffer may be an acetate, citrate, succinate or phosphate buffer, depending on the pH desired. One p of the liquid preparation of the invention
H is buffered in the range of about 4 to 8, preferably about physiological pH. Preservatives, phenol, benzyl alcohol and benzethonium halides,
Chloride, for example, is a well known antimicrobial agent that can be used.

Therapeutic PRO polypeptide compositions are generally placed in a container having a sterile access port, for example, an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The formulation is preferably intravenous (iv), subcutaneous (s).
． c. ), Or as repeated injections intramuscularly (im), or as an aerosol formulation suitable for intranasal or intrapulmonary delivery (for intrapulmonary delivery, for example,
See EP 257,956). The PRO polypeptide can also be administered in the form of sustained release formulations. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, eg films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg,
Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Te.
ch., 12: 98-105 (1982), poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polyactide (US Pat. No. 3,773,9).
19, EP 58,481), a copolymer 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-glycolic acid copolymers, eg
Lupron Depot ^™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988)
including. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When the encapsulated protein remains in the body for a long time, it is exposed to water at 37 ° C,
It may be denatured or aggregated, resulting in loss of physiological activity and altered immunogenicity. Rational mechanisms are conceivable to obtain stability by such a mechanism. For example, if it was found that the aggregation mechanism was an intermolecular SS bond by thio-disulfide exchange, the sulfhydryl residue was changed, freeze-dried from an acidic solution, the water content was adjusted, and an appropriate additive was used. The stability can be guaranteed by developing a specific polymer matrix compound. A sustained-release composition of PRO polypeptide comprises liposomally entrapped PRO polypeptide. Liposomes containing PRO polypeptides can be prepared by methods known per se, eg, DE 3,218,121, Epstein et al., Proc. Natl. Acad. Sci. USA,
82: 3688-3692 (1985), Hwang et al., Proc. Natl. 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 5
8-118008, US Pat. Nos. 4,485,045 and 4,544,545, and EP 102,324. Usually, the liposome has a lipid content of about 30 mol% or more of cholesterol, and the selected ratio is very small ().
It has a unilamellar shape of about 200-800 angstrom).

A therapeutically effective amount of a PRO polypeptide or antagonist thereof will, of course, be the pathological condition to be treated (including prophylaxis), the mode of administration, the type of compound used in the treatment, and any included. It will depend on factors such as concurrent treatment, patient age, weight, general medical condition, medical history, etc., which are well determined within the skill of the attending physician. Therefore, the therapist needs to titrate the dosage and modify the route of administration to obtain the maximum therapeutic effect. Given that the PRO polypeptide has a narrow host range, it is preferred that the preparation for treatment of human patients is a human PRO polypeptide, more preferably a native sequence human PRO polypeptide. The clinician will administer the dose until the desired effect is achieved in treating the condition.
The PRO polypeptide will be administered. For example, if the goal is treatment of CHF, it is an amount that inhibits the progressive cardiac hypertrophy associated with this condition. This treatment and progress is easily monitored by echocardiography. Similarly, patients with hypertrophic cardiomyopathy can be administered the PRO polypeptide empirically. In the above guidelines, an effective dose is generally about 0.001 to about 1.0 mg / k.
g, preferably about 0.01-1.0 mg / kg, most preferably about 0.01-0.
． It is within the range of 1 mg / kg. For parenteral use in the treatment of hypertension in adults, about 0.01 to 50 mg, preferably about 0.05 to 20 mg, most preferably 1 to 20 mg of PRO polypeptide, in the form of an injection, is administered intravenously. It is advantageous to administer by injection 1 to 3 times a day. For oral administration, the PRO polypeptide-based molecule is preferably about 5 mg to 1 g / kg body weight, preferably about 10-10.
Dosage 0 mg, 1 to 3 times daily. Endotoxin contaminants should be kept at a minimum safe level, eg less than 0.5 ng / mg protein. Moreover, for human administration, the preparations are preferably sterile, pyrogenic, generally safe, and FDA Office a
Purified as required by nd biologics standards. The dosage regimen of the pharmaceutical composition containing the PRO polypeptide used for tissue regeneration depends on various factors that alter the action of the polypeptide, such as the weight of the tissue (eg bone) desired to form, the age of the patient, the sex, It will be determined by the attending physician in view of diet, severity of infection, time of administration, and other clinical factors. The dose may vary depending on the type of matrix used for reconstitution, other protein content of the pharmaceutical composition. For example, the addition of other well known growth factors such as IGF-I to the final composition further affects the dose. Progress can be monitored by periodic assessment of tissue / bone growth and / or repair, for example by X-rays, histomorphometric determinations and tetracycline labeling.

The route of PRO polypeptide or antagonist or agonist administration is according to well-known methods, for example, intravenous, intramuscular, intracerebral, intraperitoneal, intracerebrospinal, subcutaneous, intraocular, intraarticular, intrasynovial, and envelope. By injection or infusion by the oral, oral, topical or inhalation routes, or by the sustained release system described below. Further, PRO polypeptides or their antagonists are suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes, and exert local and systemic therapeutic effects. The intraperitoneal route is expected to be particularly useful in treating ovarian tumors, for example. When the peptide or small molecule is used as an antagonist or agonist,
Preferably, it is administered to a mammal orally or parenterally in liquid or solid form. Examples of pharmaceutically acceptable salts of molecules which form salts and are useful below include alkali metal salts (e.g. sodium salt, potassium salt), alkaline earth metal salts (e.g. calcium salt, magnesium salt), ammonium. Included are salts, organic base salts (eg pyridine salt, triethylamine salt), inorganic acid salts (eg hydrochloric acid, sulfate, nitrate) and organic acid salts (eg acetate, oxalate, p-toluenesulfonate). Methods of treatment in the compositions described herein and useful for bone, cartilage, tendon, or ligament regeneration include topically, systemically or locally as an implant or device.
y) administration of the composition is included. When administered, the useful therapeutic composition is in a pyrogen-free, physiologically acceptable form. Further, it is desirable that the composition be injected or encapsulated in a viscous form that is delivered to the site of damage to bone, cartilage or tissue. Topical administration is suitable for wound healing and tissue repair. Preferably, for bone and / or cartilage formation, the composition delivers the protein-containing composition to the site of bone and / or cartilage damage, preferably developing resorbable bone and cartilage into the body. It includes a matrix that can provide a structure. Such matrices are made of materials used in other implantable medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance, and surface activity. The particular use of the composition will be defined by the appropriate formulation. The latent matrix of the composition is biodegradable and may be chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid, and polyanhydride. Other potential matrices are biodegradable and biologically well defined, eg bone or dermal collagen. The further matrix consists of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. The matrix may be of any type of material described above, for example a combination of polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramic may be altered in composition, for example calcium-aluminate-phosphate, and may have undergone processes to alter pore size, particle size and biodegradation performance. In one particular embodiment, lactic acid and glycolic acid are 50:50 (molar weight) copolymers and are in the form of porous particles having diameters in the range of 150 to 800 microns. In some applications, sequestering agents such as carboxymethyl cellulose, or autologous clots are useful in protecting the composition from separation from the matrix. One suitable family of sequestrants is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and carboxymethylcellulose, preferably carboxymethylcellulose. It is a cation salt of (CMC). Other preferred sequestrants include hyaluronic acid, sodium alginate, poly (
Ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymers, and poly (vinyl alcohol). The amount of sequestrant useful herein is 0.5-20% by weight, preferably 1-10% by weight, based on the total weight of the preparation.
And prevents the desorption of the polypeptide (or its antagonist) from the polymatrix, imparts suitable operability to the composition, and further prevents the penetration of progenitor cells into the matrix, thus allowing the polypeptide to become The amount required to provide the opportunity to promote the osteogenic activity of the.

Xii. Combination therapy PRO179, PRO238, PR in the prevention or treatment of the disease in question
O364, PRO844, PRO846, PRO1760, PRO205, PR
O321, PRO333, PRO840, PRO877, PRO878, PRO
The potency of 879, PRO882, PRO885 or PRO887 polypeptides, amines agonists or antagonists thereof may be combined with other agents effective for these purposes in the same composition or in separate compositions, or the active agent may be continuous. And then administered. For example, for the treatment of cardiac hypertrophy, PRO polypeptide therapy may be performed using well-known inhibitors of myocardial myocyte hypertrophy, such as inhibitors of α-adrenergic agonists such as phenylephrine; endothelin-I inhibitors such as BOSENTAN ^T
M and MOXONODIN ^™ ; inhibitors for CT-1 (US Pat. No. 5,679,545)
The administration of an inhibitor against LIF; an ACE inhibitor; a des-asparatate-angiotensin I inhibitor (US Pat. No. 5,773,415) and an angiotensin II inhibitor can be combined. For the treatment of cardiac hypertrophy associated with hypertension, the PRO polypeptide is administered with a β-adrenergic receptor blocking agent such as propranolol, timolol, tartarolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, Or carvedil rolls;
ACE inhibitors such as quinapril, captopril, enalapril, ramipril, benazepril, fosinopril or lisinopril; diuretics such as chlorothiazide, hydrochlorothiazide, hydroflumetazide, methylclothiazide, benzthiazide, dichlorophenamide, acetazolamide,
Alternatively, it can be administered in combination with indapamide; and / or a calcium channel blocker such as diltiazem, nifedipine, verapamil or nicardipine. The therapeutic drug identified here by the general name is commercially available as a gene pharmaceutical composition, and the therapeutic drug is administered according to the manufacturer's instructions such as dosage, administration method, side effects, contraindications, and the like. For example, Physicians'Desk Reference (Medical Economics Data Production
Co .: Montvale, NJ, 1997), 51th edition.

Preferred candidates for combination therapy in the treatment of cardiac hypertrophy are β-adrenergic receptor blocking agents (propranolol, timolol, tartarolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carbe). Dirroll), verapamil, difedipine, or diltiazem. For the treatment of hypertrophy associated with hypertension, calcium channel blockers such as diltiazem, nifedipine, verapamil and nicardipine; β-adrenergic receptor blockers; diuretics such as chlorothiazide, hydrochlorothiazide, hydroflumetazide, methylclothiazide, benzthiazide. , Dichlorophenamide, acetazolamide,
Or indapamide; and / or the use of anti-hypertensive agents using ACE inhibitors such as quinapril, captopril, enalapril, ramipril, benazepril, fosinopril or lisinopril. For other indications, PRO polypeptides or their antagonists may be combined with other agents useful in the treatment of bone and / or cartilage defects, wounds or tissues in the problem. Such agents include various growth factors such as EGF, PDGF,
Included are TGF-α or TGF-β, IGF, FGF, and CTGF. In addition, the PRO polypeptides or their antagonists used in the treatment of cancer are combined with cytotoxic, chemotherapeutic or growth inhibitory agents as identified above. Also, for the treatment of cancer, the PRO polypeptide or antagonists thereof is suitably administered sequentially or in combination with radiological treatment, with or without irradiation or administration of radioactive material. The effective amount of therapeutic agent administered in combination with the PRO polypeptide or its antagonists is at the discretion of the physician or veterinarian. Dosage and adjustments are made to achieve maximal therapeutic effect on the condition being treated. For example, in the treatment of hypertension, these amounts ideally take into account the use of diuretics or digital and hypertension or hypotension, renal damage, etc. The dose depends on such factors as the particular patient being treated and the type of therapeutic agent used. Typically, the amount used is
The same dose as when not administered with O polypeptide.

Xiii. Articles of manufacture PRO179, PRO238, PRO3 useful for the diagnosis or treatment of the above mentioned diseases
64, PRO844, PRO846, PRO1760, PRO205, PRO3
21, PRO333, PRO840, PRO877, PRO878, PRO87
An article of manufacture such as a kit containing a 9, PRO882, PRO885 or PRO887 polypeptide or antagonist thereof comprises at least one container and label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be formed from various materials such as glass or plastic. The container holds a composition useful for diagnosing or treating a condition and may have a sterile access port (eg, the container may be an intravenous bag or vial with a hypodermic needle pierceable stopper). ). The active agents in the composition are PRO179, PRO23.
8, PRO364, PRO844, PRO846, PRO1760, PRO20
5, PRO321, PRO333, PRO840, PRO877, PRO878
, PRO879, PRO882, PRO885 or PRO887 polypeptides or agonists or antagonists thereof. 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 containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. In addition, other materials desirable from a commercial and user standpoint may be provided, including other buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. The article of manufacture may also include a second or third container containing the other activator described above.

C. Antibodies Some of the many useful drug candidates of the invention are the generation of the genes identified herein,
Or antibodies and antibody fragments that inhibit the gene product and / or reduce the activity of the gene product. i. Polyclonal Antibodies Methods of preparing polyclonal antibodies are known to those of skill in the art. Polyclonal antibodies in mammals can be generated by one or more injections of, for example, 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 intraperitoneal injections. The immunizing agent is PRO179, PRO238, PRO364, PRO844,
PRO846, PRO1760, PRO205, PRO321, PRO333,
PRO840, PRO877, PRO878, PRO879, PRO882, P
It may include a RO885 or PRO887 polypeptide or a fusion protein thereof.
It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins are not limited,
Includes keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin and soybean trypsin inhibitor. Examples of adjuvants that may be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol will be selected by one of ordinary skill in the art without undue experimentation.

Ii. Monoclonal antibody, anti-PRO179, anti-PRO238, anti-PRO364, anti-PRO
844, anti-PRO846, anti-PRO1760, anti-PRO205, anti-PRO3
21, anti-PRO333, anti-PRO840, anti-PRO877, anti-PRO878
The anti-PRO879, anti-PRO882, anti-PRO885 or anti-PRO887 antibody may be a monoclonal antibody. Monoclonal antibodies are Kohler and Mi
It can be prepared using the hybridoma method as described in Lstein, Nature, 256: 495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are typically immunized with an immunizing agent to produce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Induce. The lymphocytes can also be immunized in vitro. The immunizing agent will typically be the target PRO179, PRO238, PRO36.
4, PRO844, PRO846, PRO1760, PRO205, PRO32
1, PRO333, PRO840, PRO877, PRO878, PRO879
, PRO882, PRO885 or PRO887 polypeptides or fusion proteins thereof. Generally, when human-derived cells are desired, peripheral blood lymphocytes (“P
BL ") is used, or spleen cells or lymph node cells are used if a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells. 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 used. The hybridoma cells are preferably cultured in a suitable medium that contains one or more substances that inhibit the survival or growth of the unfused, immortalized cells. For example, when the parental cell lacks the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the hybridoma's medium typically contains hypoxatin, aminoputilin and thymidine ("HAT medium"). , This substance blocks 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. A more preferred immortalized cell line is the mouse myeloma line, which is available, for example, from the Salk Institute Cell Distribution Center in San Diego, Calif. And the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse for producing human monoclonal antibodies-
Human xenomyeloma cell lines are also disclosed. Kozbor, J. Immunol., 133: 3001 (1
984), Brodeur et al., Monoclonal Antibody Production Techniques and Applicati
ons, (Marcel Dekker, Inc., New York, 1987) pp. 51-63. Next, the culture medium in which the hybridoma cells are cultured is changed to PRO179, PRO2.
38, PRO364, PRO844, PRO846, PRO1760, PRO2
05, PRO321, PRO333, PRO840, PRO877, PRO87
Assay for the presence of monoclonal antibodies against the 8, PRO879, PRO882, PRO885 or PRO887 polypeptides. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is measured by immunoprecipitation or an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunoassay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody is, for example, Munson and Pollard,
It can be measured by the Scatchard analysis method by Anal. Biochem., 107: 220 (1980).

After the desired hybridoma cells have been identified, clones can be subcloned by a limiting dilution step and grown by standard methods. Goding, supra. Suitable media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and R
Includes PMI-1640 land. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal. The monoclonal antibody secreted by the subclone is, for example, protein A
-Isolated or purified from culture medium or ascites fluid by conventional immunoglobulin purification methods such as Sepharose method, hydroxylapatite chromatography method, gel electrophoresis method, dialysis method or affinity chromatography. Monoclonal antibodies are also prepared by recombinant DNA methods, such as US Pat. No. 4,816,567.
It can be prepared by the method described in No. The DNA encoding the monoclonal antibody of the present invention can be easily prepared using a conventional method (for example, using an oligonucleotide probe capable of specifically binding to the genes encoding the heavy and light chains of mouse antibody). Can be isolated and sequenced. The hybridoma cells of the invention are a preferred source of such DNA. Once isolated, DNA
Can be placed in an expression vector, which is transfected into a host cell, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, resulting in recombinant host cell Monoclonal antibody can be synthesized with. The DNA may also be non-immunized with immunoglobulin coding sequences, eg, by substituting the coding sequences for human heavy and light chain constant domains in place of homologous mouse sequences (US Pat. No. 4,816,567; Morrison et al., Supra). It can be modified by covalently linking part or all of the coding sequence for the globulin polypeptide. Such non-immunoglobulin polypeptides can be substituted for the constant domain of the antibodies of the invention or for the variable domain of one of the antigen binding sites of the antibodies of the invention to produce chimeric, bivalent antibodies. The antibody may be a monovalent antibody. 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 cysteine residue is replaced or deleted with another amino acid residue to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Production of fragments thereof, particularly Fab fragments, by digestion of the antibody can be accomplished using conventional techniques known in the art.

Iii. Human and humanized antibodies anti-PRO179, anti-PRO238, anti-PRO364, anti-PRO844, anti
-PRO846, anti-PRO1760, anti-PRO205, anti-PRO321, anti-
PRO333, anti-PRO840, anti-PRO877, anti-PRO878, anti-PR
The O879, anti-PRO882, anti-PRO885 or anti-PRO887 antibodies further include humanized or human antibodies. Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′, F (ab ′) ₂ or other antigen-binding subsequences of antibodies). It contains a minimal sequence derived from non-human immunoglobulin. Humanized antibodies are human immunoglobulins (recipe) in which the CDR residues have been replaced by the CDR residues of a non-human species (donor antibody) having the desired specificity, affinity and capacity, such as mouse, rat or rabbit. Ent antibody). In some examples, human immunoglobulin Fv framework residues are replaced by corresponding non-human residues.
Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has at least one, and typically no, at least one, where all or almost all CDR regions correspond to those of a non-human immunoglobulin and all or almost all FR regions are of human immunoglobulin consensus sequences. Contains substantially all of the two variable domains. The humanized antibody optimally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al., Nat
ure, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Pr.
esta, 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 typically result from an "import" variable domain. Humanization is basically accomplished by substituting the relevant sequences of human antibodies with rodent CDRs or CDR sequences by Winter and coworkers [Jones et al., Nature, 321: 522-525 (1986); Riechmann. Et al, Nature, 332: 323-32
7 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)] by substituting a rodent CDR or CDR sequence with the corresponding sequence of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially less than the intact human variable domain has been replaced with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and optionally some FR residues have been replaced by residues from similar sites in rodent antibodies. Human antibodies can also be made using various methods known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 2
27: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Also, Cole and B
Methods such as Oerner can also be used to prepare human monoclonal antibodies. Co
le et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss. p. 77 (1985).
And Boerner et al., J. Immunol., 147 (1): 86-95 (1991). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, eg, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon administration, production of human antibodies is observed that is very similar to that found in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in US Pat. Nos. 5,545,807; 5,545,806.
No. 5,569,825; No. 5,625,126; No. 5,633,425; No. 5,661,016;
And the following scientific literature: Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al.
Nature 368 856-859 (1994); Morrison, Nature 368, 812-813 (1994); Fishwi
ld et al., Nature Biotechnology 14, 845-851 (1996); Neuberger, Nature Biotech
nology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93.
(1995).

Iv. Bispecific Antibodies Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. In this case, one of the binding specificities is PRO179, PRO238, PRO364,
PRO844, PRO846, PRO1760, PRO205, PRO321,
PRO333, PRO840, PRO877, PRO878, PRO879, P
Against the RO882, PRO885 or PRO887 polypeptides, the other against any other antigen, preferably a cell surface protein or receptor or receptor subunit. Methods of making bispecific antibodies are well known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, where the two heavy chains have different specificities. Milstein and Cuello, Nature,
305: 537-539 (1983). To randomly arrange the heavy and light chains of immunoglobulins,
These hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually accomplished by affinity chromatography steps. A similar procedure is described in WO 93/08829 published May 13, 1993, and Traunecker et al., EMBO J., 10: 3655-36.
56 (1991). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain, comprising at least the hinge region, and portions of the CH2 and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion, and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and cotransfected into a suitable host organism. For further details on making bispecific antibodies, see, eg, Suresh et al., Methods in Enzymology, 121: 210 (1986).

V. Heteroconjugate Antibodies Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies are used, for example, to target cells of the immune system to unwanted cells (US Pat.
, 676,980) and for the treatment of HIV infection (WO 91/00360; WO 92/200373; EP 0
3089) Proposed. It is believed that this antibody can be prepared in vitro using known methods in synthetic protein chemistry, including those associated with cross-linking agents. For example, immunotoxins can be made using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in US Pat. No. 4,6767,980. vi. Effector Function Processing It is desirable to modify the antibody of the present invention with respect to effector function, eg to improve the effectiveness of the antibody in treating cancer. For example, cysteine residue (s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus produced has improved internalization capacity and / or
Or it may have increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, BJ I.
See mmunol. 148: 2918-2922 (1992). Allodimeric antibodies with improved anti-tumor activity can also be prepared using heterobifunctional crosslinks as described by Wolff et al., Cancer research 53: 2560-2565 (1993). Alternatively, the antibody can be engineered to have two Fc regions, thereby improving complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).

Vii. Immunoconjugates The present invention also relates to chemotherapeutic agents, cytotoxic agents such as toxins (eg, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioisotopes (ie, radioconjugates). It also relates to an immunoconjugate comprising the conjugated antibody. Chemotherapeutic agents useful in the formation of such immune complexes have been described above. Enzymatically active toxins and fragments thereof that can be used are diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain. , Alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII
, And PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis
(sapaonaria oficinalis) inhibitor, gelonin, mitogellin
ellin), restrictocin, phenomycin, enomycin and trichothecene. Various radionucleotides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ^{1 31} I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re. Conjugates of antibodies and cytotoxic agents are labeled with various bifunctional protein coupling agents,
For example, N-succinimidyl-3- (2-pyridyldithiol) propionate (S
PDP), iminothiolane (IT), bifunctional derivative of imide ester (dimethyl adipimidate HCl etc.), active ester (disuccinimidyl suberate etc.), aldehyde (glutaraldehyde etc.), bis-azide compound (bis (p-azidobenzoyl) hexanediamine, etc.), bis-diazonium derivative (bis- (p-diazoniumbenzoyl) -ethylenediamine, etc.), diisocyanate (triene 2)
, 6-diisocyanate, etc.) and bis-active fluorine compounds (1,5-difluoro-
2,4-dinitrobenzene etc.). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026. In other embodiments, the antibody may be conjugated to a "receptor" (such as streptavidin) for use in tumor pre-targeting, and the antibody-receptor complex is administered to the patient, followed by a clarifier. It is used to remove unbound complex from the circulation and then administer a "ligand" (such as avidin) conjugated to a cytotoxic drug (such as a radionucleotide). viii. Immunoliposomes The antibodies disclosed herein may also be prepared as immunoliposomes. Liposomes containing antibodies are described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985).
Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat.
Prepared by methods known in the art, such as those described in 485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes are phosphatidylcholine, cholesterol and PEG.
-Produced by the reverse phase evaporation method with a lipid composition containing derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined size pores to produce liposomes with the desired size. F of the antibody of the present invention
Ab 'fragments can be conjugated to liposomes via a disulfide exchange reaction as described by Martin et al., J. Biol. Chem. 257: 286-288 (1982).
A chemotherapeutic agent (such as doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81 (19) 1484 (1989).

Ix. Antibody Pharmaceutical Compositions PRO179, PRO238, PRO364, PRO844 identified herein
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
Antibodies that specifically bind to PRO885 or PRO887 polypeptides, as well as other molecules identified in the screening assays disclosed above, may be used in the form of pharmaceutical compositions for the treatment of various disorders described above and below. It can be administered. PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
Internalized antibodies are preferred when the PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptide is intracellular and whole antibodies are used as inhibitors. However, lipofections or liposomes are also antibodies,
Alternatively, it can be used to introduce antibody fragments into cells. When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules can be designed that retain the ability to bind to target protein sequences based on the variable region sequences of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893 (1993). The formulation herein comprises one or more active compounds as necessary for the particular indication being treated,
Those having complementary activities that preferably do not adversely affect each other may also be included. Alternatively, or in addition, the composition may include a cytotoxic drug, cytokine or growth inhibitor. These molecules are suitably present in combination in amounts that are effective for the purpose intended. Also, the active ingredient may be incorporated into a colloidal drug delivery system (eg , Liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules) or microemulsions. These techniques are disclosed in Remington's Pharmaceutical Science, supra. The formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through a sterile filtration membrane. Sustained-release preparations may be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg films, or microcapsules. Examples of sustained release matrices are polyester hydrogels (eg poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,
919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT (trade name) (injectable globules of lactic acid-glycolic acid copolymer and leuprolide acetate) Includes degradable lactic acid-glycolic acid copolymer, poly- (D) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When the encapsulated antibodies remain in the body for a long time, they are denatured or aggregated by exposure to water at 37 ° C, resulting in reduced biological activity and possible altered immunogenicity. . Rational methods can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism was found to be intermolecular SS bond formation through thio-disulfide exchange, stabilization could be modification of sulfhydryl residues, lyophilization from acidic solution, control of water content, appropriate Can be achieved by the addition of various additives and the development of specific polymer matrix compositions.

X. Treatment Method Using Antibodies PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
It is anticipated that antibodies against the PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptides may be used in the treatment of the various cardiovascular, endothelial and angiogenic diseases mentioned above. The antibody is administered to a mammal, preferably a human, by a well-known method, for example, intravenous administration as a bolus or by continuous infusion over a predetermined time, intramuscular, intraperitoneal, cerebrospinal, subcutaneous, interarticular, intrasynovial, It is administered by intrathecal, oral, topical, or inhalation routes.
Intravenous administration of the antibody is preferred. Other therapeutic regimens may be combined with, for example, administration of the antibodies of the invention. For example, when treating cancer with antibodies, patients treated with such antibodies may receive radiation therapy. Alternatively, or in addition, the patient may be administered a chemotherapeutic agent. The method of preparation and dose schedule for such chemotherapeutic agents will be used according to the manufacturer's instructions or empirically determined by the skilled practitioner. Preparations and dose schedules for such chemotherapy are also available in Chemotherapy Service MC.
Perry, (Williams & Wilkins, Baltimore, MD, 1992). The chemotherapeutic agent may be administered prior to, or following the administration of the antibody, or it may be administered concurrently therewith. Antibodies include anti-estrogen compounds such as tamoxifen or EVIST ^™ or anti-progesterone such as onapristone (see EP 616812)
May be combined with known doses of those molecules. Also, when the antibody is used to treat cancer, antibodies against other tumor associated antigens, such as one or more ErbB2, EGFR, ErbB3, ErbB4, or VEGF.
It is also preferable to administer an antibody that binds to the receptor. It also includes the drugs mentioned above. The antibody is suitably administered sequentially, or in combination with radiological treatment, with or without irradiation or administration of radioactive material. Alternatively, or additionally, two or more antibodies disclosed herein and binding to the same or two or more different antigens may be co-administered to the patient. Sometimes it is also advantageous to administer one or more cytokines to the patient. In a preferred embodiment, the antibody herein is co-administered with a growth inhibitor. For example, the growth inhibitor is administered first, followed by the antibody of the invention. However, it is also conceivable to co-administer or to administer the anti-cancer agent of the invention first. Appropriate doses for growth inhibitors are those currently used but may be reduced by the combined (synergistic) effect of the growth inhibitor and this antibody.

In one embodiment, tumor angiogenesis is attacked in combination therapy. Anti
-PRO polypeptide antibodies and other antibodies (eg, anti-VEGF) are administered to patients with tumors in therapeutically effective amounts, eg, determined to show necrosis of the tumor or metastatic lesions. This treatment is continued until a favorable effect is observed or there is no evidence of tumor or any metastatic lesions. TNF is then treated with an adjuvant, 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 microvascular coagulation promoting agents in tumors, such as anti-protein C antibody, anti-
Protein S antibody or C4b-binding protein (WO91 / 01753 published Feb. 21, 1991)
). Adjuvants depend on their effectiveness and it is desirable to compare their impact on tumors by a matrix screened in a convenient manner. The administration of anti-PRO polypeptide antibody and TNF is repeated until the desired clinical effect is achieved. The anti-PRO polypeptide antibody is also administered with TNF and, optionally, adjuvant. In instances where solid tumors are found in the extremities or other locations that allow isolation from the general circulatory system, the therapeutic agents described herein are administered to the isolated tumor or organ. In other embodiments, an FGF or PDGF antagonist, such as an anti-FGF or anti-PDGF neutralizing agent, is administered to the patient with the anti-PRO polypeptide antibody. Treatment with the anti-PRO polypeptide antibody is preferably discontinued during the period of wound healing or desired neovascularization. Suitable doses of the antibodies herein for the prevention or treatment of cardiovascular, endothelial and angiogenic diseases are the type of disease treated, the severity and course of the disease, the prevention or treatment as defined above. Whether or not the drug is administered for the purpose, prior treatment, patient clinical history and response to antibodies, and the discretion of the attending physician. The antibody is suitably administered to the patient once or over a series of treatments. For example, depending on the type and severity of the disease, about 1 μg / kg to 50 mg / kg (
For example, 0.1-20 mg / kg of antibody is the first candidate dose for administration to a patient, eg, in one or more separate doses or continuous infusion. A typical daily dosage might range from about 1 μg / kg to 10 depending on the factors mentioned above.
It may be 0 mg / kg or higher. For repeated administration over several days,
Depending on the condition, treatment is continued until the desired suppression of symptoms of the disease appears. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays such as X-ray tumor imaging.

Xi. Articles of Manufacture Also provided is an article of manufacture containing a container containing the antibody and a label. Such articles of manufacture have been described above, wherein the active agents are anti-PRO179, anti-PRO238, anti-P.
RO364, anti-PRO844, anti-PRO846, anti-PRO1760, anti-PR
O205, anti-PRO321, anti-PRO333, anti-PRO840, anti-PRO8
77, anti-PRO878, anti-PRO879, anti-PRO882, anti-PRO885
Alternatively, it is an anti-PRO887 antibody. xii. Diagnosis and Prognosis of Tumors Using Antibodies When the indication for which the antibodies are used is cancer, cell surface proteins such as growth receptors that are overexpressed in certain tumors can be used as candidates for drug or tumor (eg, cancer) treatment. Although an excellent target, the same proteins as the PRO polypeptide find additional use in tumor diagnosis and prediction. For example, antibodies directed against the PRO polypeptide can be used in tumor diagnosis and prognosis. For example, antibodies, including antibody fragments, can be used for qualitative or quantitative detection of expression of genes, including genes encoding PRO polypeptides. The antibody is preferably provided with a detectable, eg fluorescent, label and binding can be monitored by light microscopy, flow cytometry, fluorometry, or other techniques known in the art. Such binding assays are performed substantially as described above. In situ detection of antibodies that bind to the marker gene product can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a labeled antibody is applied to it by removing a histological sample from the patient and preferably overlaying the biological sample with the antibody. This approach also allows the distribution of the marker gene product in the tissues tested to be determined. It will be apparent to those skilled in the art that a wide range of histological methods are readily available for in situ detection.

The following examples are provided by way of illustration only and are not intended to limit the scope of the invention in any way. The entire disclosure of all patents and literature references cited in the present specification are hereby incorporated by reference. EXAMPLES Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The sources of cells identified by the ATCC Registry Number throughout the examples and throughout the specification are American Type Culture Collection,
Manassas, VA. Unless otherwise stated, the present invention used standard techniques of recombinant DNA technology, such as those described above and in the textbooks below: Samb, supra.
rook et al .; Ausubel et al., Current Protocols in Molecular Biology (Green Publish
ing Associates and Wiley Interscience, NY, 1989); Innis et al., PCR Protoc
ols: A Guide to Methods and Applications (Academic Press, Inc .; NY., 1
990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Pres
s: 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 Extracellular domain homology screen to identify novel polypeptides and their encoding cDNAs Extracellular domains (ECD) from approximately 950 known secreted proteins from the Swiss-Prot public database. The sequence (including the secretory signal sequence, if any)
It was used to search the T database. The EST database is a public database (
For example, GenBank) and corporate databases (eg, LIFESEQ (trade name), Incyte
Pharmaceuticals, Palo Alto, CA). Search the computer program B
LAST or BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996))
Was used as a comparison with the 6-frame translation of the EST sequence of the ECD protein sequence. Comparables that do not encode a known protein and have a BLAST score of 70 (sometimes 90) or higher are known as programs “phrap” (Phil Green, Univers
City of Washington, Seattle, WA) to build consensus DNA sequences. This extracellular domain homology screen was used to construct consensus DNA sequences against other identified EST sequences using phrap. Furthermore, the consensus DNA sequences obtained are often (but not always) BLAST or BLAST-2 and
Elongated using repeated cycles of phrap and consensus sequences discussed above E
The ST sequence source was used to extend as much as possible. Based on the consensus sequence obtained as described above, oligonucleotides are then synthesized, and PCR is used to identify a cDNA library containing the sequence of interest and a clone of the full-length coding sequence for the PRO polypeptide is isolated. Used as a probe. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give PCR products of about 100-1000 bp in length. The probe sequence is typically 40-55b
It is p-long. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. To screen several libraries for full length clones, DNA from the libraries was
Screened by PCR with PCR primer pairs as in Subel et al., Current Protocols in Molecular Biology. The positive library was then used to isolate clones encoding the gene of interest using one of the probe oligonucleotides and the primer pair. The cDNA library used for the isolation of the cDNA clone is Invitrogen, San Di
Prepared by standard methods using commercially available reagents such as those from ego, CA. cd
NA was primed with an oligo dT containing a NotI site, blunt-ended with a SalI hemikinase adapter, cut with NotI, appropriately sized by gel electrophoresis, and a suitable cloning vector (pRK5B or pRK5D, etc .;
pRK5B is a precursor of pRK5D that does not contain an SfiI site; Holmes et al., Sc
ience, 253: 1278-1280 (1991)) at the unique XhoI and NotI sites.

Example 2 Isolation of cDNA clones by amylase screening 1. Preparation of oligo dT primed cDNA library mRNA was isolated from human tissues of interest using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2). This RNA is called Life Tec
Oligo dT primed cD in vector pRK5D using reagents and protocols from hnologies, Gaithersburg, MD (Super Script Plasmid System)
Used to generate NA. In this method, the double-stranded cDNA was size-classified to over 1000 bp and the SalI / NotI binding cDNA was cloned into the XhoI / NotI cut vector. pRK5D was cloned into a vector with an sp6 transcription start site, followed by an SfiI restriction enzyme site, and an XhoI / NotI cDNA cloning site. 2. Preparation of Random Primed cDNA Library A secondary cDNA library was created to preferably represent the 5'end of the primary cDNA clone. Sp6 RNA was generated from the primary library (described above) and this RNA was used as the vector pSST-AMY. It was used to generate a random primed cDNA library using reagents and protocols from Life Technologies at 0 (Super Script Plasmid System referenced above). In this method, double-stranded cDNA was sized to 500-1000 bp, blunt-ended with a NotI adapter, cut at the SfiI site, and cloned into a SfiI / NotI cut vector. pSST-AMY. 0 is a cloning vector that has a yeast alcohol dehydrogenase promoter in front of the cDNA cloning site and a mouse amylase sequence (mature sequence without a secretory signal) followed by the yeast alcohol dehydrogenase transcription terminator after the cloning site. That is,
CDNA cloned into this vector fused in frame with an amylase sequence
A will lead to the secretion of amylase from appropriately transfected yeast colonies.

3. Transformation and Detection DNA from the library described in paragraph 2 above was chilled on ice and electrocompetent DH10B bacteria (Life Technoligies, 20 ml) were added. The mixture of bacteria and vector was then electroporated as recommended by the manufacturer. SOC medium (Life Technplogies, 1 ml) was then added and the mixture was incubated at 37 ° C for 30 minutes. The transformants were then plated on 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37 ° C). Positive colonies were discarded from the plate and DNA was isolated from the bacterial pellet using standard methods, eg CsCl-gradient. The purified DNA was then loaded into the yeast protocol below. Yeast methods can be divided into three categories: (1) transformation of yeast with plasmid / cDNA binding vectors; (2) detection and isolation of amylase secreting yeast clones; and (3) direct from yeast colonies. PCR amplification of inserts and DNA purification for sequencing and further analysis. The yeast strain used was HD56-5A (ATCC-90785). This strain has the following genotypes: MAT alpha, ura3-52, leu2-3, leu2-112, h
It has is3-11, his3-15, MAL ⁺ , SUC ⁺ , and GAL ⁺ . Preferably, a yeast mutant having an incomplete post-translational pathway can be used. Such mutants have translocation-deficient alleles at sec71, sec72, and sec62, with truncated sec71 being most preferred. Alternatively, antagonists (including antisense nucleotides and / or ligands) that inhibit the normal manipulation of these genes, other proteins involved in this post-translational pathway (eg, SEC61p, S
EC72p, SEC62p, SEC63p, TDJ1p or SSA1p-4p)
Alternatively, complex formation of these proteins is also preferably used in combination with the amylase-expressing yeast. Transformation was performed based on the protocol outlined in Gietz et al., Nucl. Acid. Res., 20: 1425 (1992). Transformed cells are then agar to YEPD
Seed in complex medium broth (100 ml) and grow overnight at 30 ° C. YEPD Broth, Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, C
Prepared as described in old Spring Harbor, NY, p. 207 (1994). The overnight medium was then diluted to approximately 2x10 ⁶ cells / ml (approximately OD ₆₀₀ = 0.1) in fresh YEPD broth (500 ml) and 1x10 ⁷ cells / ml (approximately OD _{60 0} = 0.4-0. It was regrown until 5).

The cells are then harvested, transferred to a GS3 rotor bottle in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, discarded for supernatant and then prepared for transformation by resuspending in sterile water and 50 ml. Again in a Beckman GS-6KR centrifuge at 3,500 rpm in a Falcon tube. Discard the supernatant and transfer the cells to LiAc / TE (10 ml
, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li ₂ OOCCH ₃ ) followed by a wash and resuspended in LiAc / TE (2.5 ml). For the transformation, the prepared cells (100 μl) were transferred to fresh denatured single-stranded salmon sperm DNA (Lofstrand Labs, Gaitherburg, MD) and transformed DNA in a microtube.
It was caused by mixing with (1 μg. Vol. <10 μl). The mixture was vortexed briefly and then 40% PEG / TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li ₂ OOCCH ₃ , pH 7.5) was added. The mixture was gently agitated and incubated at 30 ° C. with agitation for 30 minutes. The cells were then heat shocked at 42 ° C. for 15 minutes, the reaction vessel was centrifuged in a microtube at 12,000 rpm for 5-10 seconds, decanted and TE (500 μl, 10 mM).
Of Tris-HCl, 1 mM EDTA pH 7.5) followed by centrifugation. The cells were then diluted in TE (1 ml) and an aliquot (200 μl) was added to a 150 mm growth plate (VW
R) was spread on a selective medium prepared in advance. Alternatively, the transformations were performed in one large reaction rather than multiple small reactions, but the amount of reagents was scaled up accordingly. The selection medium used was Kaiser et al., Methods in Yeast Genetics, Cold Spring Har.
Uracil-free synthetic complete dextrose agar (SCD-Ura) prepared as described in Bor Press, Cold Spring Harbor, NY, p. 208-210 (1994). Transformants were grown at 30 ° C for 2-3 days. Detection of colonies secreting amylase was performed by the inclusion of red starch in the selective growth medium. Starch was coupled to a red dye (reactive Red-120, Sigma) according to the method described by Biely et al., Anal. Biochem., 172: 176-179 (1988). Bound starch on SCD-Ura agar plates to a final concentration of 0.15% (w / v)
And buffered to pH 7.0 with potassium phosphate (final concentration 50-100 mM). Positive colonies were picked and streaked on fresh selection medium (150 mm plate) to give well isolated and identifiable single colonies. Well-isolated colonies positive for amylase secretion were detected by direct introduction of the red dye group into buffered SCD-Ura agar. Positive colonies were determined by their ability to degrade starch and form visible halos directly around the positive colonies.

4. Isolation of DNA by PCR Amplification When positive colonies were isolated, a portion was picked with a toothpick and diluted in sterile water (30 μl) in a 96-well plate. At this point, positive colonies are either frozen and either stored for subsequent analysis or immediately amplified.
Aliquots of cells (5 μl) into 0.5 μl of Klentaq (Clontech, Palo Alto, CA);
4.0 μl 10 mM dNTP (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clonte
ch); 0.25 μl forward oligo 1; 0.25 μl reverse oligo 2; used as template for PCR reaction in a 25 μl volume containing 12.5 μl distilled water. The sequence of Forward Oligonucleotide 1 was: 5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3 '(SEQ ID NO: 33). The sequence of reverse oligonucleotide 2 was: 5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3 '(SEQ ID NO: 34). PCR was then performed as follows: a. Denaturation 92 ° C, 5 minutes b. Next 3 cycles: Denaturation 92 ° C, 30 seconds Annealing 59 ° C, 30 seconds Extension 72 ° C, 60 seconds c. Next 3 cycles: Denaturation 92 ° C, 30 seconds Anneal 57 ° C, 30 seconds Extension 72 ° C, 60 seconds d. Next 25 cycles: denaturation 92 ° C, 30 seconds Anneal 55 ° C, 30 seconds Extension 72 ° C, 60 seconds e. The regions underlined at 4 ° C. are annealed to the ADH promoter region and the amylase region, respectively, and the vector pSST-AMY. Amplify the 307 bp region from 0. Typically, the first 18 nucleotides at the 5'end of these oligonucleotides contained the sequence primer annealing site. That is, the total product of the PCR reaction from the empty vector was 343 bp. However, the signal sequence fusion cDNA resulted in a rather long nucleotide sequence. Following PCR, an aliquot (5 μl) of the reaction was tested by agarose gel electrophoresis using a Tris-borate-EDTA (TBE) buffer system in a 1% agarose gel as described by Sambrook et al., Supra. . Clones yielding single, strong PCR products larger than 400 bp were cloned into a 96 Qiaquick PCR clean column (Qiagen Inc).
, Chatsworth, CA) and further analyzed by DNA sequence.

Example 3 Isolation of cDNA Clones Using Signal Algorithm Analysis Various polypeptide-encoding nucleic acid sequences are described in Genentech, Inc. (South San
The proprietary sequence-finding algorithms developed by Francisco, CA) are publicly (eg, GenBank) and / or private (LIFESEQ®, Incyte Pharmaceuticals
, Inc., Palo Alto, Calif.) Database and identified by applying to the EST fragments assembled and assembled. The signal sequence algorithm is
A secretory signal score is calculated based on the nature of the DNA nucleotides surrounding the first and optionally the second methionine codon (ATG) at the 5'-end of the sequence or sequence fragment under consideration. The nucleotides following the first ATG must encode at least 35 unambiguous amino acids with no stop codon. First
If the ATG of A has the required amino acids, the second is not tested. If neither met the requirements, the candidate sequences were not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA surrounding the ATG codon and the corresponding amino acid sequence were analyzed using a set of seven sensors (evaluation parameters) known to be associated with the secretory signal. Used to score. The use of this algorithm has led to the identification of many polypeptide-encoding nucleic acid sequences.

Example 4 Isolation of a cDNA clone encoding human PRO179 cDNA was isolated by screening as described in Example 2 above. The cDNA sequence isolated in the above screen was found to have sequence homology with the nucleotide sequence encoding the angiopoietin protein by BLAST and FastA sequence alignment. This cDNA sequence is designated herein as DNA10028 and / or DNA25250. A probe was prepared from the sequence of the DNA10028 molecule based on sequence homology,
It was used to screen a human fetal liver library (LIB6) prepared as described in paragraph 1 of Example 2 above. The cloning vector is pRK5B (pRK5B is a precursor of pRK5D that does not contain an SfiI site; Holmes et al.
, Science, 253: 1278-1280 (1991)), cDNA size cut is 2800b
It was less than p. The DNA sequence of the clone isolated as described above was PRO179
And the derived protein sequence of PRO179. DN
The full-length nucleotide sequence of A16451-1388 is Fig1 (SEQ ID NO: 1).
Shown in. Clone DNA16451-1388 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 37-39 and a stop signal at nucleotide positions 1417-1419 (Fig1). The predicted polypeptide precursor is 460 amino acids long (Fig2, SEQ ID NO:
2), the full-length PRO179 protein shown in Fig2 has a calculated molecular weight of approximately 53,637 daltons and an estimated pI of approximately 6.61. Fi2 (SEQ ID NO: 2) analysis of the full-length PRO179 sequence revealed that Fi
The existence of various important polypeptide domains as shown in g2 has been revealed, and the positions given to those important polypeptide domains are approximately as described above. Analysis of the full-length PRO179 sequence (Fig2, SEQ ID NO: 2) revealed the presence of the following: a signal peptide of about amino acids 1 to about amino acids 16; about amino acids 120 to about amino acids 142 and about amino acids 127 to about amino acids 149. Leucine zipper pattern; about amino acid 23 to about amino acid 27, about amino acid 1
N-glycosylation sites from 15 to about amino acids 119, about amino acids 296 to about amino acids 300, and about amino acids 357 to about amino acids 361; and about amino acids 2
71 to about amino acid 310, about amino acid 312 to about amino acid 322, about amino acid 331 to about amino acid 369, and about amino acid 393 to about amino acid 424, the fibrinogen β and γ chain C-terminal domains. Clone DNA16451-1388 was deposited with the ATCC on April 14, 1998 and was assigned ATCC deposit no. 209776. With respect to sequence, it is understood that the inserted clone contains the exact sequence and that the sequence provided herein is based on known sequencing techniques. Analysis of the amino acid sequence of the full-length PRO179 sequence suggests that it has significant similarity to angiopoietin family proteins and thus PRO179 is a novel angiopoietin family member. More specifically, Da
Analysis of the yhoff database (version 35.45 Swiss Prot 35) revealed significant homology between the PRO179 amino acid sequence and the following Dayhoff sequence: AF004.
326_1, P_R94605, HSU83508_1, P_R94603, P_R94317, AF025818_1, HSY16132_1,
P_R65760, I37391 and HUMRS C192_1.

Example 5 Isolation of a cDNA clone encoding human PRO238 As described in Example 1 above, conrap was used to assemble consensus DNA sequences for other ESTs. This consensus sequence is now DNA3090
Name it 8. Based on the DNA30908 consensus sequence, 1) to identify a cDNA library containing the sequence of interest by PCR, and 2) PRO2
For use as a probe to isolate 38 full length coding sequence clones,
Oligonucleotides were synthesized. In addition, a synthetic oligonucleotide hybridization probe was made from the consensus DNA30908 sequence. To screen several libraries for full-length clones, the DNA from the libraries was submitted above by Ausubel et al., Current Protocols in Molecular Biol.
Screened by PCR amplification with PCR primer pairs according to Ogy.
The positive library was then used to isolate clones encoding the PRO238 gene using the probe oligonucleotide and one of the primer pairs. PCR primers (forward and reverse) were synthesized: Forward PCR primer 1: 5'-GGTGCTAAACTGGTGCTCTGTGGC-3 '(SEQ ID NO: 35) Forward PCR primer 2: 5'-CAGGGCAAGA TGAGCATTCC-3' (SEQ ID NO: 36). Reverse PCR primer: 5'-TCATACTGTTCCATCTCGGCACGC-3 '(SEQ ID NO: 37) Hybridization probe: 5'-AATGGTGGGGCCCTAGAAGAGCTCATCAGAGAACTCACCGCTTCTCATGC-3' (SEQ ID NO: 3)
8) RNA for the preparation of cDNA library was isolated from human fetal liver tissue. The cDNA library used to isolate the cDNA clones is Invitrogen
And standard reagents using commercially available reagents such as those from San Diego, CA. The cDNA was primed with an oligo dT containing a NotI site, ligated at the blunt end of the SalI hemikinase adapter, cut with NotI, appropriately size-divided by gel electrophoresis, and cloned into a suitable cloning vector (pRKB) in the defined orientation. Or pRKD etc .; pRK5B is a precursor of pRK5D that does not have an SfiI site; see Holmes et al., Science, 253: 1278-1280 (1991)).
It was cloned at the 1 and NotI sites. DNA sequencing of the clones isolated as described above revealed the full-length PRO2
Full-length DNA sequence for 38 [herein designated as DNA35600-1162] (Fig.
3, SEQ ID NO: 3) and its derived protein sequence for PRO238 was obtained. The entire nucleotide sequence of DNA35600-1162 is shown in Fig3 (SEQ ID NO: 3
). Clone DNA35600-1162 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 134-136,
And has an apparent stop codon at nucleotide positions 1064-1066 (Fi
g3). The predicted polypeptide precursor is 310 amino acids long (Fig4, SEQ ID NO: 4), has a calculated molecular weight of approximately 33,524 daltons and an estimated p of approximately 9.55.
I have. Fi4 (SEQ ID NO: 4) analysis of the full-length PRO238 sequence revealed that Fi
The presence of various important polypeptide domains as shown in g4 has been revealed and the positions given to those important polypeptide domains are approximately as described above. By analysis of full-length PRO238 polypeptide (Fig4, SEQ ID NO: 4)
, The presence of the following: a signal sequence from about amino acid 1 to about amino acid 21; a transmembrane domain from about amino acid 102 to about amino acid 119 and about amino acid 278 to about amino acid 292; an N from about amino acid 228 to about amino acid 232. -Glycosylation site; glycosaminoglycan attachment site from about amino acid 47 to about amino acid 51; tyrosine kinase phosphorylation site from about amino acid 145 to about amino acid 153; about amino acid 44 to about amino acid 50, about amino acid 105 to about amino acid 111
, About amino acid 238 to about amino acid 244, about amino acid 242 to about amino acid 2
48 and about an amino acid 291 to about amino acid 297 N-myristoylation site; about amino acid 265 to about amino acid 269 amidation site; and about amino acid 6 to about amino acid 17 a prokaryotic membrane detachment protein lipid attachment site. Cloned DNA
35600-1162 deposited with the ATCC on October 16, 1997,
C deposit number 209370 has been assigned. Analysis of the amino acid sequence of the full-length PRO238 polypeptide suggests that part of it has significant homology to reductases, especially oxidoreductases, which results in P
It was shown that RO238 could be a novel reductase.

Example 6 Isolation of a cDNA Clone Encoding Human PRO364 Expression Sequence Tag (EST) DNA Database (LIFESEQ®, Incyte Ph)
armaceuticals, Palo Alto, CA) and searched for tumor necrosis factor receptor (TNFR)
). An EST (Incyte EST no. 3003460) encoding a polypeptide showing homology with a member of the family polypeptide was identified. BLAST (Altschul et al., Methods in Enzymology 266: 460-480 (1996)) and "ph
rap "(Phil Green, University of Washington, Seattle, Washington) was used to construct consensus DNA sequences for Incyte EST no. 3003460 and other EST sequences. The consensus sequence is designated herein as "<consen01>" and is also referred to herein as DNA44825. 1) PC based on DNA44825 and the "<consen01>" consensus sequence
To identify a cDNA library containing the sequence of interest by R, and 2) P
Oligonucleotide probes were synthesized for use as probes to isolate clones of the RO364 full length coding sequence. Forward and reverse PCR primers generally range from 20 to 30 nucleotides, often about 100-1.
Designed to give a PCR product of 000 bp length. The probe sequence is typically 40-55 bp long. To screen several libraries for full-length clones, the DNA from the libraries was submitted by Ausubel et al., Current Pro.
Screened by PCR amplification with PCR primer pairs according to tocols in Molecular Biology. The positive library was then used to isolate clones encoding the gene of interest using probe oligonucleotides and one of the primer pairs. The oligonucleotide sequences used were as follows: Forward PCR primer (44825.f1): 5'-CACAGCACGGGGCGATGGG-3 '(SEQ ID NO: 39) Forward PCR primer (44825.f2): 5'-GCTCTGCGTTCTGCTCTG-3' (SEQ ID NO: 40) Forward PCR primer (44825.GITR.f): 5'-GGCACAGCACGGGGCGATGGGCGCGTTT-3 '(SEQ ID NO: 41) Reverse PCR primer (44825.r1): 5'-CTGGTCACTGCCACCTTCCTGCAC-3' (sequence No .: 42) Reverse PCR primer (44825.r2): 5'-CGCTGACCCAGGCTGAG-3 '(SEQ ID NO: 43) Forward PCR primer (44825.GITR.r): 5'-GAAGGTCCCCGAGGCACAGTCGATACA-3' (SEQ ID NO: 44) Furthermore, a synthetic oligonucleotide hybridization probe was prepared from the DNA44825 consensus sequence, which contained the following nucleotide sequence: Had columns: Hybridization probe (44825.p1): 5'-GAGGAGTGCTGTTCCGAGTGGGACTGCATGTGTGTCCAGC-3 '(SEQ ID NO: 45) Hybridization probe (44825.GITR.p): 5'-AGCCTGGGTCAGCGCCCCACCGGGGGTCCCGGGTGCGGCC-3' (SEQ ID NO: 45). : 46) To screen several libraries for a source of full length clones, DNA from the libraries was PCR amplified with the PCR primer pair identified above. The positive library is then loaded with probe oligonucleotides and PCR.
It was used to isolate clones encoding the PRO364 gene with one of the primer pairs. RNA for the construction of the cDNA library was isolated from human bone marrow tissue. cd
The cDNA library used to isolate the NA clones is Invitrogen, San
Formed by standard methods using commercially available reagents such as those from Diego, CA. cd
NA was primed with an oligo dT containing a NotI site, ligated at the blunt end of the SalI hemikinase adapter, digested with NotI, appropriately size-divided by gel electrophoresis, and cloned into a suitable cloning vector (pRKB) in the defined orientation. Or pRK
D et al .; pRK5D is a precursor of pRK5D that does not have an SfiI site; Holm
es et al., Science, 253: 1278-1280 (1991)) and cloned at the unique Xhol and NotI sites. The cDNA clone was identified and fully sequenced. DNA47365-1
The full length nucleotide sequence of 206 is shown in Figure 5 (SEQ ID NO: 5). Clone DNA47365-1206 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 121-123 and a stop codon at nucleotide positions 844-846 (Fig5; SEQ ID NO: 5). The predicted polypeptide precursor is 241 amino acids long, has a calculated molecular weight of approximately 26,000 daltons and an estimated pI of approximately 6.34. F full-length PRO364 protein
ig6 (SEQ ID NO: 6).

By analysis of the full-length PRO364 sequence shown in Fig6 (SEQ ID NO: 6), Fi
The existence of various important polypeptide domains such as those shown in g6 has been revealed, and the positions given to those important polypeptide domains are approximately as described above. Analysis of the full length PRO364 sequence (Fig6; SEQ ID NO: 6) revealed the presence of the following: a signal peptide from about amino acid 1 to about amino acid 25; a transmembrane domain from about amino acid 162 to amino acid 180; from about amino acid 146. N-glycosylation site of about amino acid 150; about amino acid 5 to about amino acid 11, about amino acid 8 to about amino acid 14, about amino acid 25 to about amino acid 31, about amino acid 30 to about amino acid 36, about amino acid 33 to about amino acid 39, About amino acid 1
18 to about amino acid 124, about amino acid 122 to about amino acid 128, about amino acid 156 to about amino acid 162 N-myristoylation site; about amino acid 166 to about amino acid 177 prokaryotic membrane protein lipid binding site; and about amino acid 171.
To a leucine zipper pattern of about amino acid 193. Clone DNA47365-1206 was deposited with the ATCC on November 7, 1997 and was assigned ATCC deposit no. 209436. Analysis of the amino acid sequence of the full-length PRO364 polypeptide suggests that some of it has significant homology with members of the tumor necrosis factor receptor family, thus indicating that PRO364 is a novel member of the tumor necrosis factor receptor family. Shows. The intracellular domain of PRO364 was shown to be similar to the motif (amino acid 2) of the CD30 receptor minimal domain shown to be required for TRAF2 binding.
(In the region of 07-214), which is also present in TNFR2. TN
There are three apparent extracellular cysteine-rich domains characteristic of the FR family (see Naismith and Sprang, Trends Biochem. Sci., 23: 74-79 (1998)), the third CRD of which is the TNFR family. It has three rather than the more typical four or six cysteines. Compared to mouse CRD1 (described below), PRO364 amino acid sequence is C compared to 5 cysteines in mouse GITR CRD1.
It has 8 cysteines in RD1 and 4 potential N-linked glycosylation sites in mouse GITR, whereas there is one potential N-linked glycosylation site in ECD. Detailed examination of the amino acid sequence of the full-length PRO364 polypeptide and the nucleotide sequence encoding the amino acid sequence has been performed by Nocentini et al., Proc. Natl. Acad. S.
Ci. USA, 94: 6216-6221 (1997) revealed sequence homology with the mouse GITR (mGITR) protein. Therefore, PRO364 represents the human counterpart of the mouse GITR protein reported in Nocentini et al.

Example 7 Isolation of cDNA Clone Encoding Human PRO844 Expression Sequence Tag (EST) DNA Database (LIFESEQ (trade name), Incyte Phar
maceuticals, Palo Alto, CA) and confirmed that EST has sequence identity with LP. Based on the information and findings obtained here, this EST clone, the in situ clone 2657496 from the cancerous lung library (309-LUNGTUT09), was further investigated. The cDNA clone was identified and fully sequenced. DNA59838-1
The full length nucleotide sequence of 462 is shown in Figure 7 (SEQ ID NO: 7). Clone DNA59838-1462 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 5-7 and nucleotide position 338.
It has a stop codon at -340 (Fig7; SEQ ID NO: 7). The predicted polypeptide precursor is 111 amino acids long, has a calculated molecular weight of approximately 12,050 daltons and an estimated pI of approximately 5.45. The full-length PRO844 protein is shown in FIG.
It is shown in SEQ ID NO: 8). By analysis of the full-length PRO844 sequence shown in Fig8 (SEQ ID NO: 8), Fi
The existence of various important polypeptide domains as shown in g8 was revealed, and the positions given to those important polypeptide domains are approximately as described above. Analysis of the full length PRO844 sequence (Fig8; SEQ ID NO: 8) revealed the presence of the following: a signal peptide from about amino acid 1 to about amino acid 19; about amino acid 23 to about amino acid 29,
About amino acid 27 to about amino acid 33, about amino acid 32 to about amino acid 38, about amino acid 102 to about amino acid 108 N-myristoylation site; and about amino acid 4
WAP-type "4-disulfide core" domain signature from 9 to about amino acid 63. Clone DNA59838-1462 was deposited with the ATCC on June 16, 1998 and was assigned ATCC deposit no. 209976. The deposited clone has virtually the correct sequence than the representation provided here. Analysis of the amino acid sequence of the full-length PRO844 polypeptide showed significant homology with serine protease inhibitors, thus suggesting that PRO844 is a novel proteinase inhibitor. More specifically, analysis of the Dayhoff database (version 35.45 Swiss Prot 35) revealed significant homology between the PRO844 amino acid sequence and the following Dayhoff sequences: ALK1_HUMAN, P_P82403, P_P
82402, ELAF_HUMAN and P_P60950.

Example 8 Isolation of a cDNA Clone Encoding Human PRO846 As described in Example 1 above, a consensus DNA sequence was constructed against other EST sequences using phrap. This consensus sequence is now DNA3
9949 and "<consen1322>". Based on the DNA39949 consensus sequence and "<consen1322>", 1) cD containing the sequence of interest by PCR
Oligonucleotides were synthesized to identify NA libraries and 2) to be used as probes to isolate clones of the PRO846 full length coding sequence. PCR primers (forward and reverse) were DNA39949 and "<consen1
322> ”based on the consensus sequence. In addition, a synthetic nucleotide hybridization probe is constructed from the consensus DNA30908 sequence. To screen several libraries for full-length clones, the DNA from the libraries was submitted above by Ausubel et al., Current Protocols in Molecular Biol.
Screened by PCR amplification with PCR primer pairs according to Ogy.
The positive library was then used to isolate clones encoding the PRO846 gene using the probe oligonucleotide and one of the PCR primer pairs. The following nucleotide sequence was obtained by the above method: Forward PCR primer (39949.fl): 5'-CCCTGCAGTGCACCTACAGGGAAG-3 '(SEQ ID NO: 47) Reverse PCR primer (39949.rl): 5'- CTGTCTTCCCCTGCTTGGCTGTGG-3 ′ (SEQ ID NO: 48) Furthermore, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus DNA39949 sequence: Hybridization probe (39949.pl): 5′-GGTGCAGGAAGGGTGGGATCCTCTTCTCTCGCTGCTCTGGCCACATC-3 ′ (sequence No .: 49) RNA for the construction of the cDNA library was isolated from human fetal kidney tissue (LIB227). The cDNA library used to isolate the cDNA clones was made by standard methods, eg, using commercially available reagents from Invitrogen, San Diego, CA. The cDNA was primed at the NotI site, ligated at the blunt end of the SalI hemikinase adaptor, cut with NotI, appropriately size-resolved by gel electrophoresis, and cloned into a suitable cloning vector (pRKB or pRKD etc. PRK5B is a precursor of pRK5D that does not have an SfiI site; Holmes et al.
, Science, 253: 1278-1280 (1991)) at unique Xhol and NotI sites. DNA sequencing of the clones isolated as described above revealed that the full-length DNA sequence of PRO846 polypeptide [herein designated as DNA44196-1353] (F
ig9, SEQ ID NO: 9) and the derived protein sequence of PRO846 polypeptide was obtained. The full length nucleotide sequence of DNA44196-1353 is Fig9 (SEQ ID NO:
9). Clone DNA44196-1353 has a single open reading frame with an apparent translational initiation site at nucleotide positions 25-27 and a stop signal at nucleotide positions 1021-1023 (Fig9). The predicted polypeptide precursor is 332 amino acids long, has a calculated molecular weight of approximately 36,143 daltons and an estimated pI of approximately 5.89 (Fig10, SEQ ID NO: 10). Analysis of the full-length PRO846 sequence shown in Figure 10 (SEQ ID NO: 10) demonstrated the presence of various important polypeptide domains shown in Figure 10,
The positions provided for those important polypeptide domains are approximately as described above. Analysis of the full-length PRO846 sequence (Fig10; SEQ ID NO: 10) demonstrated the presence of the following: a signal peptide from about amino acid 1 to about amino acid 17; a transmembrane domain from about amino acid 248 to about amino acid 269; N-glycosylation site at amino acid 100; fibrogen β and γ chain C-terminal domain from about amino acid 104 to about amino acid 114; Ig-like V-type domain from about amino acid 13 to about amino acid 128. Clone DNA44196-1353
Was deposited with the ATCC on May 6, 1998 and was assigned ATCC deposit no.

Example 9 Isolation of a cDNA clone encoding human PRO1760 The EST cluster sequence was identified from the Incyte database by using the signal sequence algorithm described in Example 3 above. This EST cluster sequence is then compared to various expressed sequence tag (EST) databases, including public databases (eg GenBank) and corporate EST DNA databases (LIFESEQ ™, Incyte Pharmaceuticals, Palo Alto, CA). , Identified the existing homology. One or more ESTs were obtained from a prostate cancer library. The homology search is performed by the computer program BLAST or BLAST2 (Altschul et al., Methods in Enzymology 2
66: 460-480 (1996)). BLAS without coding for known proteins
Comparables with a T-score of 70 (sometimes 90) or higher are available in the program "p
hrap ”(Phil Green, University of Washington, Seattle, Washington) to construct a consensus DNA sequence. The consensus sequence obtained therefrom is herein designated DNA58798. Given the sequence homology between the DNA58798 sequence and the sequence contained in Incyte's EST clone 3358745, a clone containing this EST was purchased and c
The DNA insert was obtained and sequenced. Here, the insert was found to encode the full length protein. The sequence of this cDNA insert is shown in Figure 11 (SEQ ID NO: 11) and is herein designated as DNA76532-1702. Clone DNA76532-1702 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 60-62 and ending at the stop codon at nucleotide positions 624-626 (Fig11). The predicted polypeptide precursor is 188 amino acids long (Figure 12, SEQ ID NO: 1).
2). Fig. The full-length PRO1760 protein shown in 12 has approximately 21,042.
It has a calculated molecular weight of Daltons and an estimated pI of about 5.36. Analysis of the full-length PRO1760 sequence shown in Figure 12 (SEQ ID NO: 12) has demonstrated the presence of various important polypeptide domains shown in Figure 12, but the positions given to those important polypeptide domains are About the above. Analysis of the full-length PRO1760 sequence demonstrated the presence of:
A signal peptide from about amino 1 to about amino acid 20; an N-glycosylation site from about amino acid 121 to about amino acid 125 and about amino acid 171 to about amino acid 175; about amino acid 54 to about amino acid 60 and about amino acid 160 to about amino acid 1
66 N-myristoylation sites. Clone DNA76532-1702 was deposited with the ATCC on Nov. 17, 1998, ATCC Deposit No. 203473.
Was granted. The Dayhoff database (version 35) using the WU-BLAST2 sequence alignment analysis method of the full-length sequence shown in FIG. 12 (SEQ ID NO: 12).
.45 SwissProt 35) analysis of the amino acid sequence of PRO1760 and the following Dayh
off array: CELT07F12_2, T22J18_16, ATF1C12_3, APE3_YEAST, P_W22471, SAU569
Sequence identity with 08_1, SCPA_STRPY, ATAC00423817, SAPURCLUS_2 and AF041468_9 was revealed.

Example 10 Cardiac Neonatal Hypertrophy Stimulation (Assay 1) This assay is designed to measure the ability of PRO polypeptides to stimulate neonatal cardiac hypertrophy. It is expected that PRO polypeptides that test positive in this assay will be useful in the therapeutic treatment of various heart failure diseases. Myocytes were obtained from 1-day-old Harlan Sprague Dawley rats. Cells (7.5x10 ⁴ / ml
96 μl, serum <0.1%, freshly isolated) with DMEM / F12 + 4% FCS 96
Add to well plate on day 1. Test PRO polypeptide or growth medium only
Test samples containing (negative control) (20 μL / well) were added directly to the wells on day 1. Then, on the second day, PGF (20) was added so that the final concentration was 10 ⁻⁶ M.
μL / well) is added. Cells were then stained on day 4 and visually scored on day 5, where cells that did not increase in size (compared to the negative control) were scored as 0.0, indicating a small to medium increase in size. The cells shown (compared to the negative control) are scored as 1.0 and the cells showing a large increase in size (compared to the negative control) are scored as 2.0. A positive result in this assay is a score of 1.0 or higher. PRO882 tested positive in this assay, as shown in Table 7 below.

Example 11 Inhibition of Vascular Endothelial Growth Factor (VEGF) Stimulated Endothelial Cell Growth and Proliferation (Assay 9) The ability of various PRO polypeptides to inhibit VEGF stimulated proliferation of endothelial cells was tested. Polypeptides that test positive in this assay serve to inhibit mammalian endothelial cell growth and have a beneficial effect, for example, in inhibiting tumor growth. In particular, bovine adrenal cortex capillary endothelial cells (ACE) (from primary culture, up to 12
-14 passages) were plated at 500 cells / well per 100 microliter in 96-well plates. The assay medium contains low glucose DMEM, 10% calf serum (not fetal calf), 2 mM glutamine, and 1X penicillin / streptomycin / fungizone. Control wells include: (1) no ACE cells added; (2) ACE cells only; (3) ACE cells + 5 ng / ml FGF; (4) ACE cells + 3 ng / ml VEGF; (5) ACE cells + 3 ng / ml VEGF + 1 ng / ml TGT. -Beta; and (6) ACE cells + 3ng / ml VEGF + 5ng / ml LIF. The test sample, poly-His tagged PRO polypeptide (100 microliter volume) was then added to the wells (1%, 0.1% and 0.01% dilutions, respectively). The cell culture was incubated at 37 ° C / 5% CO ₂ for 6-7 days. After incubation, the medium in the wells was aspirated and the cells were washed 1X with PBS. The acid phosphatase reaction mixture (100 microliters, 0.1 M sodium acetate, pH 5.5, 0.1% Triton X-100, 10 mM p-nitrophenyl phosphate) was then added to each well. After incubation for 2 hours at 37 ° C., the reaction was stopped by the addition of 10 microliters of 1N NaOH. Optical density (O
D) was measured at 405 nm in microtiter plates. The activity of the PRO polypeptide was calculated as the percent inhibition of VEGF (3 ng / ml) stimulated growth (as measured by acid phosphatase activity at OD405nm) versus unstimulated cells. TGF-beta is 70 for VEGF-stimulated ACE cell proliferation.
TGF-beta was used as an activity control at 1 ng / ml to block -90%. The results shown in Table 8 below show that PRO in the treatment of cancer, especially in inhibiting tumor angiogenesis.
Shows the utility of the polypeptide. The values shown in Table 8 (relative inhibition) are known to calculate the percent inhibition of VEGF stimulated proliferation by PRO polypeptide relative to unstimulated cells and then inhibit 70-90% of VEGF stimulated cell proliferation. There is one
It is determined by dividing the percent by the percent inhibition obtained with ng / ml TGF-β. The results show that PRO polypeptide stimulates VEGF stimulation of endothelial cell growth.
A case of 30% or more inhibition (30% or more relative inhibition) was considered positive.

Example 12 Induction of c-fos in Endothelial Cells (Assay 34) This assay is designed to determine whether PRO polypeptides show the ability to induce c-fos in endothelial cells. PRO polypeptides tested positive in this assay are useful in the therapeutic treatment of conditions and diseases, their angiogenesis is eg wound healing, and the like (such as agonists of these PRO polypeptides). Is useful for those that include. PR tested positive in this assay
O polypeptides are expected to be useful in the therapeutic treatment of cancerous tumors. Growth medium (50% Ham's F12 w / o GHT: low glucose, and 50% DMEM without glycine: NaHCO ₃ , 1% glutamine, 10 mM HEPES, 10% FB.
Human umbilical vein endothelial cells (HUVEC, Cell Systems) in S, 10 ng / ml bFGF)
Were plated in 96-well macrotiter plates at a cell density of 1x10 ⁴ cells / well. The day after plating, the growth medium was removed and the cells were incubated with 100 μl / well of test sample and control (positive control: growth medium; negative control: 10 mM HEPES, 140 mM NaCl, 4% (w / v) mannitol,
Cells were starved by treatment with pH 6.8). Cells were incubated for 30 minutes at 37 ° C. in 5% CO ₂ . The sample was removed and the first part of the bDNA kit protocol (Chiron Diagnostics, cat. # 6005-037) was followed. here,
Each capitalized reagent / buffer below was available from the kit. Briefly, the amount of TM Lysis Buffer and probe required for the test was calculated based on the information provided by the manufacturer. Appropriate amount of thaw probe was added to TM lysis buffer. Capture hybridization buffer (Capture
Hybridization Buffer) was warmed to room temperature. Set up bDNA strips in metal strip holders and 100 μl of capture hybridization buffer for each b-DN required
Add to well A and incubate for at least 30 minutes. The intracellular test plate was removed from the incubator and the medium was gently removed using a vacuum manifold. Lysis hybridization buffer with 100 μl of probe in each well of the microtiter plate was quickly pipetted into each b-DNA well. The plates were then incubated for 15 minutes at 55 ° C. Removed from incubator, placed the plate in a vortex mixer equipped with a microtiter adapter head and vortexed at setting # 2 for 1 minute. 80 μl of lysate was removed and added to bDNA wells containing capture hybridization buffer and pipetted up and down to mix. The plates were incubated at 53 ° C for at least 16 hours.

The next day, the second part of the bDNA kit protocol was followed. That is, the plate was removed from the incubator, placed on the bench and cooled for 10 minutes. The volume of addition required was calculated based on the information applied by the manufacturer. Amplifier Concentration (Amplifier) at 1: 100 dilution in AL Hybridization Buffer
An amplifier working solution was prepared by preparing Concentrate) (20 fm / μl). The hybridization mixture was washed twice with Wash A. 50μ
l of amplifier working fluid is added to each well, which is
Incubated at 30 ° C. for 30 minutes. The plate was then removed from the incubator and cooled for 10 minutes. Labeled probe working solution was prepared by making a labeled concentrate (40 pmoles / μl) at a dilution of 1: 100 in AL hybridization buffer. After a 10 minute cooling period, the amplifier hybridizing mixture was removed and the plate washed twice with detergent A. 50 μl of labeled probe working solution was added to each well and the wells were incubated for 15 minutes at 53 ° C. After cooling for 10 minutes, the substrate was warmed to room temperature. 3 μl of substrate enhancer was added to each mole of substrate required for the assay, the plate was cooled for 10 minutes, the labeling hybridization mixture was removed, and the plate was washed twice with detergent A and three times with detergent D. Substrate solution with 50 μl enhancer was added to each well. Incubate the plate for 30 minutes at 37 ° C and
The LU was read with a suitable luminometer. The replicates were averaged to determine the coefficient of variation. Negative control (HEP described above
Measurement of fold-increase activity versus ES buffer) value was indicated by chemiluminescence units (RLU). The results are shown in Table 9 below, and samples showing at least twice the negative control values were considered positive. Negative control = 1.00
1.00 RLU with% dilution. Positive control = 8.39 RLU at 1.00% dilution.

[0130]

Example 13 Enhancement of Cardiac Neonatal Hypertrophy Induced by F2a (Assay 37) This assay is designed to measure the ability of PRO polypeptides to stimulate neonatal cardiac hypertrophy. PRO polypeptides tested positive in this assay are expected to be useful in a variety of heart failure diseases and therapeutic treatments. Myocytes were obtained from 1-day-old Harlan Sprague Dawley rats. Cells (7.5x10 ⁴ / ml
96 μl, serum <0.1%, freshly isolated) with DMEM / F12 + 4% FCS 96
Add to well plate on day 1. Test PRO polypeptide (20 μl / well)
The test sample containing the was added directly to the well on day one. Then, two days later, PGF (
20 μl / well) was added at a final concentration of 10 ⁻⁶ M. The cells were then stained on day 4,
Scores were given on the fifth day. The visual score is based on cell size, scoring cells that do not show an increase in size as compared to the negative control with 0.0 and cells showing a small to moderate increase in size as compared to the negative control with 1.0. Cells that showed a large increase in size compared to the negative control were scored as 2.0. A score of 1.0 or higher was considered positive. PBS is not included because the Ca concentration is important for the assay reaction. The plate is DM
Coated with EM / F12 + 4% FCS (200 μl / well). Assay media include: DM
EM / F12 (containing 2.44 gm bicarbonate), 10 μg / ml transferrin, 1 μg / ml insulin, 1 μg / ml aprotinin, 2 mmol / L glutamine, 100 U / ml penicillin G, 100 μg / ml streptomycin. . Protein buffer containing mannitol (4%) gave a positive signal (score 3.5) at 1/10 (0.4%) and 1/100 (0.04%) but not at 1/1000 (0.004%). Therefore, the test sample buffer containing mannitol was not run. The PRO205, PRO882 and PRO887 polypeptides tested positive in this assay.

Example 14 Inhibition of Cardiac Adult Hypertrophy (Assay 42) This assay is designed to measure inhibition of cardiac adult hypertrophy. PRO polypeptides that test positive in this assay find use in the therapeutic treatment of heart diseases associated with cardiac hypertrophy. Ventricular myocytes were newly isolated from adult (250 g) Harlan Sprague Dawley rats,
Cells were plated at 2000 cells / 180 μl well volume. On day 2, test sample containing PRO polypeptide (20 μl) was added. Cells were fixed on day 5 and then stained. Increased ANP message can also be measured by PCR from cells after several hours. Results are based on visual score of cell size: 0 = no inhibition, -1 = small inhibition, -2 = large inhibition. Scores less than 0 were considered positive. The activity reference corresponds to 0.1 mM phenylephine (PE) as a positive control. A score of 2 is considered very positive. The assay medium contains: 100 mM insulin, 0.2% BSA, 5 mM cretin, 2 mM L-carnitine, 5 mM taurine, 1
00 U / ml penicillin G, M199 (modified) -glutamine free, NaHCO ₃ , phenol red suspended in 100 μg / ml streptomycin (CCT medium). 9
Only the inner 60 wells of the 6-well plate were used. Of these, 6 wells were reserved for negative and positive (PE) controls. The PRO878 polypeptide showed a score less than 0 in the above assay.

Example 15 Induction of Endothelial Cell Apoptosis (Assay 73) The ability of PRO polypeptides to induce apoptosis in endothelial cells was tested in human venous umbilical vein endothelial cells (HUVEC, Cell Systems). A positive result in the assay indicates the utility of the PRO polypeptide in the therapeutic treatment of tumors such as vascular disease and is beneficial in inducing endothelial cell apoptosis. The ability of PRO polypeptides to induce apoptosis in endothelial cells was determined by 96
Well type was used to test human umbilical vein endothelial cells (HUVEC, Cell Systems) in 0% serum medium supplemented with 100 ng / ml VEGF. (HUVEC cells should be easily removed from the plate surface and pipetting in all wells should be done as gently as possible.) Media was aspirated and cells were washed once with PBS. 5 ml 1x trypsin was added to the cells in a T-175 flask and the cells were left undisturbed until released from the plate
(About 5-10 minutes). Trypsin treatment was stopped by the addition of 5 ml growth medium. The cells were spun at 1000 rpm for 5 minutes at 4 ° C. The medium was aspirated and the cells were resuspended in 10 ml of 10% serum-supplemented medium (Cell Systems), 1x penicillin / streptomycin. 96-well microtiter plate (Amersham Life Science, cytos
Tar-T scintillating microplates, sterile, tissue culture treated, individually wrapped) were plated in 10% serum (CSG-vehicle, Cell Systems) at a density of 2x10 ⁴ cells per well in a total volume of 100 μl. On day two, test samples containing PRO polypeptide were added in 3 steps of 1%, 0.33% and 0.11% dilutions. Wells without cells were used as a blank and wells with cells were used as a negative control. 1 of 3x stock of staurosporine as a positive control
Three serial dilutions of 50 μl were used. The ability of PRO polypeptides to induce apoptosis was determined by processing 96 wells to detect apoptosis for the detection of annexin V, a member of the calcium and phospholipid binding proteins. 0.2 ml of annexin V-biotin stock solution (100 μg / ml) was added to 4.6 ml of 2x.
Diluted in Ca ²⁺ binding buffer and 2.5% BSA (1:25 dilution). 50 μl of diluted Annexin V-Biotin solution was added to each well (except control) to a final concentration of 1.0 μg / ml. Samples were incubated with Annexin-Biotin for 10-15 minutes prior to direct addition of ³⁵ S-streptavidin. ³⁵ S-streptavidin was diluted in 2 x Ca ²⁺ combined buffer, 2.5% BSA to give a final concentration of 3 x 10 ⁴ c.
Add to all wells until pm / well. The plate is then sealed and 1000
It was centrifuged at rpm for 15 minutes and placed on an orbital shaker for 2 hours. Analysis is 1450 M
It was performed with icrobeta Trilux (Wallac). The results are shown in Table 10 below and represent the percentage amount of counts per minute where the percent above background exceeds the negative control. Those above 30% above background are considered positive. PRO333, PRO364 and PRO879 scored positive results in the above assay.

Example 16 Suppression of Cardiac Neonatal Hypertrophy Induced by LIF and Endothelin-1 (ET-1) (Assay 74) This assay demonstrates that the PRO polypeptides of the invention are LIF and endothelin-1 (
It is designed to determine whether it exhibits the ability of PRO polypeptides to suppress ET-1) -induced neonatal cardiac hypertrophy. Test compounds that show a positive response in this assay are expected to be useful in the therapeutic treatment of heart diseases or disorders characterized or associated with unwanted hypertrophy of the myocardium. Myocytes from 1-day-old Harlan Sprague Dawley rats (180 μl at 7.5 × 10 ⁴ / ml,
Serum <0.1%, freshly isolated) is added to DMEM / F12 + 4% FCS pre-coated 96-well plates on day 1. Then, on day 2, test PRO polypeptide sample or growth medium alone (negative control) is added directly to the wells in a volume of 20 μl. LIF + ET-1 is then added to the wells on day three. Two more days later,
Cells are stained with medium and scored visually the next day. An assay is positive if the myosites treated with PRO polypeptide are visually small on average or low in number compared to untreated myocytes. The PRO238 and PRO1760 polypeptides tested positive in this assay.

Example 17 Endothelial Tube Formation-Stimulation of Bud Formation (Assay 86) This assay is designed to determine whether PRO polypeptides demonstrate the ability to promote endothelial vacuole and lumen formation. P tested positive in this assay
RO polypeptides are useful for therapeutic treatment of disorders where endothelium vacuolation and / or lumen formation are beneficial, including beneficial effects on pinocytosis, ion pumping, vascular permeability and / or junction formation, for example. Is expected to be. HUVEC cells (passage less than 8 from inception) were mixed with type I rat tail collagen (final concentration 2.6 mg / ml) to give a density of 6 × 10 ⁵ cells / ml and 1% F.
50 μl per well of M199 medium supplemented with 1 μM 6-FAM-FITC dye for staining vacuoles in the presence of PRO polypeptide during formation with BS was plated. The cells are incubated at 37 ° C / 5% CO ₂ for 48 hours and at room temperature for 10 minutes 3
． Fix with 7% formalin, wash 5 times with M199 medium, then at 4 ° C. overnight,
Ph-Phalloidin was used for staining, and nuclear staining was performed with 4 μM of DAPI. A positive result in the assay is less than or equal to 2 [1 = all cells are round, 2 = cells are long,
3 = cells form tubes with some connections, 4 = cells form a complex tubular network]. The PRO179 polypeptide tested positive in this assay.

Example 18 Induction of Endothelial Cell Apoptosis (ELISA) (Assay 109) The ability of PRO polypeptides to induce apoptosis in endothelial cells was determined to be 100 n.
Human venous umbilical vein endothelial cells (HUVEC, Cell System) using a 96-well format in 0% serum medium supplemented with g / ml VEGF, 0.1% BSA, 1X penn / strep.
s) tested in. The 96-well plate used was manufactured by Falcon (No. 3072). A 96-well coating was prepared by gelatinizing with 100 μl of 0.2% gelatin in PBS solution for> 30 minutes. After aspirating the gelatin mixture thoroughly, a final concentration of 2x10 ⁴ cells / ml in 10% serum-containing medium-per well
Plated in 100 μl volume. The cells were allowed to grow for 24 hours before adding the test sample containing the PRO polypeptide of interest. All wells received 100 μl in 0% serum medium supplemented with 100 ng / ml VEGF, 0.1% BSA, 1X penn / strep. 1% of test sample containing PRO polypeptide
, 0.33% and 0.11% dilutions in triplicate. Wells without cells were used as blanks and wells with cells only were used as negative controls. As a positive control, 50 μl of a 1: 3 serial dilution of staurosporine in 3x stock solution was used. Cells were incubated for 24-35 hours prior to ELISA. ELISA, Boehringer manual [Boehringer, cell death detection ELISA plus,
Catalog No. 1920685] and used to determine the level of apoptosis preparation solution. Sample preparation: 96 well plate was spun down at 1 krpm for 10 minutes (200
g), the supernatant was removed by high speed inversion and the plate was placed upside down on a paper towel to remove the remaining liquid. To each well, 200 μl of IX lysis buffer was added and incubated for 30 minutes at room temperature without shaking. Plates were spun down at 1 krpm for 10 minutes and 20 μl of lysate (cytoplasmic fraction) transferred to streptavidin coated MTP. 80 μl of immunoreagent mixture was added to 20 μl lysate in each well. The MTP was covered with a sticky foil and incubated for 2 hours at room temperature by placing it on an orbital shaker (200 rpm). After 2 hours, remove the supernatant by aspiration and remove 2 per well.
Wells were rinsed 3 times with 50 μl of 1X incubation buffer (removed by aspiration). Substrate solution was added to each well (100 μl) and placed on an orbital shaker at room temperature for 25
Incubated at 0 rpm until sufficient color was developed for photometric analysis (approximately 10-20
Minute after). A 96 well reader was used to read the plate at 405 nm with a reference wavelength of 492 nm. 100% of the level obtained for PIN32 (control buffer)
Set to. Samples with levels> 130% were considered positive for induction of apoptosis. PRO846 and PRO844 tested positive in this assay.

Example 19 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 includes, for example, identification of gene expression sites, analysis of tissue distribution of transcripts, identification and localization of viral infections, specific mRN
Useful for tracking changes in A synthesis and assisting in chromosome mapping. In situ hybridization was performed by Lu and Gillett, Cell Vision 1: 169-176 (1
Following an optimized version of the protocol 994), using PCR-generated ^{33 P-} labeled riboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized and stained with proteinase K (20 g / ml) for 15 minutes.
Deproteinize at ℃ and hybridize in situ as described by Lu and Gillett, supra. ( ^33- P) UTP-labeled antisense riboprobe was
Produce from R product and hybridize at 55 ° C. overnight. Immerse slides in Kodak NTB2 ^™ Nuclear Track Emulsion and expose for 4 weeks. ³³ P-riboprobe synthesis 6.0 μl (125 mCi) of ³³ P-UTP (Amersham BF 1002, SA <2000 Ci / mmol)
Was speed vacuum dried. The following components were added to each tube containing dry ³³ P-UTP: 2.0 μl 5x transcription buffer 1.0 μl DTT (100 mM) 2.0 μl NTP mixture (2.5 mM: 10 μl each 10 mM GTP, CTP and ATP + 10 μl) H ₂ O
) 1.0 μl UTP (50 μM) 1.0 μl RNAsin 1.0 μl DNA template (1 μg) 1.0 μl H ₂ O 1.0 μl RNA polymerase (T3 = AS, T7 = S, usually for PCR products) Tubes at 37 ° C Incubated for 1 hour, added 1.0 μl of RQ1 DNase, then incubated at 37 ° C. for 15 minutes. 90μl TE (10mM Tris pH7.6 / 1m
M EDTA pH 8.0) was added and the mixture was pipetted onto DE81 paper. The remaining solution is Mi
The crocon-50 ^™ ultrafiltration unit was loaded and spun using program 10 (6 minutes). The filtration unit was converted to a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 μl TE was added. 1 μl of final product was pipetted onto DE81 paper and counted in 6 ml BIOFLUOR II ^™ . The probe was run on a TBE / urea gel. 1-3 μl probe or 5 μl RN
A MrkIII was added to 3 μl of loading buffer. 3 to 95 ° C on a heating block
After heating for minutes, the gel was immediately placed on ice. Flush the gel well,
The sample was loaded and run at 180-250 volts for 45 minutes. The gel was wrapped in Saran wrap and exposed to XAR film with a reinforcing screen in a -70 ° C freezer for 1 hour to overnight.

³³ P-Hybridization A. Frozen Section Pretreatment Slides were removed from the freezer, placed in aluminum trays and thawed at room temperature for 5 minutes. The trays were placed in a 55 ° C. incubator for 5 minutes to reduce condensation.
The slides were fixed for 5 minutes in ice 4% paraformaldehyde in the fume hood, and washed at room temperature for 5 minutes with 0.5xSSC (25ml 20xSSC + 975ml SQ H 2 O). 0.5μ
After deproteinization in g / ml proteinase K for 10 minutes at 37 ° C. (12.5 μl of a 10 mg / ml stock in 250 ml preheated RNase buffer without RNase), sections were washed with 0.5 × SSC for 10 minutes at room temperature. Sections were dehydrated in 70%, 95%, and 100% ethanol for 2 minutes each. B. Pre-treatment of paraffin-embedded sections: Slides were deparaffinized, placed in SQ H ₂ O and rinsed twice in 2 × SSC at room temperature for 5 min each. 20 μg / ml of proteinase K (500 μl of 10 mg / ml in 250 ml RNase buffer without RNase; 37 ° C., 15 minutes)
Human embryos or 8x proteinase K (100 μl in 250 ml RNase buffer,
(37 ° C, 30 minutes) -Deproteinized with formalin tissue. Subsequent rinsing with 0.5 × SSC and dehydration were performed as described above. C. Prehybridization: Slides were placed in plastic boxes lined with Box buffer (4xSSC, 50% formamide) -saturated filter paper. Tissues were coated with 50 μl of hybridization buffer (3.75 g dextran sulfate + 6 ml SQ H ₂ O), vortexed, uncapped and heated in microwave for 2 min. 18.75 ml after cooling on ice
Formamide, 3.75 ml 20 × SSC and 9 ml SQ H ₂ O were added, the tissue was vortexed well and incubated at 42 ° C. for 1-4 hours. D. Hybridization: 1.0 × 10 ⁶ cpm probe per slide and 1.0 μl tRNA (50 mg / ml stock) were heated at 95 ° C. for 3 minutes. Cool slides on ice, 48 per slide
μl of hybridization buffer was added. After vortexing, 50 μl of 33
The P mix was added to 50 μl of prehybrid on slides. Slide at 55 ℃
Incubate overnight. E. Washing: Washing was carried out at room temperature in 2xSSC, EDTA for 2x10 minutes (400 ml of 20xSSC +
16 ml of 0.25 M EDTA, V _f = 4 L) followed by RNase A treatment for 30 minutes at 37 ° C. (500 μl = 20 μg / ml 10 mg / ml in 250 ml RNase buffer). The slides were washed 2x10 minutes with 2xSSC, EDTA at room temperature. Stringent wash conditions are as follows: 55 ° C. for 2 hours, 0.1 × SSC, EDTA (20 ml 20 × SSC + 16 ml
EDTA, V _f = 4 L).

F. Oligonucleotides In situ analysis was performed on two of the DNA sequences disclosed herein. The oligonucleotides used for these analyzes are as follows: (1) DNA47365-1206 (PRO364) (TNF receptor homolog) p1: 5'-GGATTCTAATACGACTCACTATAGGGCAACCCGAGCATGGCACAGCAC-3 '(SEQ ID NO: 50) p2: 5'-CTATGAAATTAACCCTCACTAAAGGGATCTCCCAGCCGCCCCTTCTC-3 '(SEQ ID NO: 51) (2) DNA30868 (PRO205) (Hollistatin homolog) p1: 5'-GGATTCTAATACGACTCACTATAGGGCAGAGACAGGGCAAGCAGAAACT-3' (SEQ ID NO: 52) p2: 5 ' SEQ ID NO: 53) (3) DNA41374 (PRO333) (CD33 homolog) p1: 5'-GGATTCTAATACGACTCACTATAGGGCCTCCACAGAACCTCGCCATCA-3 '(SEQ ID NO: 54) p2: 5'-CTATGAAATTAACCCTCACTAAAGGGATGGGGCAAGACTCACAAGCAG: -3' (sequence)

G. Results In situ analysis was performed on the above two DNA sequences disclosed herein.
The results from these analyzes are as follows: (1) DNA47365-1206 (PRO364) (TNF receptor homologue) Expression was observed in the fetus in the fascia lining the anterior surface of the vertebral bodies. Expression was also found throughout the fetal retina. However, low levels of expression occurred throughout fetal neurons. All other tissues were negative. (2) DNA30868 (PRO205) (follistatin homolog) In fetal tissues, there was expression in the spinal cord, autonomic ganglia, enteric nerves, sacral plexus, peripheral and cranial nerves. All other fetal and adult tissues were negative. Fetal tissues tested (12-16 weeks) include: placenta, umbilical cord, liver, kidney, adrenal gland, thyroid, lung, heart, aorta, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye,
Spinal cord, body wall, pelvis and lower extremities. Adult tissues include: liver, kidneys, adrenal glands, myocardium, aorta, spleen, lymph nodes,
Pancreas, lungs and skin.

(3) DNA41374 (PRO333) (CD33 homolog) This molecule has been shown to be immunostimulatory (enhancing T lymphocyte proliferation in one-way mixed lymphocyte reaction in a T lymphocyte co-stimulation assay). It was The distribution pattern of this molecule was evaluated by a limited tissue screen that included the tissues available at the start of this study. Weak nucleic acid expression was detected in thymic T lymphocytes in many assayed tissues
(Spleen and lymph nodes were not tested). This result was confirmed in subsequent in situ hybridization studies. The results of that study showed similarly low levels of expression in non-human primate thymus and human tonsils of T lymphocyte specific areas. The restricted distribution pattern is suggested to be expressed by cells (such as dendritic cell population) closely related to T lymphocytes such as T lymphocytes and antibody-presenting cells. Expression of areas containing large numbers of T lymphocytes in inflamed human tissues (inflammatory bowel disease and chronic lymphocytic interstitial pneumonia / bronchitis) in the presence of significant lymphoid inflammation and reactive follicle formation Is not detected. Differences in variance (eg, expression in thymus and tonsil lymphatic regions other than those with T lymphocytic inflammation) suggest the following possibilities: Having selective / restricted expression on a subset of T lymphocytes present in thymus and tonsil lymph nodes other than inflamed cells. Immature, non-functioning T lymphocytes are present in both the thymus and tonsils, but are probably not the major population in chronically inflamed tissue. 2. Weak expression in T lymphocytes and the differential detection in T lymphocyte bearing tissues is a reflection of the RNA qualities of those tissue parts, rather than a reflection of the different types of T lymphocyte populations. That means. 3. Expression in thymus and tonsils is not in lymphocytes, but rather in specific cell populations deeply involved in T lymphocytes and not detected in inflamed lungs and intestines.
Such a possibility is a dendritic cell population. In non-human primates, there is a weak divergent expression of thymic lymphocytes. Subsequent studies reported the following results: Inflamed lung: (chronic lymphocytic and granulomatous lung): A weak negative signal was observed in the stroma as compared to the control sense probe. There was weak expression in normal chimpanzee thymus (human thymus not available) and human tonsils. Later expression was prominent in the T lymphocyte region of this structure including the perifollicular zone and in the cortex. No expression was detected in the following human tissues: inflammatory bowel disease (8 patient specimens), chronic inflammatory and normal lung (6 patient specimens), chronic sclerosing nephritis (1 patient specimen), acute inflammatory and sclerosis. Liver (10 patient specimens), normal and psoriatic skin, and peripheral lymph nodes (non-reactive)
.

Example 20 PRO179, PRO238, PRO364, PRO844, PRO846, P
RO1760, PRO205, PRO321, PRO333, PRO840, P
RO877, PRO878, PRO879, PRO882, PRO885 or P
Use of RO887 as a Hybridization Probe The following method was performed using PRO179, PRO238, PRO364, PRO844,
PRO846, PRO1760, PRO205, PRO321, PRO333,
PRO840, PRO877, PRO878, PRO879, PRO882, P
The use of nucleotide sequences encoding RO885 or PRO887 as hybridization probes is described. (Figures 1, 3, 5, 7, 7, 9, 11, 13, 15, 17, 19, 21 respectively)
, 23, 25, 27, 29, and 31, respectively, SEQ ID NOs: 1, 3, 5, 7, 9
, 11, 13, 15, 17, 17, 19, 21, 23, 25, 27, 29, and 31) full length or mature PRO179, PRO238, PRO364, P.
RO844, PRO846, PRO1760, PRO205, PRO321, P
RO333, PRO840, PRO877, PRO878, PRO879, PR
D containing the coding sequence of O882, PRO885 or PRO887 or a fragment thereof
NA is a homologous DN of a human tissue cDNA library or a human tissue genomic library.
A (PRO179, PRO238, PRO364, PRO844, PRO846
, PRO1760, PRO205, PRO321, PRO333, PRO840
, PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887, etc.). Hybridization and washing of filters containing either library DNA is performed under the following high stringency conditions. PRO179, PRO238, PRO3
64, PRO844, PRO846, PRO1760, PRO205, PRO3
21, PRO333, PRO840, PRO877, PRO878, PRO87
Hybridization to a filter of a radiolabeled probe derived from a gene encoding a 9, PRO882, PRO885 or PRO887 polypeptide was carried out using 50% formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM.
It was carried out at 42 ° C. for 20 hours in a solution of sodium phosphate, pH 6.8, 2 × Denhard's solution, and 10% dextran sulfate. Wash the filter with 0.1x SSC and 0.1% SDS
Carried out in aqueous solution at 42 ° C. DNAs with the desired identity to the DNA encoding the full length native sequence can then be identified using standard techniques known in the art.

Example 21 PRO179, PRO238, PRO364, PRO844, P in E. coli
RO846, PRO1760, PRO205, PRO321, PRO333, P
RO840, PRO877, PRO878, PRO879, PRO882, PR
Expression of O885 or PRO887 This example demonstrates recombinant expression of E. coli in a non-glycosylated form of PRO17.
9, PRO238, PRO364, PRO844, PRO846, PRO176
0, PRO205, PRO321, PRO333, PRO840, PRO877
, PRO878, PRO879, PRO882, PRO885 or PRO887.
The preparation of is illustrated. PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 13, 15, respectively)
DNA sequences encoding 7, 19, 21, 23, 25, 27, 29, or 31) were first amplified using selected PCR primers. The primer must have a restriction enzyme site corresponding to the restriction enzyme site of the selected expression vector. Various expression vectors are used. An example of a suitable vector is pBR322 (
It was derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) and contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzymes and dephosphorylated. The PCR amplified sequence is then ligated into the vector. The vector is preferably an antibiotic resistance gene, trp promoter, poly his leader (including the first 6 STII codons, poly his sequence, and enterokinase cleavage site), PRO179, PRO238, PRO364, P.
RO844, PRO846, PRO1760, PRO205, PRO321, P
RO333, PRO840, PRO877, PRO878, PRO879, PR
It contains the O882, PRO885 or PRO887 coding region, the lambda transcription terminator, and the argU gene. The ligation mixture is then used to transform selected E. coli using the method described by Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and then antibiotic resistant clones are selected.
Plasmid DNA is isolated and confirmed by restriction analysis and DNA sequencing. Selected clones can be grown overnight in liquid media such as LB broth supplemented with antibiotics. The overnight medium is subsequently used to seed large-scale medium.
The cells are then grown at optimal density during which the expression promoter operates. After culturing for a few more hours, cells can be harvested by centrifugation. The cell pellet obtained by centrifugation is solubilized using various reagents known in the art, and then solubilized PRO179, PRO238, PRO364, PRO844, PRO84.
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
Alternatively, the PRO887 protein can be purified under conditions that allow tight binding of the polypeptide using a metal chelation column. PRO238, PRO364 and PRO1760 are poly-Hi as described above.
It was successfully expressed in E. coli in the s-tag form.

Example 22 PRO179, PRO238, PRO364, PRO84 in mammalian cells
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
Expression of Nucleic Acids Encoding P., PRO885 or PRO887 This example demonstrates that recombinantly expressed mammalian cells in potentially glycosylated forms of PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
The preparation of PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 is exemplified. The vector pRK5 (see EP 307,247 published on Mar. 15, 1989) was used as an expression vector. In some cases, PRO179, PRO238, PRO364, P
RO844, PRO846, PRO1760, PRO205, PRO321, P
RO333, PRO840, PRO877, PRO878, PRO879, PR
Restriction of O882, PRO885 or PRO887 DNA with pRK5
And PRO17 using the ligation method as described in Sambrook et al., Supra.
9, PRO238, PRO364, PRO844, PRO846, PRO176
0, PRO205, PRO321, PRO333, PRO840, PRO877
, PRO878, PRO879, PRO882, PRO885 or PRO887.
Insert the coding DNA. The resulting vector was pRK5- (PRO179, P
RO238, PRO364, PRO844, PRO846, PRO1760, P
RO205, PRO321, PRO333, PRO840, PRO877, PR
O878, PRO879, PRO882, PRO885 or PRO887 encoding DNA). In one embodiment, the host cell selected can be 293 cells. Human 293 cells (ATCC CCL 1573) were grown to confluence in tissue culture plates in media such as DMEM supplemented with fetal calf serum and optionally nutrients or antibiotics. About 10 μg of pRK5- (PRO179, PRO238, PR
O364, PRO844, PRO846, PRO1760, PRO205, PR
O321, PRO333, PRO840, PRO877, PRO878, PRO
879, PRO882, PRO885 or PRO887 encoding DNA) about 1 μg
DNA encoding the VA RNA gene of E. coli [Thimmappaya et al., Cell, 31: 543 (1982
))], And mixed with 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M C.
It was dissolved in aCl2. To this mixture, drop-wise, 500 μl of 50 mM HEPES (pH
7.35), 280 mM NaCl, 1.5 mM NaPO ₄ was added to form a precipitate at 25 ° C. for 10 minutes. The precipitate was suspended, added to 293 cells, and fixed at 37 ° C. for about 4 hours. The culture medium was aspirated and 2 ml of 20% glycerol in PBS was added for 30 seconds. Two
The 93 cells were then washed with serum free medium, fresh medium was added and the cells were incubated for about 5 days. Approximately 24 hours after transfection, remove the culture medium and culture medium (only) or 200 μCi /
The medium was replaced with culture medium containing ³⁵ S-cysteine and 200 μCi / ml of ³⁵ S-methionine. After a 12 hour incubation, the conditioned medium was harvested, concentrated on a spin filter and loaded on a 15% SDS gel. The treated gel is dried and PRO1
79, PRO238, PRO364, PRO844, PRO846, PRO17
60, PRO205, PRO321, PRO333, PRO840, PRO87
7, PRO878, PRO879, PRO882, PRO885 or PRO88
The film was exposed for a selected time that revealed the presence of the 7 polypeptide. The medium containing the transformed cells was subjected to further incubation (in serum-free medium) and the medium was tested in the selected bioassay.

Alternative techniques include PRO179, PRO238, PRO364, PRO.
844, PRO846, PRO1760, PRO205, PRO321, PRO
333, PRO840, PRO877, PRO878, PRO879, PRO8
82, PRO885 or PRO887 encoding genes are described in Somparyac et al., Pro
c. Natl. Acad. Sci., 12: 7575 (1981), the dextran sulfate method may be used to transiently introduce into 293 cells. 293 cells were grown to maximum density in spinner flasks and 700 μg of pRK5- (PRO179, PRO238, PR
O364, PRO844, PRO846, PRO1760, PRO205, PR
O321, PRO333, PRO840, PRO877, PRO878, PRO
879, PRO882, PRO885 or PRO887 encoding DNA) is added. The cells were first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate was incubated on the cell pellet for 4 hours. Cells were treated with 20% glycerol for 90 seconds, washed with tissue culture medium and reintroduced into spinner flasks containing tissue culture medium, 5 μg / ml bovine insulin and 0.1 μg / ml bovine transferrin. After about 4 days, the conditioned medium was centrifuged and filtered,
Cells and cell debris were removed. Then PRO179, PRO238, PRO364
, PRO844, PRO846, PRO1760, PRO205, PRO321
, PRO333, PRO840, PRO877, PRO878, PRO879,
The sample containing the expressed gene encoding PRO882, PRO885 or PRO887 was concentrated and purified by a selected method such as dialysis or column chromatography. In other embodiments, PRO179, PRO238, PRO364, PRO84.
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, PRO885 or PRO887 encoding genes can be expressed in CHO cells. pRK5- (PRO179, PRO238, PRO364, PRO
844, PRO846, PRO1760, PRO205, PRO321, PRO
333, PRO840, PRO877, PRO878, PRO879, PRO8
82, PRO885 or PRO887 encoding DNA) nucleic acid is CaPO ₄ or D
CHO cells can be transfected with known reagents such as EAE-dextran. As described above, the cell culture medium can be incubated and the medium replaced with culture medium (alone) or medium containing a radiolabel such as ³⁵ S-methionine. PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
After identifying the presence of the PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptide, the culture medium may be replaced with serum free medium. Preferably, the medium is incubated for about 6 days and then the conditioned medium is collected. Then, the expressed PRO179, PRO238, PRO364, PR
O844, PRO846, PRO1760, PRO205, PRO321, PR
O333, PRO840, PRO877, PRO878, PRO879, PRO
Media containing 882, PRO885 or PRO887 can be concentrated and purified by the method of choice.

In addition, PRO179, PRO238, PRO364, PRO844, PRO8
46, PRO1760, PRO205, PRO321, PRO333, PRO8
40, PRO877, PRO878, PRO879, PRO882, PRO88
An epitope tag gene encoding the 5 or PRO887 polypeptide may be expressed in host CHO cells. PRO179, PRO238, PRO
364, PRO844, PRO846, PRO1760, PRO205, PRO
321, PRO333, PRO840, PRO877, PRO878, PRO8
The gene encoding 79, PRO882, PRO885 or PRO887 may be subcloned from the pRK5 vector. Subclone insert is PC
Upon R amplification, it can be fused in frame with a selected epitope tag such as a poly-his tag into a baculovirus expression vector. Poly-his tag PRO17
9, PRO238, PRO364, PRO844, PRO846, PRO176
0, PRO205, PRO321, PRO333, PRO840, PRO877
, PRO878, PRO879, PRO882, PRO885 or PRO887.
The gene insert encoding DHFR was then cloned into DHFR for selection of stable clones.
Can be subcloned into an SV40-inducible vector containing a selectable marker such as Finally, CHO cells were transfected with the SV40 inducible vector (as above). Labeling may be performed as described above to confirm expression. Expressed poly-hi
s tag PRO179, PRO238, PRO364, PRO844, PRO84
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
Alternatively, the medium containing the expressed gene encoding PRO887 can then be concentrated and purified by a selected method such as Ni ²⁺ -chelate affinity chromatography. PRO179, PRO364, PRO840, PRO844, PRO846 and PRO205 were stably expressed in CHO cells by the method described above. Furthermore, PRO364 and PRO846 were stably expressed in CHO cells by the transient method.

Example 23 PRO179, PRO238, PRO364, PRO844, PRO in yeast
846, PRO1760, PRO205, PRO321, PRO333, PRO
840, PRO877, PRO878, PRO879, PRO882, PRO8
Expression of Nucleic Acid Encoding 85 or PRO887 The following method is used to determine PRO179, PRO238, PRO364, P in yeast.
RO844, PRO846, PRO1760, PRO205, PRO321, P
RO333, PRO840, PRO877, PRO878, PRO879, PR
Recombinant expression of the gene encoding O882, PRO885 or PRO887 is described. First, PRO179, PRO23 from the ADH2 / GAPDH promoter
8, PRO364, PRO844, PRO846, PRO1760, PRO20
5, PRO321, PRO333, PRO840, PRO877, PRO878
, A yeast expression vector for intracellular production or secretion of PRO879, PRO882, PRO885 or PRO887. PRO179, PRO238,
PRO364, PRO844, PRO846, PRO1760, PRO205,
PRO321, PRO333, PRO840, PRO877, PRO878, P
DNA encoding RO879, PRO882, PRO885 or PRO887
And promoter into the appropriate restriction enzyme sites of the selected plasmid
179, PRO238, PRO364, PRO844, PRO846, PRO1
760, PRO205, PRO321, PRO333, PRO840, PRO8
77, PRO878, PRO879, PRO882, PRO885 or PRO8
Directs intracellular expression of the gene encoding 87. For secretion, PRO179
, PRO238, PRO364, PRO844, PRO846, PRO1760
, PRO205, PRO321, PRO333, PRO840, PRO877,
A plasmid encoding DNA encoding PRO878, PRO879, PRO882, PRO885, or PRO887 was added to the ADH2 / GAPDH promoter, native PRO179, PRO238, PRO364, PRO844, PRO84.
6, PRO1760, PRO205, PRO321, PRO333, PRO84
0, PRO877, PRO878, PRO879, PRO882, PRO885
Or a DNA encoding a PRO887 signal polypeptide or other mammalian signal peptide, or eg a yeast alpha factor secretion signal / leader sequence,
And (if necessary) PRO179, PRO238, PRO364, PRO844
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
It can be cloned with a linker sequence for the expression of PRO885 or PRO887. Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatant can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining the gel with Coomassie blue stain. Subsequently, recombinant PRO179, PRO238, PRO364, PRO844, P
RO846, PRO1760, PRO205, PRO321, PRO333, P
RO840, PRO877, PRO878, PRO879, PRO882, PR
O885 or PRO887 can be isolated and purified by removing yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. PRO179, PRO238, PRO364, PRO8
44, PRO846, PRO1760, PRO205, PRO321, PRO3
33, PRO840, PRO877, PRO878, PRO879, PRO88
The concentrate containing 2, PRO885 or PRO887 may be further purified using a selected column chromatography resin.

Example 24 PRO179, PRO238, PRO364 in Baculovirus-Infected Insect Cells
, PRO844, PRO846, PRO1760, PRO205, PRO321
, PRO333, PRO840, PRO877, PRO878, PRO879,
Expression of Nucleic Acid Encoding PRO882, PRO885 or PRO887 The following method describes recombinant expression in baculovirus infected insect cells. PRO179, PRO238, PRO364, PRO844, PRO846,
PRO1760, PRO205, PRO321, PRO333, PRO840,
The sequence encoding PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 was fused upstream of an epitope tag contained in a baculovirus expression vector. Such an epitope tag is poly-His
Include tags and immunoglobulin tags (such as the Fc region of IgG). pVL1393
Various plasmids can be used, including plasmids derived from commercially available plasmids such as (Navogen). Briefly, PRO179, PRO23
8, PRO364, PRO844, PRO846, PRO1760, PRO20
5, PRO321, PRO333, PRO840, PRO877, PRO878
, PRO879, PRO882, PRO885 or PRO887 or PRO17
9, PRO238, PRO364, PRO844, PRO846, PRO176
0, PRO205, PRO321, PRO333, PRO840, PRO877
, PRO878, PRO879, PRO882, PRO885 or PRO887.
A predetermined portion of [such as a sequence encoding the extracellular domain of a transmembrane protein] is 5
Amplified by PCR with primers complementary to the'and 3'regions. The 5'primer may include flanking (selected) restriction enzyme sites. The product is then digested with the selected restriction enzymes and subcloned into the expression vector. Recombinant baculovirus is the above plasmid and BaculoGold ™ virus D
NA (Pharmingen) with Spodoptera frugiperda (“Sf9”) cells (ATCC CRL
1711) with lipofectin (commercially available from GIBCO-BRL). After incubation at 28 ° C for 4-5 days, released virus was collected and used for further amplification. Viral infection and protein expression are
illey et al., Baculovirus expression vectors: A laboratory Manual, Oxford: Ox
Performed as described in ford University Press (1994). Then, the expressed poly-His tag- [PRO179, PRO238, PRO
364, PRO844, PRO846, PRO1760, PRO205, PRO
321, PRO333, PRO840, PRO877, PRO878, PRO8
79, PRO882, PRO885 or PRO887] is purified, for example, by Ni ²⁺ -chelate affinity chromatography as follows. Extracts were prepared from virus infected recombinant Sf9 cells as described in Rupert et al., Nature, 362: 175-179 (1993). Briefly, Sf9 cells were washed and sonicated buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCl ₂ ; 0.1 mM
EDTA; 10% glycerol; 0.1% NP-40; 0.4M KCl) and sonicated twice on ice for 20 seconds. The sonicate was clarified by centrifugation and the supernatant was loaded with loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.
It was diluted 50 times with 8) and filtered through a 0.45 μm filter. A Ni ²⁺ -NTA agarose column (commercially available from Qiagen) was prepared with a total volume of 5 ml, washed with 25 ml water and equilibrated with 25 ml loading buffer. The filtered cell extract was loaded onto the column at 0.5 ml / min. The column was washed with loading buffer to the baseline of A280, which is the point where fraction collection begins. The column is then loaded with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, non-specifically eluting the bound protein).
It was washed with pH 6.0). After reaching the A280 baseline again, the column was developed with a 0 to 500 mM imidazole gradient in secondary wash buffer. Fractions of 1 ml were collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni ²⁺ -NTA conjugated to alkaline phosphatase (Qiagen). Eluted His1
0-tags PRO179, PRO238, PRO364, PRO844, PRO8
46, PRO1760, PRO205, PRO321, PRO333, PRO8
40, PRO877, PRO878, PRO879, PRO882, PRO88
Fractions containing 5 or PRO887 were pooled and dialyzed against loading buffer.

Alternatively, IgG tag (or Fc tag) -PRO179, PRO238, PR
O364, PRO844, PRO846, PRO1760, PRO205, PR
O321, PRO333, PRO840, PRO877, PRO878, PRO
Purification of 879, PRO882, PRO885 or PRO887 can be carried out using known chromatographic techniques including, for example, protein A or protein G column chromatography. Expression was actually on the 0.5-2L scale, but easily larger (eg 8L)
Scale up to preparation. Protein is an IgG product (immunoadhesin)
Where the protein extracellular region is fused to an IgG1 constant region sequence containing hinge, CH2 and CH3 domains and / or poly-His tagged forms. Following PCR amplification, the corresponding coding sequences were baculovirus expression vector (pb.PH.IgG for IgG fusion and pb.PH.His.c for poly-His tag).
The vector and Baculogold (registered trademark) baculovirus DNA (Pharmingen) were subcloned into Spodoptera frugiperda (Spodoptera frugi).
perda) (“Sf9”) cells (ATCC CRL 1711) and lipofectin (Gibco BRL)
Was used to co-transfect. pb.PH.IgG and PH.His.c are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), and His or Fc.
It has a polylinker region modified to include the tag sequence. Cells with 10% FB
The cells were grown in Hink's TNM-FM medium supplemented with S (Hyclone). 28 ° C for cells
And incubated for 5 days. The supernatant was collected, followed by Hink with 10% FBS
Used for initial viral amplification with an infection efficiency (MOI) of about 10 by infection of Sf9 cells in TNM-FH medium. Cells were incubated at 28 ° C for 3 days. The supernatant was collected and the expression of the construct in the baculovirus expression vector was expressed in 25 ml of Ni2 ⁺ -NTA beads (QIAGEN) for histidine tag protein or protein A Sepharose CL-4B beads for IgG tag protein in 1 ml of supernatant. (Pharmacia
), Followed by SDS-PAGE analysis comparing known concentrations of protein standards by Coomassie blue staining. The first amplified viral supernatant was used to infect Sf9 cells grown in ESF-921 medium (Expression System LLC) with a spinner medium (500 ml) at an MOI of about 0.1. Cells were incubated at 28 ° C for 3 days. The supernatant was collected and filtered.
Batch binding and SDS-PAGE were repeated as needed until expression of spinner medium was confirmed. The conditioned medium (0.5-3 L) from the transfected cells is centrifuged to remove the cells and 0.2
Recovered by filtration through a 2 micron filter. For poly-His tag constructs, the protein containing sequence was purified using a Ni ²⁺ -NTA column (Qiagen). Before purification, imidazole was added to the conditioned medium to a concentration of 5 mM. Conditioned medium containing 20 mM Hepes, p containing 0.3 M NaCl and 5 mM imidazole.
A 6 ml Ni ²⁺ -NTA column equilibrated with H7.4 buffer was pumped at 4 ° C. at a flow rate of 4-5 ml / min. After loading, the column was further washed with equilibration buffer and the protein was eluted with equilibration buffer containing 0.25M imidazole. The highly purified protein was subsequently desalted with 25 ml G25 Superfine (Pharmacia) in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8 and at -80 ° C. Stored in.

The immunoadhesin (Fc-containing) construct of the protein was purified from conditioned medium as follows. Conditioned medium was loaded onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer and then eluted with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions in a tube containing 275 ml 1 M Tris buffer, pH 9. The highly purified protein is then
-Desalted in storage buffer as described above for His-tagged proteins. Protein homogeneity was determined by SDS polyacrylamide gel (PEG) electrophoresis and Edman
It can be evaluated by N-terminal amino acid sequencing by (Edman) degradation. PRO205, PRO321, PRO840, PRO846, PRO885 and PRO887 were expressed in Sf9 insect cells infected with baculovirus by the method described above. Alternatively, the modified baculovirus method may be used for uptake of high5 cells. In this method, the DNA encoding the desired sequence may be amplified in a suitable system such as Pfu (Stratagene), or may be fused (5'-) upstream of the epitope tag contained in the baculovirus expression vector. Good. Such an epitope tag is
It includes a poly-His tag and an immunoglobulin tag (such as the Fc region of IgG). A variety of plasmids can be used, including plasmids derived from commercially available plasmids such as pIE1-1 (Novagen). The pIE1-1 and pIE1-2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus ie1 promoter in stably transformed insect cells. This plasmid differs only in the orientation of multiple cloning sites, and all promoter sequences known to be important for ie1 mediated gene expression in uninfected insect cells as well as hr5.
Contains enhancer components. pIE1-1 and pIE1-2 contain the IE translation initiation site and can be used to produce fusion proteins. Briefly, the desired sequence or a 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 incorporate flanking (selected) restriction enzyme sites. The product is then digested with selected restriction enzymes and subcloned into an expression vector. For example, pI
The derivative of El-1 is the Fc region of human IgG (pb.PH.IgG) or 8 histidine (pb.P.
H.His) tag downstream (3'-) can be included. Preferably, the vector construct is sequenced for confirmation.

High-5 cells were grown to 50% confluence under conditions of 27 ° C., no CO _2, no pen / strep. For each 150 mm plate, pIE-based vector containing 30 μg of PRO polypeptide was added to 1 ml of Ex-cell medium (vehicle: Ex-cell 401 + 1/100).
L-Glu JRH Biosciences # 14401-78P (note: this medium is light sensitive)) and in a separate tube, 100 μl of Cellfectin (CellFECTIN (Gibco BRL # 10362-
010) (mixed by vortex)) was mixed with 1 ml Ex-cell medium. The two solutions were mixed and incubated at room temperature for 15 minutes. Add 8 ml of Ex-Cell Medium to 2 ml of D
Layers were formed on high-5 cells that were added to the NA / Cellfectin mixture and washed once with Ex-cell medium. The plates were then incubated at room temperature in the dark. The DNA / CellFECTIN mixture was then aspirated and the cells were washed once with Ex-cells to remove excess Cellfectin. Add 30 ml of fresh Ex-Cell Medium and add 2 cells.
Incubated at 8 ° C for 3 days. The supernatant was collected and the expression of the PRO polypeptide in the baculovirus infection vector was determined using 1 mL of supernatant of 25 mL of Ni ²⁺ -NTA beads (QIAGEN) for histidine tag protein or Protein A Sepharose CL for IgG tag protein. -4B beads (Pharmacia) by batch binding followed by SDS-PAGE comparing known concentrations of protein standards by Coomassie blue staining
It was measured by analysis. The conditioned medium (0.5-3 L) from the transfected cells is centrifuged to remove the cells and 0.2
Recovered by filtration through a 2 micron filter. For poly-His tag constructs, protein constructs were purified using a Ni ²⁺ -NTA column (Qiagen). Before purification, imidazole was added to the conditioned medium to a concentration of 5 mM. Conditioned medium is 20 mM Hepes, pH 7.4 containing 0.3 M NaCl and 5 mM imidazole.
A 6 ml Ni ²⁺ -NTA column equilibrated with buffer was pumped at 48 ° C. at a flow rate of 4-5 ml / min. After loading, the column was further washed with equilibration buffer and the protein was eluted with equilibration buffer containing 0.25M imidazole. The highly purified protein was subsequently desalted with 25 ml G25 Superfine (Pharmacia) in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8 and at -80 ° C. Stored in. The immunoadhesin (Fc-containing) construct of the protein was purified from conditioned medium as follows. Conditioned medium was loaded onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer and then eluted with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions in a tube containing 275 ml 1 M Tris buffer, pH 9. The highly purified protein is then
-Desalted in storage buffer as described above for His-tagged proteins. PRO
Polypeptide homogeneity can be assessed by SDS-polyacrylamide gel and N-terminal amino acid sequencing by Edman degradation and other analytical techniques as desired or required. PRO179, PRO205, PRO321, PRO333, PRO364,
PRO844, PRO846, PRO877, PRO879, PRO882, P
RO885 or PRO1760 was expressed in high5 cells by the method described above.

Example 25 PRO179, PRO238, PRO364, PRO844, PRO846, P
RO1760, PRO205, PRO321, PRO333, PRO840, P
RO877, PRO878, PRO879, PRO882, PRO885 or P
Preparation of Antibodies that Bind RO887 This example describes PRO179, PRO238, PRO364, PRO844,
PRO846, PRO1760, PRO205, PRO321, PRO333,
PRO840, PRO877, PRO878, PRO879, PRO882, P
The preparation of a monoclonal antibody capable of specifically binding to RO885 or PRO887 is exemplified. Techniques for the production of monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be used are PRO179, PR
O238, PRO364, PRO844, PRO846, PRO1760, PR
O205, PRO321, PRO333, PRO840, PRO877, PRO
Purified PRO179, PRO238, PRO364, PRO844, PRO846, including 878, PRO879, PRO882, PRO885 or PRO887.
PRO1760, PRO205, PRO321, PRO333, PRO840,
PRO877, PRO878, PRO879, PRO882, PRO885 or PRO887 fusion protein, recombinant PRO179, PRO238 on the cell surface,
PRO364, PRO844, PRO846, PRO1760, PRO205,
PRO321, PRO333, PRO840, PRO877, PRO878, P
Includes cells expressing the gene encoding RO879, PRO882, PRO885 or PRO887. The choice of immunogen can be made by one of ordinary skill in the art without undue experimentation. Mice such as Balb / c were emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally at 1-100 micrograms PRO179, PRO238, P.
RO364, PRO844, PRO846, PRO1760, PRO205, P
RO321, PRO333, PRO840, PRO877, PRO878, PR
Immunize with O879, PRO882, PRO885 or PRO887 immunogen.
Alternatively, the immunogen is replaced with MPL-TDM adjuvant (Ribi Immunochemical Resear
h, Hamilton, MT) and injected into the hind footpads of animals. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. The mice are then boosted with additional immunization injections for several weeks. Anti-PRO179, Anti-PRO238, Anti-PRO364, Anti-PRO844
, Anti-PRO846, anti-PRO1760, anti-PRO205, anti-PRO321,
Anti-PRO333, Anti-PRO840, Anti-PRO877, Anti-PRO878, Anti-
Serum samples from retroorbital bleeds may be taken periodically from mice for testing in an ELISA assay for detection of PRO879, anti-PRO882, anti-PRO885 or anti-PRO887 antibodies.

After a suitable antibody titer has been detected, animals that are “positive” for the antibody are given PRO17
9, PRO238, PRO364, PRO844, PRO846, PRO176
0, PRO205, PRO321, PRO333, PRO840, PRO877
, PRO878, PRO879, PRO882, PRO885 or PRO887.
Can be the last infusion of an intravenous injection of After 3 to 4 days, the mice were sacrificed and spleen cells were removed. The spleen cells were then (using 35% polyethylene glycol) P3X63AgU.P3 available from ACTT under the number CRL1597. 1
And selected mouse myeloma cell lines. Hybridoma cells were generated by the fusion and then plated on 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit the growth of unfused cells, myeloma hybrids, and spleen cell hybrids. The hybridoma cells are PRO179, PRO238, PRO364, PRO.
844, PRO846, PRO1760, PRO205, PRO321, PRO
333, PRO840, PRO877, PRO878, PRO879, PRO8
82, PRO885 or PRO887 screened by ELISA for reactivity. Desired PRO179, PRO238, PRO364, PR
O844, PRO846, PRO1760, PRO205, PRO321, PR
O333, PRO840, PRO877, PRO878, PRO879, PRO
The determination of "positive" hybridoma cells secreting a monoclonal antibody against 882, PRO885 or PRO887 is within the skill of the art. Positive hybridoma cells were injected intraperitoneally into syngeneic Balb / c mice,
Anti-PRO179, Anti-PRO238, Anti-PRO364, Anti-PRO844, Anti-
PRO846, anti-PRO1760, anti-PRO205, anti-PRO321, anti-P
RO333, anti-PRO840, anti-PRO877, anti-PRO878, anti-PRO
Ascites fluid containing 879, anti-PRO882, anti-PRO885 or anti-PRO887 monoclonal antibodies is generated. Alternatively, hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibody produced in the ascites can be performed using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the affinity of the antibody for protein A or protein G can be used.

Deposit of Materials The following cell lines have been deposited with American Type Culture Collection, 10801 University Boulevard, Manassas, VA20110-2209, USA (ATCC): Material ATCC Deposit No. Deposit Date DNA16451-1388 209776 1998 4 14th March DNA35600-1162 209370 16th October 1997 DNA47365-1206 209436 7th November 1997 DNA59838-1462 209976 16th June 1998 DNA44196-1353 209847 6th May 1998 DNA76532-1702 203473 11th 1998 17th March DNA34433 209719 31st March 1998 DNA53987 209858 12th May 1998 . This guarantees that the viable culture of the deposit will be maintained for 30 years from the date of deposit.
Deposits may be obtained from the ATCC in accordance with the terms of the Budapest Treaty and in accordance with the agreement between Genentech and ATCC, whichever is first, at the time of issuance of the relevant U.S. patent or at any time. At the time of publication of a U.S. or foreign patent application, it is guaranteed that the progeny of the deposited culture will be permanently and unrestrictedly available, and will be subject to United States Patent Law Section 122 and the Patent Office Commissioner Regulations accordingly (especially reference numeral 886OG638).
It guarantees that the descendants will be available to those who have been determined by the US Patent Office Commissioner to have rights in accordance with 7 CFR Article 1.14). The assignee of the present application agrees that if the deposited culture dies or is lost or destroyed if cultured under the proper conditions, the material will be replaced immediately with the same at the time of notification. To do. The availability of deposited material is not to be construed as a license to practice the invention in breach of any rights granted under the authority of any government pursuant to patent law. The written specification given above is considered sufficient to enable one skilled in the art to practice the invention. The deposited embodiment is intended as an illustration of one of the aspects of the invention and any functionally equivalent construct is within the scope of the invention, so that the deposited deposits are within the scope of the invention. It is not limited. The deposit of matter herein is not an admission that the written description contained therein is sufficient to enable the practice of any aspect of the invention, including its best mode. It is not to be construed as limiting the scope of the claims to the particular illustrations presented. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of the native sequence PRO179 cDNA (SEQ ID NO: 1), SEQ ID NO: 1 is the clone herein designated “DNA16451-1388”.

FIG. 2 shows the amino acid sequence (SEQ ID NO: 2) derived from the coding sequence of SEQ ID NO: 1 shown in FIG.

FIG. 3 is a diagram showing the nucleotide sequence of the native sequence PRO238 cDNA (SEQ ID NO: 3), wherein SEQ ID NO: 3 is herein referred to as “DNA35600-116”.
2 ”is a clone named.

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

FIG. 5 shows the nucleotide sequence of the native sequence PRO364 cDNA (SEQ ID NO: 5), wherein SEQ ID NO: 5 is herein “DNA47365-120”.
6 ”is a clone named.

FIG. 6 shows the amino acid sequence (SEQ ID NO: 6) derived from the coding sequence of SEQ ID NO: 5 shown in FIG.

FIG. 7 shows the nucleotide sequence of native sequence PRO844 cDNA (SEQ ID NO: 7), wherein SEQ ID NO: 7 is herein referred to as “DNA59838-146.
2 ”is a clone named.

FIG. 8 shows the amino acid sequence (SEQ ID NO: 8) derived from the coding sequence of SEQ ID NO: 7 shown in FIG.

FIG. 9 shows the nucleotide sequence (SEQ ID NO: 9) of the native sequence PRO846 cDNA, wherein SEQ ID NO: 9 is herein “DNA44196-135”.
3 ”is a clone.

FIG. 10 shows the amino acid sequence (SEQ ID NO: 10) derived from the coding sequence of SEQ ID NO: 9 shown in FIG.

FIG. 11 shows the nucleotide sequence of native sequence PRO1760 cDNA (SEQ ID NO: 11), wherein SEQ ID NO: 11 is herein “DNA76532”.
-1702 ”.

FIG. 12 shows the amino acid sequence (SEQ ID NO: 12) derived from the coding sequence of SEQ ID NO: 11 shown in FIG. 11.

FIG. 13 shows the nucleotide sequence of native sequence PRO205 cDNA (SEQ ID NO: 13), wherein SEQ ID NO: 13 is “DNA30868”.
Is a clone named.

FIG. 14 shows the amino acid sequence (SEQ ID NO: 14) derived from the coding sequence of SEQ ID NO: 13 shown in FIG. 13.

FIG. 15 shows the nucleotide sequence (SEQ ID NO: 15) of native sequence PRO321 cDNA, wherein SEQ ID NO: 15 is “DNA34433” herein.
Is a clone named.

FIG. 16 shows the amino acid sequence (SEQ ID NO: 16) derived from the coding sequence of SEQ ID NO: 15 shown in FIG.

FIG. 17 shows the nucleotide sequence of native sequence PRO333 cDNA (SEQ ID NO: 17), wherein SEQ ID NO: 17 is herein “DNA41374”.
Is a clone named.

FIG. 18 shows the amino acid sequence (SEQ ID NO: 18) derived from the coding sequence of SEQ ID NO: 17 shown in FIG.

FIG. 19 shows the nucleotide sequence (SEQ ID NO: 19) of native sequence PRO840 cDNA, wherein SEQ ID NO: 19 is herein “DNA53987”.
Is a clone named.

FIG. 20 shows the amino acid sequence (SEQ ID NO: 20) derived from the coding sequence of SEQ ID NO: 19 shown in FIG.

FIG. 21 shows the nucleotide sequence of native sequence PRO877 cDNA (SEQ ID NO: 21), wherein SEQ ID NO: 21 is “DNA58120”.
Is a clone named.

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

FIG. 23 shows the nucleotide sequence of native sequence PRO878 cDNA (SEQ ID NO: 23), wherein SEQ ID NO: 23 is “DNA58121”.
Is a clone named.

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

FIG. 25 shows the nucleotide sequence of native sequence PRO879 cDNA (SEQ ID NO: 25), wherein SEQ ID NO: 25 is herein “DNA58122”.
Is a clone named.

FIG. 26 shows the amino acid sequence (SEQ ID NO: 26) derived from the coding sequence of SEQ ID NO: 25 shown in FIG. 25.

FIG. 27 shows the nucleotide sequence (SEQ ID NO: 27) of native sequence PRO882 cDNA, wherein SEQ ID NO: 27 is herein “DNA58125”.
Is a clone named.

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

FIG. 29 shows the nucleotide sequence of native sequence PRO885 cDNA (SEQ ID NO: 29), wherein SEQ ID NO: 29 is “DNA58128”.
Is a clone named.

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

FIG. 31 shows the nucleotide sequence of native sequence PRO887 cDNA (SEQ ID NO: 31), wherein SEQ ID NO: 31 is herein “DNA58130”.
Is a clone named.

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

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 9/04 4C084 A61P 9/04 9/10 4C085 9/10 17/02 4C086 17/02 27/02 4H045 27/02 C07K 14/47 C07K 14/47 16/18 16/18 19/00 19/00 C12N 1/19 C12N 1/19 1/21 1/21 7/00 5/10 C12P 21 / 02 C 7/00 C12Q 1/02 15/09 ZNA G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/02 33/53 D G01N 33/15 M 33/50 C12P 21/08 33/53 A61K 37/02 C12N 15/00 ZNAA // C12P 21/08 5/00 B (31) Priority claim number US9912252 (32) Priority date June 2, 1999 (1999.6.2) (33) Priority right Claiming country United States (US) (31) Priority claim number 60/144 758 (32) Priority date July 20, 1999 (July 20, 1999) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 145,698 (32) Priority date 1999 July 26, 1999 (July 26, 1999) (33) Priority claiming country United States (US) (31) Priority claim number US9920111 (32) Priority date September 1, 1999 (September 1, 1999) (33) Priority claiming United States (US) (31) Priority claiming number US9921090 (32) Priority date September 15, 1999 (1999.15.1999) (33) Priority claiming United States (US) ( 31) Priority claim number US9928313 (32) Priority date November 30, 1999 (November 30, 1999) (33) Country of priority claim United States (US) (31) Priority claim number US9928409 (32) Priority date November 30, 1999 (November 30, 1999) (33) Priority claiming country United States (US) (31) Priority claim number US9928565 (32) Priority date December 2, 1999 (December 1999. 2) (33) Priority claiming countries United States (US) (31) Priority claim number US0000219 (32) Priority date January 5, 2000 (1.5.2000) (33) Priority claim country United States (US) (31) Priority claim number US0004341 (32) Priority date 2000 February 18, 2000 (February 18, 2000) (33) Priority claiming country United States (US) (31) Priority claim number US0004342 (32) Priority date February 18, 2000 (February 18, 2000) (33) Priority claiming United States (US) (31) Priority claiming number US0004414 (32) Priority date February 22, 2000 (February 22, 2000) (33) Priority claiming United States (US) ( 81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, Z, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Baker, Kevin, Pea. United States Maryland 20878, Darnestown, Indian Land Live 14006 (72) Inventor Ferrara, Napoleon United States California 94109, San Francisco, Pacific Avenue 2090,704 (72) Inventor Gerber, Hans Peter United States California 94107, San Francisco , Tennessee Street 1121, Room 5 (72) Inventor Geritzen, Marie, E. United States California 94402, San Mateo, Parrot Drive 541 (72) Inventor Goddard, Audrey United States California 94131, San Francisco, Congo Street 110 (72) Inventor Gurney, Austin, El. United States California 94002, Belmont, Debbie Lane 1 (72) Inventor Hiran, Kenneth, J .. United States California 94114, San Francisco, Seward Street 64 (72) Inventor Marsters, Scott, A .. United States California 94070, San Carlos, Cherry Street 990 (72) Inventor Paoni, Nicholas, F. USA California 94002, Belmont, Terrace Drive 1756 (72) Inventor Pitti, Robert, Em. United States California 94530, El Cerrito, Liberty Street 1110 (72) Inventor Watanabe, Colin, Kay. United States California 94556 Moraga, Corris Drive 128 (72) Inventor Williams, P. , Mickey USA California 94019, Half Moon Bay, Alt Avenue 509 (72) Inventor Wood, William, Ai. United States California 94010, Hillsborough, Southdown Court 35F Term (Reference) 2G045 AA34 AA35 BB10 BB14 BB20 BB24 BB41 BB46 BB48 BB50 BB51 CB01 DA13 DA36 FA18 FB02 FB03 FB07 4B024 AA01 AA11 BA53 BA80 GA17 DA14 DA12 DA03 DA14 DA12 DA03 4B063 QA05 QQ08 QQ79 QQ91 QR51 QR77 QS31 QX10 4B064 AG27 BJ12 CA02 CA06 CA10 CA19 CA20 CC24 DA03 DA05 DA13 4B065 AA26X AA72X AA90X AA90Y AA97A4 A40A40 A14A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A14 A40 AA01 AA02 AA03 EA16 MA02 NA14 ZA40 ZB26 4H045 AA10 AA20 AA30 BA10 BA41 BA54 CA40 DA75 DA76 EA28 EA34 EA51 FA72 FA73 FA74 GA26

Claims (80)

[Claims] 1. PRO179, PRO238, PRO364, PRO844
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
A composition comprising a PRO885 or PRO887 polypeptide, or an agonist or antagonist thereof, in admixture with a pharmaceutically acceptable carrier. 2. The composition of claim 1, comprising a therapeutically effective amount of the polypeptide, or the agonist or antagonist thereof. 3. The agonist is anti-PRO179, anti-PRO238, anti-P.
RO364, anti-PRO844, anti-PRO846, anti-PRO1760, anti-PR
O205, anti-PRO321, anti-PRO333, anti-PRO840, anti-PRO8
77, anti-PRO878, anti-PRO879, anti-PRO882, anti-PRO885
Alternatively, the composition according to claim 1, which is an anti-PRO887 antibody. 4. The antagonist is anti-PRO179, anti-PRO238, anti
-PRO364, anti-PRO844, anti-PRO846, anti-PRO1760, anti-
PRO205, anti-PRO321, anti-PRO333, anti-PRO840, anti-PR
O877, anti-PRO878, anti-PRO879, anti-PRO882, anti-PRO8
The composition of claim 1, which is 85 or an anti-PRO887 antibody. 5. The composition of claim 1, further comprising a cardiovascular, endothelial, angiogenic or angiostatic agent. 6. PRO179, PRO238, PRO364, PRO844
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
The method for producing the composition according to claim 1, which comprises mixing the PRO885 or PRO887 polypeptide, or an agonist or antagonist thereof with a pharmaceutically acceptable carrier. 7. (1) (a) PRO179, PRO238, PRO364,
PRO844, PRO846, PRO1760, PRO205, PRO321,
PRO333, PRO840, PRO877, PRO878, PRO879, P
RO882, PRO885 or PRO887 polypeptide, (b) PRO179
, PRO238, PRO364, PRO844, PRO846, PRO1760
, PRO205, PRO321, PRO333, PRO840, PRO877,
An agonist of a PRO878, PRO879, PRO882, PRO885 or PRO887 polypeptide, or (c) PRO179, PRO238, PRO36.
4, PRO844, PRO846, PRO1760, PRO205, PRO32
1, PRO333, PRO840, PRO877, PRO878, PRO879
, PRO882, PRO885 or PRO887 polypeptide antagonist in admixture with a pharmaceutically acceptable carrier; (2) a container containing the composition; and (3) cardiovascular, endothelium of the composition, and An article of manufacture comprising a label affixed to the container or a packaging insert contained in the container, the label indicating use in the treatment of angiogenic disorders. 8. The agonist is anti-PRO179, anti-PRO238, anti-PRO
-PRO364, anti-PRO844, anti-PRO846, anti-PRO1760, anti-
PRO205, anti-PRO321, anti-PRO333, anti-PRO840, anti-PR
O877, anti-PRO878, anti-PRO879, anti-PRO882, anti-PRO8
The manufactured product according to claim 7, which is 85 or anti-PRO887 antibody. 9. The antagonist is anti-PRO179, anti-PRO238.
, Anti-PRO364, anti-PRO844, anti-PRO846, anti-PRO1760,
Anti-PRO205, Anti-PRO321, Anti-PRO333, Anti-PRO840, Anti-
PRO877, anti-PRO878, anti-PRO879, anti-PRO882, anti-PR
The product according to claim 7, which is an O885 or anti-PRO887 antibody. 10. The article of manufacture of claim 7, wherein the composition comprises a therapeutically effective amount of the polypeptide or agonist or antagonist thereof, mixed with the pharmaceutically acceptable carrier. 11. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, PRO885 or PRO887 polypeptides are identified by: (a) the cells and test compounds to be screened are PRO179, PRO2.
38, PRO364, PRO844, PRO846, PRO1760, PRO2
05, PRO321, PRO333, PRO840, PRO877, PRO87
Contacting the cells with a test compound under conditions suitable for eliciting a cellular response usually elicited by a 8, PRO879, PRO882, PRO885 or PRO887 polypeptide; and (b) measuring the induction of said cellular response. , Determining whether the test compound is an effective agonist, wherein eliciting the cellular response indicates that the test compound is an effective agonist. 12. The cellular response normally elicited by the polypeptide is
The method according to claim 11, which is stimulation of cell proliferation. 13. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
In the method for identifying a compound that inhibits the activity of a PRO885 or PRO887 polypeptide, a test compound is contacted with the polypeptide under conditions and for a time sufficient for the test compound to interact with the polypeptide, A method comprising determining whether activity is inhibited. 14. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, A method of identifying a compound that inhibits the activity of a PRO885 or PRO887 polypeptide: (a) a cell and a test compound to be screened for a cellular response that is normally elicited by said polypeptide Contacting under conditions suitable for induction; and (b) measuring the induction of said cellular response to determine whether the test compound is an effective antagonist. 15. The cellular response normally elicited by the polypeptide is
The method according to claim 14, which is stimulation of cell proliferation. 16. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, PRO885 or PRO887 polypeptide, which is a method for identifying a compound that inhibits the expression of the polypeptide in a cell that normally expresses the polypeptide, wherein the method is suitable conditions for expressing the cell and a test compound in the polypeptide. A method comprising contacting under and determining whether expression of said polypeptide is inhibited. 17. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, Agonists of the PRO885 or PRO887 polypeptides. 18. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, An antagonist of the PRO885 or PRO887 polypeptides. 19. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, A compound that inhibits the expression of the PRO885 or PRO887 polypeptide in a mammalian cell that expresses the polypeptide. 20. The compound of claim 19, wherein the compound is an antisense oligonucleotide. 21. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
, An isolated antibody that binds to a PRO885 or PRO887 polypeptide. 22. The antibody according to claim 21, which is a monoclonal antibody. 23. The antibody according to claim 21, which is an antibody fragment. 24. The antibody according to claim 21, which is a single chain antibody. 25. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
A method of diagnosing a disease or susceptibility to a disease associated with a mutation in a PRO-885 or PRO887 polypeptide-encoding nucleic acid sequence, comprising determining the presence or absence of said mutation in said polypeptide-encoding nucleic acid sequence. , A method in which the presence or absence of the mutation indicates the presence of the disease or susceptibility to the disease. 26. A method for diagnosing cardiovascular, endothelial or angiogenic disease in a mammal, comprising: (a) a test sample of tissue cells obtained from the mammal; and (b)
) PRO179, PRO2 in a control sample of known normal tissue cells of the same cell type
38, PRO364, PRO844, PRO846, PRO1760, PRO2
05, PRO321, PRO333, PRO840, PRO877, PRO87
8. Analyzing the expression level of a gene encoding a 8, PRO879, PRO882, PRO885 or PRO887 polypeptide, wherein the higher or lower expression level in a test sample when compared to a control sample is cardiovascular in said mammal. ,
A method of indicating the presence of an endothelial or angiogenic disorder. 27. In a method of diagnosing cardiovascular, endothelial or angiogenic disease in a mammal, PRO1 in a test sample of tissue cells obtained from said mammal.
79, PRO238, PRO364, PRO844, PRO846, PRO17
60, PRO205, PRO321, PRO333, PRO840, PRO87
7, PRO878, PRO879, PRO882, PRO885 or PRO88
7 detecting the presence or absence of a polypeptide, wherein the presence or absence of said polypeptide in said test sample is indicative of the presence of cardiovascular, endothelial or angiogenic disease in said mammal. 28. In a method of diagnosing cardiovascular, endothelial or angiogenic disease in a mammal, (a) an anti-PR test sample of tissue cells obtained from the mammal.
O179, anti-PRO238, anti-PRO364, anti-PRO844, anti-PRO8
46, anti-PRO1760, anti-PRO205, anti-PRO321, anti-PRO33
3, anti-PRO840, anti-PRO877, anti-PRO878, anti-PRO879,
Contacted with anti-PRO882, anti-PRO885 or anti-PRO887 antibody, and (b) said antibody in the test sample and PRO179, PRO238, PRO364.
, PRO844, PRO846, PRO1760, PRO205, PRO321
, PRO333, PRO840, PRO877, PRO878, PRO879,
Comprising detecting complex formation with a PRO882, PRO885 or PRO887 polypeptide, the formation of said complex being cardiovascular in said mammal,
A method of indicating the presence of an endothelial or angiogenic disorder. 29. PRO179, PRO238, PRO364, P in the sample
RO844, PRO846, PRO1760, PRO205, PRO321, P
RO333, PRO840, PRO877, PRO878, PRO879, PR
In a method for determining the presence of O882, PRO885 or PRO887 polypeptide, a sample suspected of containing the polypeptide is treated with anti-PRO179, anti-
PRO238, anti-PRO364, anti-PRO844, anti-PRO846, anti-PR
O1760, Anti-PRO205, Anti-PRO321, Anti-PRO333, Anti-PRO
840, anti-PRO877, anti-PRO878, anti-PRO879, anti-PRO88
2. Contacting with an anti-PRO885 or anti-PRO887 antibody and measuring the binding of said antibody to a component of said sample. 30. Anti-PRO179, anti-PRO238, anti-PRO364,
Anti-PRO844, Anti-PRO846, Anti-PRO1760, Anti-PRO205, Anti
-PRO321, Anti-PRO333, Anti-PRO840, Anti-PRO877, Anti-P
RO878, anti-PRO879, anti-PRO882, anti-PRO885 or anti-PR
A diagnostic kit for cardiovascular, endothelial or angiogenic diseases, which comprises the O887 antibody and a carrier in a suitable package. 31. A method of treating cardiovascular, endothelial or angiogenic disorders in a mammal, comprising PRO179, PRO238, PRO364, PRO844.
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
A method comprising administering to a mammal a therapeutically effective amount of a PRO885 or PRO887 polypeptide, or an agonist or antagonist thereof. 32. The method of claim 31, wherein the mammal is a human. 33. The method of claim 32, wherein the human has myocardial infarction. 34. The method of claim 32, wherein the human has cardiac hypertrophy, trauma, cancer, or age-related macular degeneration. 35. The method of claim 34, wherein cardiac hypertrophy is characterized by the presence of elevated levels of PGF _2α . 36. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
32. The method of claim 31, wherein the PRO885 or PRO887 polypeptide is administered with a cardiovascular, endothelial or angiogenic agent. 37. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
35. The method of claim 34, wherein the PRO885 or PRO887 polypeptide is administered following primary angioplasty. 38. The method of claim 31, wherein the cardiovascular, endothelial or angiogenic disease is cancer. 39. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
39. The method of claim 38, wherein the PRO885 or PRO887 polypeptide is administered in combination with a chemotherapeutic agent, growth inhibitory agent or cytotoxic agent. 40. The agonist is anti-PRO179, anti-PRO238, anti
-PRO364, anti-PRO844, anti-PRO846, anti-PRO1760, anti-
PRO205, anti-PRO321, anti-PRO333, anti-PRO840, anti-PR
O877, anti-PRO878, anti-PRO879, anti-PRO882, anti-PRO8
32. The method of claim 31, which is 85 or anti-PRO887 antibody. 41. The antagonist is anti-PRO179, anti-PRO238.
, Anti-PRO364, anti-PRO844, anti-PRO846, anti-PRO1760,
Anti-PRO205, Anti-PRO321, Anti-PRO333, Anti-PRO840, Anti-
PRO877, anti-PRO878, anti-PRO879, anti-PRO882, anti-PR
32. The method of claim 31, which is an O885 or anti-PRO887 antibody. 42. A method of treating cardiovascular, endothelial or angiogenic disorders in a mammal, comprising PRO179, PRO238, PRO364, PRO844.
, PRO846, PRO1760, PRO205, PRO321, PRO333
, PRO840, PRO877, PRO878, PRO879, PRO882,
A method comprising administering to a mammal a nucleic acid molecule encoding a PRO885 or PRO887 polypeptide, or an agonist or antagonist thereof. 43. The agonist is anti-PRO179, anti-PRO238, anti-
-PRO364, anti-PRO844, anti-PRO846, anti-PRO1760, anti-
PRO205, anti-PRO321, anti-PRO333, anti-PRO840, anti-PR
O877, anti-PRO878, anti-PRO879, anti-PRO882, anti-PRO8
43. The method of claim 42, which is 85 or an anti-PRO887 antibody. 44. The antagonist is anti-PRO179, anti-PRO238.
, Anti-PRO364, anti-PRO844, anti-PRO846, anti-PRO1760,
Anti-PRO205, Anti-PRO321, Anti-PRO333, Anti-PRO840, Anti-
PRO877, anti-PRO878, anti-PRO879, anti-PRO882, anti-PR
43. The method of claim 42, which is an O885 or anti-PRO887 antibody. 45. The method of claim 42, wherein the mammal is a human. 46. The method of claim 42, wherein the nucleic acid molecule is administered via ex vivo gene therapy. 47. (1) Promoter, (2) PRO179, PRO238,
PRO364, PRO844, PRO846, PRO1760, PRO205,
PRO321, PRO333, PRO840, PRO877, PRO878, P
A retroviral vector comprising a nucleic acid encoding a RO879, PRO882, PRO885 or PRO887 polypeptide or an agonist or antagonist thereof, and (3) a retroviral vector consisting essentially of a signal sequence for cellular secretion of the polypeptide. Is a recombinant retroviral particle with a retroviral structural protein. 48. A retrovirus structural protein is expressed, (1) promoter, (2) PRO179, PRO238, PRO364, PRO844, PRO.
846, PRO1760, PRO205, PRO321, PRO333, PRO
840, PRO877, PRO878, PRO879, PRO882, PRO8
85 or a PRO887 polypeptide or an agonist or antagonist thereof, and (3) an ex vivo producing cell comprising a retroviral vector nucleic acid structure consisting essentially of a signal sequence for cellular secretion of the polypeptide, comprising: A producer cell which comprises a retroviral vector with a structural protein to produce a recombinant retroviral particle. 49. A method of inhibiting endothelial cell growth in a mammal, comprising:
A method of inhibiting endothelial cell growth in a mammal, comprising administering a PRO333, PRO364, PRO877, PRO879, PRO882 or PRO885 polypeptide or an agonist thereof to the mammal. 50. A method of stimulating endothelial cell growth in a mammal, comprising:
PRO179, PRO321, PRO840, PRO844, PRO846, P
A method of stimulating endothelial cell growth in a mammal, comprising administering a RO878 or PRO879 polypeptide or agonist thereof to the mammal. 51. A method of inhibiting endothelial cell growth in a mammal, comprising:
PRO179, PRO321, PRO840, PRO844, PRO846, P
A method of inhibiting endothelial cell growth in a mammal comprising administering to said mammal an antagonist of RO878 or PRO879 polypeptide. 52. A method of stimulating endothelial cell growth in a mammal, comprising:
A method of stimulating endothelial cell growth in a mammal, comprising administering to said mammal an antagonist of PRO333, PRO364, PRO877, PRO879, PRO882 or PRO885 polypeptide. 53. A method of inducing cardiac hypertrophy in a mammal comprising administering to said mammal a PRO205, PRO882 or PRO887 polypeptide or agonist thereof, wherein cardiac hypertrophy is induced in said mammal. . 54. A method for suppressing cardiac hypertrophy in a mammal, comprising administering PRO238, PRO878 or PRO1760 polypeptide or an agonist thereof to the mammal, wherein cardiac hypertrophy in the mammal is suppressed. . 55. A method of inducing cardiac hypertrophy in a mammal, the method comprising:
A method of suppressing the cardiac hypertrophy in a mammal, comprising administering an O238, PRO878 or PRO1760 polypeptide or an agonist thereof to the mammal. 56. A method of inhibiting cardiac hypertrophy in a mammal, the method comprising:
A method of suppressing cardiac hypertrophy in a mammal, comprising administering an antagonist of O205, PRO882 or PRO887 polypeptide to the mammal. 57. PRO179, PRO321, PRO8 in mammals
In a method of inhibiting angiogenesis induced by a 40, PRO844, PRO846, PRO878 or PRO879 polypeptide, anti-PRO179, anti-
PRO321, anti-PRO840, anti-PRO844, anti-PRO846, anti-PR
A method of inhibiting angiogenesis, comprising administering an O878 or anti-PRO879 polypeptide antibody to the mammal. 58. PRO179, PRO321, PRO8 in a mammal
40, PRO844, PRO846, PRO878 or PRO879 polypeptide, comprising administering to said mammal a therapeutically effective amount of said polypeptide, whereby said angiogenesis is stimulated. How to do. 59. Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO: 4),
Fig6 (SEQ ID NO: 6), Fig8 (SEQ ID NO: 8), Fig10 (SEQ ID NO: 10), Fig12 (SEQ ID NO: 12), Fig14 (SEQ ID NO: 14), F
ig16 (SEQ ID NO: 16), Fig18 (SEQ ID NO: 18), Fig20 (SEQ ID NO: 20), Fig22 (SEQ ID NO: 22), Fig24 (SEQ ID NO: 24)
), FIG26 (SEQ ID NO: 26), FIG28 (SEQ ID NO: 28), FIG3
0 (SEQ ID NO: 30) and an isolated nucleic acid having at least 80% nucleic acid sequence identity with a nucleotide sequence encoding an amino acid sequence selected from the group consisting of the amino acid sequences shown in Figure 32 (SEQ ID NO: 32). . 60. FIG. 1 (SEQ ID NO: 1), FIG3 (SEQ ID NO: 3),
FIG5 (SEQ ID NO: 5), FIG7 (SEQ ID NO: 7), FIG9 (SEQ ID NO: 7)
9), Fig11 (SEQ ID NO: 11), Fig13 (SEQ ID NO: 13), Fig
15 (SEQ ID NO: 15), FIG17 (SEQ ID NO: 17), FIG19 (SEQ ID NO: 19), FIG21 (SEQ ID NO: 21), FIG23 (SEQ ID NO: 23),
FIG25 (SEQ ID NO: 25), FIG27 (SEQ ID NO: 27), FIG29 (
SEQ ID NO: 29), and an isolated nucleic acid having at least 80% nucleic acid sequence identity with a nucleotide sequence selected from the group consisting of the nucleotide sequences shown in Figure 31 (SEQ ID NO: 31). 61. FIG. 1 (SEQ ID NO: 1) and FIG3 (SEQ ID NO: 3)
, Fig5 (SEQ ID NO: 5), Fig7 (SEQ ID NO: 7), Fig9 (SEQ ID NO: 9), Fig11 (SEQ ID NO: 11), Fig13 (SEQ ID NO: 13), Fi
g15 (SEQ ID NO: 15), Fig17 (SEQ ID NO: 17), Fig19 (SEQ ID NO: 19), Fig21 (SEQ ID NO: 21), Fig23 (SEQ ID NO: 23)
, Fig25 (SEQ ID NO: 25), Fig27 (SEQ ID NO: 27), Fig29
(SEQ ID NO: 29), and an isolated nucleic acid having at least 80% nucleic acid sequence identity with a nucleotide sequence selected from the group consisting of the full length coding sequences of the nucleotide sequence shown in Figure 31 (SEQ ID NO: 31). 62. ATCC Registration Nos. 209776, 209370, 2094
36, 209976, 209847, 203473, 209719, or 209
An isolated nucleic acid having at least 80% nucleic acid sequence identity with the full length coding sequence of the DNA deposited under 858. 63. A vector comprising the nucleic acid according to any one of claims 59 to 62. 64. The vector of claim 63, operably linked to a control sequence recognized by a host cell transformed with the vector. 65. A host cell comprising the vector of claim 63. 66. The host cell of claim 65, wherein said cell is a CHO cell. 67. The host cell of claim 65, wherein said cell is E. coli. 68. The host cell of claim 65, wherein said cell is a yeast cell. 69. The host cell of claim 65, wherein said cell is a baculovirus-infected insect cell. 70. PRO179, PRO238, PRO364, PRO84
4, PRO846, PRO1760, PRO205, PRO321, PRO33
3, PRO840, PRO877, PRO878, PRO879, PRO882
66. A method for producing a PRO885 or PRO887 polypeptide.
A method comprising culturing the host cell of claim 1 under conditions suitable for expression of said polypeptide, and recovering said polypeptide from the culture medium. 71. Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO: 4),
Fig6 (SEQ ID NO: 6), Fig8 (SEQ ID NO: 8), Fig10 (SEQ ID NO: 10), Fig12 (SEQ ID NO: 12), Fig14 (SEQ ID NO: 14), F
ig16 (SEQ ID NO: 16), Fig18 (SEQ ID NO: 18), Fig20 (SEQ ID NO: 20), Fig22 (SEQ ID NO: 22), Fig24 (SEQ ID NO: 24)
), FIG26 (SEQ ID NO: 26), FIG28 (SEQ ID NO: 28), FIG3
0 (SEQ ID NO: 30), and an isolated polypeptide having at least 80% amino acid sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequences shown in Figure 32 (SEQ ID NO: 32). 72. Figure 2 (SEQ ID NO: 2), Figure4 (SEQ ID NO: 4),
Fig6 (SEQ ID NO: 6), Fig8 (SEQ ID NO: 8), Fig10 (SEQ ID NO: 10), Fig12 (SEQ ID NO: 12), Fig14 (SEQ ID NO: 14), F
ig16 (SEQ ID NO: 16), Fig18 (SEQ ID NO: 18), Fig20 (SEQ ID NO: 20), Fig22 (SEQ ID NO: 22), Fig24 (SEQ ID NO: 24)
), FIG26 (SEQ ID NO: 26), FIG28 (SEQ ID NO: 28), FIG3
An isolated polypeptide that scores at least 80% positive when compared to an amino acid sequence selected from the group consisting of 0 (SEQ ID NO: 30) and the amino acid sequence shown in Figure 32 (SEQ ID NO: 32). 73. ATCC Registration Nos. 209776, 209370, 2094
36, 209976, 209847, 203473, 209719, or 209
An isolated polypeptide having at least 80% amino acid sequence identity with the amino acid sequence encoded by the full length coding sequence of the deposited DNA under 858. 74. A chimeric molecule comprising the polypeptide of any of claims 71-73 fused to a heterologous amino acid sequence. 75. The chimeric molecule of claim 74, wherein the heterologous amino acid sequence is an epitope tag sequence. 76. The chimeric molecule of claim 74, wherein the heterologous amino acid sequence is an immunoglobulin Fc region. 77. An antibody that specifically binds to the polypeptide of any one of claims 71-73. 78. The antibody of claim 77, wherein the antibody is a monoclonal antibody, a humanized antibody or a single chain antibody. 79. (a) FIG2 (SEQ ID NO: 2) and FIG4 (SEQ ID NO :)
4), Fig6 (SEQ ID NO: 6), Fig8 (SEQ ID NO: 8), Fig10 (SEQ ID NO: 10), Fig12 (SEQ ID NO: 12), Fig14 (SEQ ID NO: 14)
), FIG16 (SEQ ID NO: 16), FIG18 (SEQ ID NO: 18), FIG2
0 (SEQ ID NO: 20), FIG22 (SEQ ID NO: 22), FIG24 (SEQ ID NO: 24), FIG26 (SEQ ID NO: 26), FIG28 (SEQ ID NO: 28), F
ig30 (SEQ ID NO: 30) and the polypeptide shown in Fig32 (SEQ ID NO: 32), which encodes a polypeptide lacking a signal sequence related thereto; (b) Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO: 4), FIG6 (SEQ ID NO: 6), FIG8 (SEQ ID NO: 8), FIG10 (SEQ ID NO: 10), F
ig12 (SEQ ID NO: 12), Fig14 (SEQ ID NO: 14), Fig16 (SEQ ID NO: 16), Fig18 (SEQ ID NO: 18), Fig20 (SEQ ID NO: 20)
), FIG22 (SEQ ID NO: 22), FIG24 (SEQ ID NO: 24), FIG2
6 (SEQ ID NO: 26), FIG28 (SEQ ID NO: 28), Fig30 (SEQ ID NO: 30), and the extracellular domain of the polypeptide shown in FIG32 (SEQ ID NO: 32), which has a signal peptide associated therewith Or (c) Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO: 4), Fig6 (SEQ ID NO: 6), Fig8 (SEQ ID NO: 8), Fig10 (SEQ ID NO: 10) ), F
ig12 (SEQ ID NO: 12), Fig14 (SEQ ID NO: 14), Fig16 (SEQ ID NO: 16), Fig18 (SEQ ID NO: 18), Fig20 (SEQ ID NO: 20)
), FIG22 (SEQ ID NO: 22), FIG24 (SEQ ID NO: 24), FIG2
6 (SEQ ID NO: 26), FIG28 (SEQ ID NO: 28), Fig30 (SEQ ID NO: 30), and the extracellular domain of the polypeptide shown in FIG32 (SEQ ID NO: 32), which has a signal peptide associated therewith An isolated nucleic acid having at least 80% nucleic acid sequence identity with the nucleotide sequence encoding the lacking one. (80) (a) Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO :)
4), Fig6 (SEQ ID NO: 6), Fig8 (SEQ ID NO: 8), Fig10 (SEQ ID NO: 10), Fig12 (SEQ ID NO: 12), Fig14 (SEQ ID NO: 14)
), FIG16 (SEQ ID NO: 16), FIG18 (SEQ ID NO: 18), FIG2
0 (SEQ ID NO: 20), FIG22 (SEQ ID NO: 22), FIG24 (SEQ ID NO: 24), FIG26 (SEQ ID NO: 26), FIG28 (SEQ ID NO: 28), F
ig30 (SEQ ID NO: 30) and the polypeptide shown in Fig32 (SEQ ID NO: 32), which lacks a signal sequence associated therewith; (b) Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO: 4) ), FIG6 (SEQ ID NO: 6), FIG8 (SEQ ID NO: 8), FIG10 (SEQ ID NO: 10), F
ig12 (SEQ ID NO: 12), Fig14 (SEQ ID NO: 14), Fig16 (SEQ ID NO: 16), Fig18 (SEQ ID NO: 18), Fig20 (SEQ ID NO: 20)
), FIG22 (SEQ ID NO: 22), FIG24 (SEQ ID NO: 24), FIG2
6 (SEQ ID NO: 26), FIG28 (SEQ ID NO: 28), Fig30 (SEQ ID NO: 30), and the extracellular domain of the polypeptide shown in FIG32 (SEQ ID NO: 32), which has a signal peptide associated therewith Or (c) Fig2 (SEQ ID NO: 2), Fig4 (SEQ ID NO: 4), Fig6 (SEQ ID NO: 6), Fig8 (SEQ ID NO: 8), Fig10 (SEQ ID NO: 10), F
ig12 (SEQ ID NO: 12), Fig14 (SEQ ID NO: 14), Fig16 (SEQ ID NO: 16), Fig18 (SEQ ID NO: 18), Fig20 (SEQ ID NO: 20)
), FIG22 (SEQ ID NO: 22), FIG24 (SEQ ID NO: 24), FIG2
6 (SEQ ID NO: 26), Fig28 (SEQ ID NO: 28), Fig30 (SEQ ID NO: 30), and the extracellular domain of the polypeptide shown in Fig32 (SEQ ID NO: 32), which has a signal peptide associated therewith Missing and at least 80
An isolated polypeptide having% amino acid sequence identity.