WO2002008284A2 - Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis - Google Patents

Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis Download PDF

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Publication number
WO2002008284A2
WO2002008284A2 PCT/US2001/021735 US0121735W WO0208284A2 WO 2002008284 A2 WO2002008284 A2 WO 2002008284A2 US 0121735 W US0121735 W US 0121735W WO 0208284 A2 WO0208284 A2 WO 0208284A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
sequence
acid sequence
amino acid
polypeptide
Prior art date
Application number
PCT/US2001/021735
Other languages
French (fr)
Other versions
WO2002008284A3 (en
Inventor
Kevin P. Baker
Napoleone Ferrara
Hanspeter Gerber
Mary E. Gerritsen
Audrey Goddard
Paul J. Godowski
Austin L. Gurney
Kenneth J. Hillan
Scot A. Marsters
James Pan
Nicholas F. Paoni
Jean-Philippe F. Stephan
Colin K. Watanabe
P. Mickey Williams
William I. Wood
Weilan Ye
Original Assignee
Genentech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2000/020710 external-priority patent/WO2001009327A2/en
Priority claimed from US09/643,657 external-priority patent/US6642024B1/en
Priority claimed from PCT/US2000/023522 external-priority patent/WO2001016319A2/en
Priority claimed from PCT/US2000/023328 external-priority patent/WO2001016318A2/en
Priority claimed from PCT/US2000/030952 external-priority patent/WO2001049715A2/en
Priority claimed from PCT/US2000/030873 external-priority patent/WO2001040465A2/en
Priority claimed from PCT/US2000/032678 external-priority patent/WO2001040466A2/en
Priority claimed from US09/747,259 external-priority patent/US6569645B2/en
Priority claimed from PCT/US2000/034956 external-priority patent/WO2001046420A2/en
Priority claimed from US09/767,609 external-priority patent/US20020042367A1/en
Priority claimed from PCT/US2001/006520 external-priority patent/WO2001068848A2/en
Priority claimed from PCT/US2001/006666 external-priority patent/WO2001066740A2/en
Priority claimed from US09/816,744 external-priority patent/US6579520B2/en
Priority claimed from US09/828,366 external-priority patent/US20020010137A1/en
Priority claimed from US09/854,208 external-priority patent/US7217412B2/en
Priority claimed from PCT/US2001/017092 external-priority patent/WO2001092331A2/en
Priority claimed from US09/866,028 external-priority patent/US6642360B2/en
Priority claimed from US09/866,034 external-priority patent/US20030170864A1/en
Priority claimed from PCT/US2001/017443 external-priority patent/WO2002016611A2/en
Priority claimed from US09/870,574 external-priority patent/US6551799B2/en
Priority claimed from PCT/US2001/017800 external-priority patent/WO2001093983A1/en
Priority claimed from PCT/US2001/019692 external-priority patent/WO2002000690A2/en
Priority to EP01951036A priority Critical patent/EP1309685A2/en
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to JP2002514188A priority patent/JP2004516013A/en
Priority to CA002416538A priority patent/CA2416538A1/en
Priority to AU2001271973A priority patent/AU2001271973A1/en
Priority to US09/918,585 priority patent/US20030060406A1/en
Priority to US09/931,836 priority patent/US7435793B2/en
Priority to CA002420193A priority patent/CA2420193A1/en
Priority to MXPA03001643A priority patent/MXPA03001643A/en
Priority to US09/938,418 priority patent/US20020161199A1/en
Priority to KR1020037002672A priority patent/KR100607611B1/en
Priority to EP01966255A priority patent/EP1311668A2/en
Priority to AU8678501A priority patent/AU8678501A/en
Priority to AU2001286785A priority patent/AU2001286785B2/en
Priority to JP2002522275A priority patent/JP2004520810A/en
Priority to PCT/US2001/026626 priority patent/WO2002016602A2/en
Priority to US09/941,992 priority patent/US20030082546A1/en
Priority to CA002421056A priority patent/CA2421056A1/en
Priority to PCT/US2001/027099 priority patent/WO2002024888A2/en
Priority to EP07022176A priority patent/EP1944317A3/en
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Priority to US13/269,984 priority patent/US20120058909A1/en
Priority to US13/593,362 priority patent/US20130064827A1/en
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Abstract

Compositions and methods are disclosed for stimulating or inhibiting angiogenesis and/or cardiovascularization in mammals, including humans. Pharmaceutical compositions are based on polypeptides or antagonists thereto that have been identified for one or more of these uses. Disorders that can be diagnosed, prevented, or treated by the compositions herein include trauma such as wounds, various cancers, and disorders of the vessels including atherosclerosis and cardiac hypertrophy. In addition, the present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

Description

COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF DISORDERS
INVOLVING ANGIOGENESIS
1. Field ofthe Invention
The present invention relates to compositions and methods useful for the modulation (e.g., promotion or inhibition) of angiogenesis and or cardiovascularization in mammals in need of such biological effect. The present invention further relates to the diagnosis and treatment of disorders involving angiogenesis (e.g. , cardiovascular as well as oncological disorders).
2. Background ofthe Invention
2.1. Angiogenesis
Angiogenesis, defined as the growth or sprouting of new blood vessels from existing vessels, is a complex process that primarily occurs during embryonic development. Under normal physiological conditions in adults, angiogenesis takes place only in very restricted situations such as hair growth and wounding healing (Auerbach,
W. and Auerbach, R., 1994, Pharmacol Ther 63(3):265-3 11; Ribatti et al.,1991, Haematologica 76(4):3 11-20; Risau, 1997, Nature 386(6626):67 1-4). Unregulated angiogenesis has gradually been recognized to be responsible for a wide range of disorders, including, but not limited to cardiovascular disease, cancer, rheumatoid arthritis, psoriasis and diabetic retinopathy (Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999, Circulation 99(13): 1653-5; Koch, 1998,ArthritisRheum4l(6) 951-62; Wahb^ l999, Rheumatology (Oxford) 38(2):^^
1997, Nat Med '3(2): 158-64).
2.2. Cardiac Disorders and Factors
Heart failure affects approximately five million Americans, and new cases of heart failure number about 400,000 each year. It is the single most frequent cause of hospitalization for people age 65 and older in the United
States. Recent advances in the management of acute cardiac diseases, including acute myocardial infarction, are resulting in an expanding patient population that will eventually develop chronic heart failure. From 1979 to 1995, hospitalizations for congestive heart failure (CHF) rose from 377,000 to 872,000 (a 130 percent increase) and CHF deaths increased 116 percent. CHF is a syndrome characterized by left ventricular dysfunction, reduced exercise tolerance, impaired quality of life, and markedly shortened life expectancy. The sine qua non of heart failure is an inability ofthe heart to pump blood at a rate sufficient to meet the metabolic needs of the body's tissues (in other words, there is insufficient cardiac output).
At least four major compensatory mechanisms are activated in the setting of heart failure to boost cardiac output, including peripheral 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. See, Eichhorn, American Journal of Medicine, 104: 163-169 (1998). Increased output from the sympathetic nervous system increases vascular tone, heart rate, and contractility. Angiotensin II elevates blood pressure by 1) directly stimulating vascular smooth muscle contraction, 2) promoting plasma volume expansion by stimulating aldosterone and antidiuretic hormone secretion, 3) stimulating sympathetic-mediated vascular tone, and 4) catalyzing the degradation of bradykinin, which has vasodilatory and natriuretic activity. See, review by Brown and Vaughan. Circulation.97: 1411-1420(1998). As noted below, angiotensin II may also have directly deleterious effects on the heart by promoting myocyte necrosis (impairing systolic function) and intracardiac fibrosis (impairing diastolic and in some cases systolic function). See, Weber, Circulation. 96: 4065-4082 (1998).
A consistent feature of congestive heart failure (CHF) is cardiac hypertrophy, an enlargement ofthe heart that is activated by both mechanical and hormonal stimuli and enables the heart to adapt to demands for increased cardiac output. Morgan and Baker, Circulation. 83: 13-25 (1991). This hypertrophic response is frequently associated with a variety of distinct pathological conditions such as hypertension, aortic stenosis, myocardial infarction, cardiomyopathy, valvular regurgitation, and intracardiac shunt, all of which result in chronic hemodynamic overload.
Hypertrophy is generally defined as an increase in size of an organ or structure independent of natural growth that does not involve tumor formation. Hypertrophy of the heart is due either to an increase in the mass of the individual cells (myocytes), or to an increase in the number of cells making up the tissue (hyperplasia), or both. While the enlargement of an embryonic heart is largely dependent on an increase in myocyte number (which continues until shortly after birth), post-natal cardiac myocytes lose their proliferative capacity. Further growth occurs through hypertrophy ofthe individual cells. Adult myocyte hypertrophy is initially beneficial as a short term response to impaired cardiac function by permitting a decrease in the load on individual muscle fibers. With severe, long-standing overload, however, the hypertrophied cells begin to deteriorate and die. Katz, "Heart Failure", in: Katz A.M. ed., Physiology ofthe Heart (New York: Raven Press, 1992) pp. 638-668. Cardiac hypertrophy is a significant risk factor for both mortality and morbidity in the clinical course of heart failure. Katz. Trends Cardiovasc. Med..5: 7-44(1995). For further details of the causes and pathology of cardiac hypertrophy see, e.g., Heart Disease. A Textbook of Cardiovascular
Medicine. Braunwald, E. ed. (W.B. Saunders Co., 1988), Chapter 14, "Pathophysiology of Heart Failure."
On a cellular level, the heart is composed of myocytes and surrounding support cells, generically called non-myocytes. While non-myocytes are primarily fibroblast/mesenchyrnal cells, they also include endothelial and smooth muscle cells. Indeed, although myocytes make up most ofthe adult myocardial mass, they represent only about 30% ofthe total cell numbers present in heart. In response to hormonal, physiological, hemodynamic, and pathological stimuli, adult ventricular muscle cells can adapt to increased workloads through the activation of a hypertrophic process. This response is characterized by an increase in myocyte cell size and contractile protein content of individual cardiac muscle cells, without concomitant cell division and activation of embryonic genes, including the gene for atrial natriuretic peptide (ANP). Chien et al., FASEB J.. 5: 3037-3046 (1991); Chien et al., Annu. Rev. Phvsiol, 55: 77-95 (1993). An increment in myocardial mass as a result of an increase in myocyte size that is associated with an accumulation of interstitial collagen within the extracellular matrix and around intramyocardial coronary arteries has been described in left ventricular hypertrophy secondary to pressure overload in humans. Caspari et al, Cardiovasc. Res.. U: 554-558 (1977); Schwarz et al., Am. J. Cardiol. 42: 895-903 (1978); Hess et al, Circulation. 63: 360-371 (1981); Pearlman et al, Lab. Invest. 46: 158-164 (1982).
It has also been suggested that paracrine factors produced by non-myocyte supporting cells may additionally be involved in the development of cardiac hypertrophy, and various non-myocyte derived hypertrophic factors, such as, leukocyte inhibitory factor (LIF) and endothelin, have been identified. Metcalf, Growth Factors.
7: 169-173 (1992); Kurzrock et al., Endocrine Reviews. 12: 208-217 (1991); Inoue et al, Proc. Natl. Acad. Sci. USA. 86- 2863-2867 (1989); Yanagisawa and Masaki, Trends Pharm. Sci.. 10: 374-378 (1989); U.S. Patent No. 5,573,762 (issued November 12, 1996). Further exemplary factors that have been identified as potential mediators of cardiac hypertrophy include cardiotrophin-1 (CT-1) (Permica et al. Proc. Nat. Acad. Sci. USA.92: 1142-1146 (1995)), catecholamines, adrenocorticosteroids, angiotensin, and prostaglandins.
At present, the treatment of cardiac hypertrophy varies depending on the underlying cardiac disease. Catecholamines, adrenocorticosteroids, angiotensin, prostaglandins, LIF, endothelin (including endothelin-1, -2, and -3 and big endothelin), and CT-1 are among the factors identified as potential mediators of hypertrophy. For example, beta-adrenergic receptor blocking drugs (beta-blockers, e.g., propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, carvedilol, etc.) and verapamil have been used extensively in the treatment of hypertrophic cardiomyopathy. The beneficial effects of beta-blockers on symptoms (e.g., chest pain) and exercise tolerance are largely due to a decrease in the heart rate with a consequentprolongation of diastole and increased passive ventricular filling. Thompson et al, BrJ Heart J..44: 488-98 (1980); Harrison et al, Circulation. 29: 84-98 (1964). Verapamil has been described to improve ventricular filling and probably reducing myocardial ischemia. Bonow et al, Circulation. 72: 853-64 (1985).
Nifedipine and diltiazem have also been used occasionally in the treatment of hypertrophic cardiomyopathy. Lorell et al, Circulation. 65: 499-507 (1982); Betocchi et al, Am. J. Cardiol.. 78: 451-457 (1996). However, because of its potent vasodilating properties, nifedipine may be harmful, especially in patients with outflow obstruction. Disopyramide has been used to relieve symptoms by virtue of its negative inotropic properties. Pollick, N. Engl. J. Med..307: 997-999 (1982). In many patients, however, the initial benefits decrease with time. Wigle et al, Circulation. 92: 1680-1692 (1995). Antihypertensive drug therapy has been reported to have beneficial effects on cardiac hypertrophy associated with elevated blood pressure. Examples of drugs used in antihypertensive therapy, alone or in combination, are calcium antagonists, e.g. , nitrendipine; adrenergic receptor blocking agents, e.g., those listed above; angiotensin converting enzyme (ACE) inhibitors such as quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, and lisinopril; diuretics, e.g., chlorothiazide, hydrochloro iazide,hydroflumemazide,memylchlot azide,benzthiazide, dichlorphenamide, acetazolamide, and indapamide; and calcium channel blockers, e.g., diltiazem, nifedipine, verapamil, and nicardipine.
For example, treatment of hypertension with diltiazem and captopril showed a decrease in left ventricular muscle mass, but the Doppler indices of diastolic function did not normalize. Szlachcic et al, Am. J. Cardiol., 63: 198-201 (1989); Shahi et al, Lancet. 336: 458-461 (1990). These findings were interpreted to indicate that excessive amounts of interstitial collagen may remain after regression of left ventricular hypertrophy. Rossi et al, Am. Heart J.. 124: 700-709 (1992). Rossi et al, supra, investigated the effect of captopril on the prevention and regression of myocardial cell hypertrophy and interstitial fibrosis in pressure overload cardiac hypertrophy, in experimental rats.
Agents that increase cardiac contractility directly (iontropic agents) were initially thought to benefit patients with heart failure because they improved cardiac output in the short term. However, all positive inotropic agents except digoxigenin have been found to result in increased long-term mortality, in spite of short-term improvements in cardiac performance. Massie, Curr. Op. in Cardiology. : 209-217 (1997); Reddy et al, Curr. Opin. Cardiol.. 12: 233-241 (1997). Beta-adrenergicreceptorblockers have recently been advocated foruseinheart failure. Evidence from clinical trials suggests that improvements in cardiac function can be achieved without increased mortality, though documented improvements of patient survival have not yet been demonstrated. See also, U.S. Pat Nos. 5,935,924, 5,624,806; 5,661,122; and 5,610,134 and WO 95/28173 regarding the use of cardiotropin-1 or antagonists thereof, or growth hormone and/or insulin-like growth factor-I in the treatment of
CHF. Another treatment modality is heart transplantation, but this is limited by the availability of donor hearts.
Endothelin is a vasoconstricting peptide comprising 21 amino acids, isolated from swine arterial endothelial culture supernatant and structurally determined. Yanagisawa et al, Nature. 332: 411-415 (1988). Endothelin was later found to exhibit various actions, and endothelin antibodies as endothelin antagonists have proven effective in the treatment of myocardial infarction, renal failure, and other diseases. Since endothelin is present in live bodies and exhibits vasoconstricting action, it is expected to be an endogenous factor involved in the regulation of the circulatory system, and may be associated with hypertension, cardiovascular diseases such as myocardial infarction, and renal diseases such as acute renal failure. Endothelin antagonists are described, for example, in U.S. Pat. No. 5,773,414; JP Pat. Publ. 3130299/1991, EP 457,195; EP 460,679; and EP 552,489. A new endothelin B receptor for identifying endothelin receptor antagonists is described in U.S. Pat. No. 5,773,223.
Current therapy for heart failure is primarily directed to using angiotensin-converting enzyme (ACE) inhibitors, such as captopril, and diuretics. These drugs improve hemodynamic profile and exercise tolerance and reduce the incidence of morbidity and mortality in patients with CHF. Kramer et al, Circulation. 67(4): 807-816 (1983); Captopril Multicenter Research Group, J.A.C.C., 2(4): 755-763 (1983); The CONSENSUS Trial Study
Group. N. Engl. J. Med..316(23V. 1429-1435 (1987): The SOLVD Investigators. N. Engl. J. Med..325(5V.293-302 (1991). Further, they are useful in treating hypertension, left ventricular dysfunction, atherosclerotic vascular disease, and diabetic nephropathy. Brown and Vaughan, supra. However, despite proven efficacy, response to ACE inhibitors has been limited. For example, while prolonging survival in the setting of heart failure, ACE inhibitors appear to slow the progression towards end-stage heart failure, and substantial numbers of patients on
ACE inhibitors have functional class III heart failure.
Moreover, improvement of functional capacity and exercise time is only small and mortality, although reduced, continues to be high. The CONSENSUS Trial Study Group. N. Engl. J. Med..316(23): 1429-1453 (1987); The SOLVD Investigators. N. Engl. J. Med..325(51: 293-302 (1991); Cohnef a N. Engl. J. Med..325(5): 303-310 (1991); The Captopril-Digoxin Multicenter Research Group, JAMA. 259(4): 539-544 (1988). Hence, ACE inhibitors consistently appear unable to relieve symptoms in more than 60% of heart failure patients and reduce mortality of heart failure only by approximately 15-20%. For further adverse effects, see Brown and Vaughan, supra.
An alternative to ACE inhibitors is represented by specific ATI receptor antagonists. Clinical studies are planned to compare the efficacy of these two modalities in the treatment of cardiovascular and renal disease. However, animal model data suggests that the ACE/Ang II pathway, while clearly involved in cardiac hypertrophy, is not the only, or even the primary pathway active in this role. Mouse genetic "knockout" models have been made to test individual components ofthe pathway. In one such model, the primary cardiac receptor for Ang II, AT sub IA, has been genetically deleted; these mice do not develop hypertrophy when Ang II is given experimentally (confirming the basic success ofthe model in eliminating hypertrophy secondary to Ang II). However, when the aorta is constricted in these animals (a model of hypertensive cardiac stress), the hearts still become hypertrophic. This suggests that alternative signaling pathways, not depending on this receptor (AT sub 1 A), are activated in hypertension. ACE inhibitors would presumably not be able to inhibit these pathways. See, Harada et al, Circulation, 97: 1952-1959 (1998). See also, Homey, Circulation, 97: 1890-1892 (1998) regarding the enigma associated with the process and mechanism of cardiac hypertrophy.
About 750,000 patients suffer from acute myocardial infarction (AMI) annually, and approximately one- fourth of all deaths in the United States are due to AMI. In recent years, thrombolytic agents, e.g., streptokinase, urokinase, and in particular tissue plasminogen activator (t-PA) have significantly increased the survival of patients who suffered myocardial infarction. When administered as a continuous intravenous infusion over 1.5 to 4 hours, t-PA produces coronary patency at 90 minutes in 69% to 90% ofthe treated patients. Topol et al, Am. J. Cardiol.. 61: 723-728 (1988); Neuhaus et al, J. Am. Coll. Cardiol. 12: 581-587 (1988); Neuhaus et al, J. Am. Coll. Cardiol., 14: 1566-1569 (1989). The highest patency rates have been reported with high dose or accelerated dosing regimens. Topol, J. Am. Coll. Cardiol.. 15: 922-924 (1 90). t-PA may also be administered as a single bolus, although due to its relatively short half-life, it is better suited for infusion therapy. Tebbe et al, Am. J. Cardiol., 64: 448-453 (1989). A t-PA variant, specifically designed to have longer half-life and very high fibrin specificity, TNK t-PA (a T103N, NI 17Q, KHRR(296-299)AAAA t-PA variant, Keyt et al, Proc. Natl. Acad. Sci. USA.91: 3670-3674 (1994)) is particularly suitable for bolus administration. However, despite all these advances, the long-term prognosis of patient survival depends greatly on the post-infarction monitoring and treatment ofthe patients, which should include monitoring and treatment of cardiac hypertrophy.
2.3. Growth Factors Various naturally occurring polypeptides reportedly induce the proliferation of endothelial cells. Among those polypeptides are the basic and acidic fibroblast growth factors (FGF) (Burgess and Maciag, Annual Rev. Biochem., 58 : 575 (1989)), latelet-derived endothelial cell growth factor (PD-ECGF) (Ishikawa etal. , Nature, 338: 557 (1989)), and vascular endothelial growth factor (VEGF). Leung et al. Science.246: 1306 (1989); Ferrara and Henzel, Biochem. Biophvs. Res. Commun., 161: 851 (1989); Tischer et al, Biochem. Biophvs. Res. Commun., 165: 1198 (1989); EP 471/754B granted July 31, 1996.
Media conditioned by cells transfected with the human VEGF (hVEGF) cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not Leung et al, Science. 246: 1306 (1989). Several additional cDNAs were identified in human cDNA libraries that encode 121-, 189-, and 206-amino acid isoforms of hVEGF (also collectively referred to as hVEGF-related proteins). The 121 -amino acid protein differs from hVEGF by virtue ofthe deletion ofthe 44 amino acids between residues 116 and 159 in hVEGF. The 189-amino acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 116 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF). The 206-amino acid protein differs from hVEGF by virtue of an insertion of 41 amino acids at residue 116 in hVEGF. Houck et al. , Mol. Endocrin.. 5: 1806 (1991); Ferrara et al, J. Cell. Biochem., 47: 211 (1991); Ferrara et al, Endocrine Reviews. 13: 18 (1992); Keck et al, Science.246: 1309 (1989); Connolly et al, J. Biol. Chem., 264: 20017 (1989); EP 370,989 published May 30, 1990. It is now well established that angiogenesis, which involves the formation of new blood vessels from preexisting endothelium, is implicated in the patliogenesis of a variety of disorders. These include solid tumors and metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular syndromes such as proliferative retinopathies, e.g. , diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis. Folkmane/α/., J. Biol. Chem., 267: 10931-10934 (1992); Klagsbrune/fl/. Annu. Rev. Physiol., 53: 217-
239 (1991); and Garner A., "Vascular diseases", In: Pathobiology of Ocular Disease. ADvnamic Approach. Gamer A., Klintworth GK, eds., 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710.
In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment for the growth and metastasis ofthe tumor. Folkman et al. , Nature, 339: 58 (1989). Theneovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells. A tumor usually begins as a single aberrant cell which can proliferate only to a size of a few cubic millimeters due to the distance from available capillary beds, and it can stay 'dormant' without further growth and dissemination for a long period of time. Some tumor cells then switch to the angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only allow for continued growth ofthe primary tumor, but also for the dissemination and recolonization of metastatic tumor cells. Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors. Weidner 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). The precise mechanisms that control the angiogenic switch is not well understood, but it is believed that neovascularization of tumor mass results from the net balance of a multitude of angiogenesis stimulators and inhibitors (Folkman, 1995, Nat Med 1(1):27-31).
The search for positive regulators of angiogenesis has yielded many candidates, including aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α, angiogenin, IL-8, etc. Folkman et al, J.B.C., supra, and Klagsbrun et al, supra.. The negative regulators so far identified include thrombospondin (Good et αl, Proc. Natl. Acad. Sci. USA.. 82: 6624- 6628 (1990)), the 16-kilodalton N-terminal fragment of prolactin (Clapp et αl, Endocrinology, 133: 1292-1299
(1993)), angiostatin (O'Reilly et αl., Cell, 79: 315-328 (1994)), and endostatin. O'Reilly et αl, Cell, 88: 277-285 (1996). Work done over the last several years has established the key role of VEGF, not only in stimulating vascular endothelial cell proliferation, but also in inducing vascular permeability and angiogenesis. Ferrara et al, Endocr. Rev.. 18: 4-25 (1997). The finding that the loss of even a single VEGF allele results in embryonic lethality points to an irreplaceable role played by this factor in the development and differentiation ofthe vascular system. Furthermore, VEGF has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders. Ferrara et al, Endocr. Rev., supra. The VEGF mRNA is overexpressed by the majority of human tumors examined. Berkman et al, J. Clin. Invest.. 91: 153-159 (1993); Brown et al, Human Pathol.. 26: 86-91 (1995); Brown etal, Cancer Res., 53: 4727-4735 (1993); Matterne β/., Brit J. Cancer, 73: 931-934 (1996); Dvorak et al, Am. J. Pathol., 146: 1029-1039 (1995). Also, the concentration levels of VEGF in eye fluids are highly correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies. Aiello et al, N. Engl. J. Med.. 331: 1480-1487 (1994). Furthermore, recent studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by AMD. Lopez et al, Invest Ophthalmol. Vis. Sci..37: 855-868 (1996). Anti- VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in nude mice
(Kim et al, Nature, 362: 841-844 (1993); Warren et al, J. Clin. Invest. 95: 1789-1797 (1995); Borgstrόm et al, Cancer Res.. 56: 4032-4039 (1996); Melnyk et al, Cancer Res.. 56: 921-924 (1996)) and also inhibit intraocular angiogenesis in models of ischemic retinal disorders. Adamis et al, Arch. Ophthalmol., 114: 66-71 (1996). Therefore, anti- VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of solid tumors and various intraocular neovascular disorders. Such antibodies are described, for example, in EP 817,648 published January 14, 1998 and in W098/45331 and W098/45332 both published October 15, 1998.
There exist several other growth factors and mitogens, including transforming oncogenes, that are capable of rapidly inducing a complex set of genes to be expressed by certain cells. Lau and Nathans, Molecular Aspects of Cellular Regulation. 6: 165-202 (1991). These genes, which have been named immediate-early- or early- response genes, are transcriptionally activated within minutes after contact with a growth factor or mitogen, independent oϊde novo protein synthesis. A group of these intermediate-early genes encodes secreted, extracellular proteins that are needed for coordination of complex biological processes such as differentiation and proliferation, regeneration, and wound healing. Ryseck et al, Cell Growth Differ., 2: 235-233 (1991).
Highly-related proteins that belong to this group include ceflO (Simmons et al, Proc. Natl. Acad. Sci. USA, 86: 1178- 1182 ( 1989)), cyr 61 , which is rapidly activated by serum- or platelet-derived growth factor (PDGF)
(O'Brien et al, Mol. Cell Biol..10: 3569-3577 (1990), human connective tissue growth factor (CTGF) (Bradham et al , J. Cell. Biol.. 114: 1285-1294 (1991)), which is secreted by human vascular endothelial cells in high levels after activation with transforming growth factor beta (TGF-β), exhibits PDGF-like biological and immunological activities, and competes with PDGF for a particular cell surface receptor, fisp~l 2 (Ryseck et al. , Cell Growth Differ., 2: 235-233 (1991)), human vascular IBP-like growth factor (VIGF) (WO 96/17931), and nov, normally arrested in adult kidney cells, which was found to be overexpressed in myeloblastosis-associated-virus-type-1 -induced nephroblastomas. Joloit et al. Mol. Cell. Biol.. 12: 10-21 (1992). The expression of these immediate-early genes acts as "third messengers" in the cascade of events triggered by growth factors. It is also thought that they are needed to integrate and coordinate complex biological processes, such as differentiation and wound healing in which cell proliferation is a common event.
As additional mitogens, insulin-like growth factor binding proteins (IGFBPs) have been shown, in complex with insulin-like growth factor (IGF), to stimulate increased binding of IGF to fibroblast and smooth muscle cell surface receptors. Clemmons et al. J. Clin. Invest..77: 1548 (1986). Inhibitory effects of IGFBP on various IGF actions in vitro include stimulation of glucose transport by adipocytes, sulfate incorporation by chondrocytes, and thymidine incorporation in fibroblast. Zap fet al, J. Clin. Invest, 63: 1077 (1979). In addition, inhibitory effects of IGFBPs on growth factor-mediated mitogen activity in normal cells have been shown.
2.4. Need for Further Treatments
In view ofthe role of vascular endothelial cell growth and angiogenesis in many diseases and disorders, it is desirable to have a means of reducing or inhibiting one or more of the biological effects causing these processes. It is also desirable to have a means of assaying for the presence of pathogenic polypeptides in normal and diseased conditions, and especially cancer. Further, in a specific aspect, as there is no generally applicable therapy for the treatment of cardiac hypertrophy, the identification of factors that can prevent or reduce cardiac myocyte hypertrophy is of primary importance in the development of new therapeutic strategies to inhibit pathophysiological cardiac growth. While there are several treatment modalities for various cardiovascular and oncologic disorders, there is still a need for additional therapeutic approaches.
3. Summary ofthe Invention
The present invention provides compositions and methods for modulating (e.g. , promoting or inhibiting) angiogenesis and/or cardiovascularization in mammals. The present invention is based on the identification of compounds ( . e. , proteins) that test positive in various cardiovascular assays that test modulation (e.g. , promotion or inhibition) of certain biological activities. Accordingly, the compounds are believed to be useful drugs and/or drug components for the diagnosis and/or treatment (including prevention and amelioration) of disorders where such effects are desired, such as the promotion or inhibition of angiogenesis, inhibition or stimulation of vascular endothelial cell growth, stimulation of growth or proliferation of vascular endothelial cells, inhibition of tumor growth, inhibition of angiogenesis-dependent tissue growth, stimulation of angiogenesis-dependent tissue growth, inhibition of cardiac hypertrophy and stimulation of cardiac hypertrophy, e.g., for the treatment of congestive heart failure. In addition, the compositions and methods ofthe invention provide for the diagnostic monitoring ofpatients undergoing clinical evaluation for the treatment of angiogenesis-related disorders, for monitoring the efficacy of compounds in clinical trials and for identifying subjects who may be predisposed to such angiogenic-related disorders. In one embodiment, the present invention provides a composition comprising a PRO polypeptide, an agonist or antagonist thereof, or an anti-PRO antibody in admixture with a pharmaceutically acceptable carrier. In one aspect, the composition comprises a therapeutically effective amount ofthe polypeptide, agonist, antagonist or antibody. In another aspect, the composition comprises a further active ingredient, namely, a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent. Preferably, the composition is sterile. The PRO polypeptide, agonist, antagonist or antibody may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability. Preserved liquid pharmaceutical formulations might contain multiple doses of PRO polypeptide, agonist, antagonist or antibody, and might, therefore, be suitable for repeated use. In a preferred embodiment, where the composition comprises an antibody, the antibody is a monoclonal antibody, an antibody fragment, a humanized antibody, or a single-chain antibody.
In a further embodiment, the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of a PRO polypeptide, agonist, antagonist or antibody with a pharmaceutically acceptable carrier.
In a still further aspect, the present invention provides an article of manufacture comprising:
(a) a composition of matter comprising a PRO polypeptide or agonist or antagonist thereof;
(b) a container containing said composition; and (c) a label affixed to said container, or a package insert included in said container referring to the use of said PRO polypeptide or agonist or antagonist thereof in the treatment of a cardiovascular, endothelial or angiogenic disorder, wherein the agonist or antagonist may be an antibody which binds to the PRO polypeptide. The composition may comprise a therapeutically effective amount of the PRO polypeptide or the agonist or antagonist thereof. In another embodiment, the present invention provides a method for identifying an agonist of a PRO polypeptide comprising:
(a) contacting cells and a test compound to be screened under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide; and
(b) determining the induction of said cellular response to determine if the test compound is an effective agonist, wherein the induction of said cellular response is indicative of said test compound being an effective agonist.
In another embodiment, the present invention provides a method for identifying an agonist of a PRO polypeptide comprising:
(a) contacting cells and a test compound to be screened under conditions suitable for the stimulation of cell proliferation by a PRO polypeptide; and
(b) measuring the proliferation of said cells to determine if the test compound is an effective agonist, wherein the stimulation of cell proliferation is indicative of said test compound being an effective agonist.
In another embodiment, the invention provides a method for identifying a compound that inhibits the activity of a PRO polypeptide comprising contacting a test compound with a PRO polypeptide under conditions and for a time sufficient to allow the test compound and polypeptide to interact and determining whether the activity ofthe PRO polypeptide is inhibited. In a specific preferred aspect, either the test compound or the PRO polypeptide is immobilized on a solid support. In another preferred aspect, the non- immobilized component carries a detectable label. In a preferred aspect, this method comprises the steps of:
(a) contacting cells and a test compound to be screened in the presence of a PRO polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide; and
(b) determining the induction of said cellular response to determine if the test compound is an effective antagonist.
In another preferred aspect, this process comprises the steps of:
(a) contacting cells and a test compound to be screened in the presence of a PRO polypeptide under conditions suitable for the stimulation of cell proliferation by a PRO polypeptide; and
(b) measuring the proliferation ofthe cells to determine if the test compound is an effective antagonist. In another embodiment, the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that normally expresses the polypeptide, wherein the method comprises contacting the cells with a test compound and determining whether the expression of the PRO polypeptide is inhibited. In a preferred aspect, this method comprises the steps of:
(a) contacting cells and a test compound to be screened under conditions suitable for allowing expression of the PRO polypeptide; and
(b) determining the inhibition ofexpression of said polypeptide.
In a still further embodiment, the invention provides a compound that inhibits the expression of a PRO polypeptide, such as a compound that is identified by the methods set forth above.
Another aspect ofthe present invention is directed to an agonist or an antagonist of a PRO polypeptide which may optionally be identified by the methods described above.
One type of antagonist of a PRO polypeptide that inhibits one or more ofthe functions or activities ofthe PRO polypeptide is an antibody. Hence, in another aspect, the invention provides an isolated antibody that binds a PRO polypeptide. In a preferred aspect, the antibody is a monoclonal antibody, which preferably has non-human complementarity-determining-region (CDR) residues and human framework-region (FR) residues. The antibody may be labeled and may be immobilized on a solid support. In a further aspect, the antibody is an antibody fragment, a single-chain antibody, or a humanized antibody. Preferably, the antibody specifically binds to the polypeptide.
In a still further aspect, the present invention provides a method for diagnosing a disease or susceptibility to a disease which is related to a mutation in a PRO polypeptide-encoding nucleic acid sequence comprising determining the presence or absence of said mutation in the PRO polypeptide nucleic acid sequence, wherein the presence or absence of said mutation is indicative ofthe presence of said disease or susceptibility to said disease.
In a still further aspect, the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises analyzing the level o expression of a gene encoding a PRO polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal tissue cells ofthe same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample is indicative ofthe presence of a cardiovascular, endothelial or angiogenic disorder in said mammal. The expression of a gene encoding a PRO polypeptide may optionally be accomplished by measuring the level of mRNA or the polypeptide in the test sample as compared to the control sample.
In a still further aspect, the present invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises detecting the presence or absence of a PRO polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of said PRO polypeptide in said test sample is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in said mammal.
In a still further embodiment, the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal comprising (a) contacting an anti-PRO antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the PRO polypeptide in the test sample, wherein the formation of said complex is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in the mammal. The detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells ofthe same cell type. A larger or smaller quantity of complexes formed in the test sample indicates the presence of a cardiovascular, endothelial or angiogenic dysfunction in the mammal from which the test tissue cells were obtained. The antibody preferably carries a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. The test sample is usually obtained from an individual suspected to have a cardiovascular, endothelial or angiogenic disorder.
In another embodiment, the invention provides a method for determining the presence of a PRO polypeptide in a sample comprising exposing a sample suspected of containing the PRO polypeptide to an anti-PRO antibody and determining binding of said antibody to a component of said sample. In a specific aspect, the sample comprises a cell suspected of containing the PRO polypeptide and the antibody binds to the cell. The antibody is preferably detectably labeled and/or bound to a solid support.
In further aspects, the invention provides a cardiovascular, endothelial or angiogenic disorder diagnostic kit comprising an anti-PRO antibody and a carrier in suitable packaging. Preferably, such kit further comprises instructions for using said antibody to detect the presence of the PRO polypeptide. Preferably, the carrier is a buffer, for example. Preferably, the cardiovascular, endothelial or angiogenic disorder is cancer.
In yet another embodiment, the present invention provides a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of a PRO polypeptide. Preferably, the disorder is cardiac hypertrophy, trauma such as wounds or bums, or a type of cancer. In a further aspect, the mammal is further exposed to angioplasty or a drug that treats cardiovascular, endothelial or angiogenic disorders such as ACE inhibitors or chemotherapeutic agents if the cardiovascular, endothelial or angiogenic disorder is a type of cancer. Preferably, the mammal is human, preferably one who is at risk of developing cardiac hypertrophy and more preferably has suffered myocardial infarction.
In another preferred aspect, the cardiac hypertrophy is characterized by the presence of an elevated level of PGF2o. Alternatively, the cardiac hypertrophy may be induced by myocardial infarction, wherein preferably the administration ofthe PRO polypeptide is initiated within 48 hours, more preferably within 24 hours, following myocardial infarction. In another preferred embodiment, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy and said PRO polypeptide is administered together with a cardiovascular, endothelial or angiogenic agent. The preferred cardiovascular, endothelial or angiogenic agent for this purpose is selected from the group consisting of an antihypertensive drug, an ACE inhibitor, an endothelin receptor antagonist and a thrombolytic agent. If a thrombolytic agent is administered, preferably the PRO polypeptide is administered following administration of such agent. More preferably, the tlirombolytic agent is recombinant human tissue plasminogen activator.
In another preferred aspect, 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, preferably wherein the mammal is further exposed to angioplasty or a cardiovascular, endothelial, or angiogenic agent.
In another preferred embodiment, the cardiovascular, endothelial or angiogenic disorder is a cancer and the PRO polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent or a cytotoxic agent. In a further embodiment, the invention provides a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of a PRO polypeptide agonist, antagonist or anti-PRO antibody. Preferably, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration. Also preferred is where the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the agonist, antagonist or anti-PRO antibody.
In still further embodiments, the invention provides a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal that suffers therefrom comprising administering to the mammal a nucleic acid molecule that codes for either (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide or (c) an antagonist of a PRO polypeptide, wherein said agonist or antagonist may be an anti-PRO antibody. In a preferred embodiment, the mammal is human. In another preferred embodiment, the gene is administered via ex vivo gene therapy. In a further preferred embodiment, the gene is comprised within a vector, more preferably an adenoviral, adeno-associated viral, lentiviral, or retroviral vector.
In yet another aspect, the invention pro vides a recombinant retroviral particle comprising a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide, or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein the retroviral vector is in association with retroviral structural proteins. Preferably, the signal sequence is from a mammal, such as from a native PRO polypeptide.
In a still further embodiment, the invention supplies an ex vivo producer cell comprising a nucleic acid construct that expresses retroviral structural proteins and also comprises a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide or
(c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion ofthe polypeptide, wherein said producer cell packages the retroviral vector in association with the structural proteins to produce recombinant retroviral particles.
In yet another embodiment, the invention provides a method for inhibiting endothelial cell growth in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or
(c) an antagonist of a PRO polypeptide, wherein endothelial cell growth in said mammal is inhibited, and wherein said agonist or antagonist may be an anti-PRO antibody. Preferably, the mammal is human and the endothelial cell growth is associated with a tumor or a retinal disorder.
In yet another embodiment, the invention provides a method for stimulating endothelial cell growth in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein endothelial cell growth in said mammal is stimulated, and wherein said agonist or antagonist may be an anti-PRO antibody. Preferably, the mammal is human.
In yet another embodiment, the invention provides a method for inhibiting cardiac hypertrophy in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is inhibited, and wherein said agonist or antagonist may be an anti-PRO antibody. Preferably, the mammal is human and the cardiac hypertrophy has been induced by myocardial infarction.
In yet another embodiment, the invention provides a method for stimulating cardiac hypertrophy in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is stimulated, and wherein said agonist or antagonist may be an anti-PRO antibody. Preferably, the mammal is human who suffers from congestive heart failure.
In yet another embodiment, the invention provides a method for inliibiting angiogenesis induced by a PRO polypeptide in a mammal comprising administering a therapeutically effective amount of an anti-PRO antibody to the mammal. Preferably, the mammal is a human, and more preferably the mammal has a tumor or a retinal disorder. In yet another embodiment, the invention provides a method for stimulating angiogenesis induced by a
PRO polypeptide in a mammal comprising administering a therapeutically effective amount of a PRO polypeptide to the mammal. Preferably, the mammal is a human, and more preferably angiogenesis would promote tissue regeneration or wound healing.
In yet another embodiment, the invention provides a method for modulating (e.g. , inhibiting or stimulating) endothelial cell growth in a mammal comprising administering to the mammal a PR021, PR0181, PRO205,
PR0214, PR0221, PR0229, PR0231, PR0238, PR0241, PR0247, PR0256, PR0258, PR0263, PR0265, PR0295, PR0321, PR0322, PR0337, PR0363, PR0365, PR0444, PR0533, PR0697, PRO720, PR0725, PR0771, PR0788, PR0791, PR0819, PR0827, PR0828, PR0836, PR0846, PR0865, PRO1005, PRO1006, PRO1007, PRO1025, PRO1029, PRO1054, PRO1071, PRO1075, PRO1079, PRO1080, PR01114, PROH31, PR01155, PRO1160, PROH84, PR01186, PRO1190, PROH92, PROH95, PR01244, PR01272, PR01273,
PR01274, PR01279, PR01283, PR01286, PRO1306, PRO1309, PR01325, PR01329, PR01347, PR01356, PR01376, PR01382, PR01411, PR01412, PR01419, PR01474, PR01477, PR01488, PRO1508, PRO1550, PR01556, PRO1760, PR01782, PR01787, PRO1801, PR01868, PR01887, PRO1890, PR03438, PR03444, PRO4302, PR04324, PR04333, PR04341, PR04342, PR04353, PR04354, PR04356, PR04371, PRO4405, PRO4408, PR04422, PR04425, PR04499, PR05723, PR05725, PR05737, PR05776, PRO6006, PRO6029, PRO6071, PR07436, PR09771, PR09821, PR09873, PRO10008, PRO10096, PRO19670, PRO20040, PRO200445 PRO21055, PRO21384 orPRO28631 polypeptide, agonist or antagonist thereof, wherein endothelial cell growth in said mammal is modulated.
In yet another embodiment, the invention provides amethod for modulating (e.g. , inliibiting or stimulating) smooth muscle cell growth in a mammal comprising administering to the mammal a PRO 162, PR0181, PRO 182, PR0195, PRO204, PR0221, PRO230, PR0256, PR0258, PR0533, PR0697, PR0725, PR0738, PR0826, PR0836,PR0840,PR0846,PR0865,PR0982,PR01025,PR01029,PR01071,PR01080,PR01083,PR01134,
PRO1160, PR01182, PR01184, PR01186, PR01192, PR01265, PR01274, PR01279, PR01283, PRO1306, PRO1308, PRO1309, PR01325, PR01337, PR01338, PR01343, PR01376, PR01387, PR01411, PR01412, PR01415, PR01434, PR01474, PR01488, PRO1550, PR01556, PR01567, PRO1600, PR01754, PR01758, PRO1760, PR01787, PR01865, PR01868, PR01917, PR01928, PR03438, PR03562, PRO4302, PR04333, PR04345, PR04353, PR04354, PRO4405, PRO4408, PRO4430, PRO4503, PR05725, PR06714, PR09771,
PRO9820,PRO9940,PRO10096,PRO21055,PRO21184 or PR021366 polypeptide, agonist or antagonistthereof, wherein endothelial cell growth in said mammal is modulated.
In yet another embodiment, the invention provides a method for modulating (e.g. , inducing or reducing) cardiac hypertrophy in a mammal comprising administering to the mammal a PR021 polypeptide, agonist or antagonist thereof, wherein cardiac hypertrophy in said mammal is modulated.
In yet another embodiment, the invention provides a method for modulating (e.g., inducing or reducing) endothelial cell apoptosis in a mammal comprising administering to the mammal a PRO4302 polypeptide, agonist or antagonist thereof, wherein cardiac hypertrophy in said mammal is modulated.
In yet another embodiment, the invention provides a method for modulating (e.g. , stimulating or inhibiting) angiogenesis in a mammal comprising administering a therapeutically effective amount of a PRO 1376 or PRO 1449 polypeptide, agonist or antagonist thereof to the mammal, wherein said angiogenesis is modulated.
In yet another embodiment, the invention provides a method for modulating (e.g., inducing or reducing) angiogenesis by modulating (e.g., inducing or reducing) endothelial cell tube formation in a mammal comprising administering to the mammal a PR0178, PR0195, PR0228, PRO301, PRO302, PR0532, PR0724, PRO730, PR0734,PR0793,PR0871,PR0938,PR01012,PR01120,PR01139,PR01198,PR01287,PR01361,PR01864,
PR01873, PRO2010, PR03579, PR04313, PR04527, PR04538, PR04553, PR04995, PRO5730, PRO6008, PR07223, PR07248 or PR07261 polypeptide, agonist or antagonist thereof, wherein endothelial cell tube formation in said mammal is modulated.
In other embodiments ofthe present invention, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment ofthe full-length amino acid sequence as disclosed herein, or (b) the complement ofthe DNA molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PRO polypeptide cDNA as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement ofthe DNA molecule of (a).
In a further aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%o, 96%, 97% or 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any ofthe human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement ofthe DNA molecule of (a).
Another aspect ofthe present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains ofthe herein described PRO polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO antibody or as antisense oligonucleotide probes. Such nucleic acid fragments are usually at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700 or 800 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence fragments) are novel. All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody.
In another embodiment, the invention provides an isolated PRO polypeptide encoded by any ofthe isolated nucleic acid sequences hereinabove identified.
In a certain aspect, the invention provides an isolatedPRO polypeptide comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment ofthe full-length amino acid sequence as disclosed herein.
In a further aspect, the invention provides an isolated PRO polypeptide comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97% or 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to an amino acid sequence encoded by any ofthe human protein cDNAs deposited with the ATCC as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and that is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes forproducing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression ofthe PRO polypeptide and recovering the PRO polypeptide from the cell culture. Another aspect of the invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated. Processes forproducing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression ofthe PRO polypeptide and recovering the PRO polypeptide from the cell culture. In yet another embodiment, the invention provides agonists and antagonists of a native PRO polypeptide as defined herein. In a particular embodiment, the agonist or antagonist is an anti-PRO antibody or a small molecule.
In a further embodiment, the invention provides a method of identifying agonists or antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide. Preferably, the PRO polypeptide is a native PRO polypeptide.
In a still further embodiment, the invention provides a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
Another embodiment ofthe present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an agonist or antagonist thereof or an anti-PRO antibody. In additional embodiments of the present invention, the invention provides vectors comprising DNA encoding any ofthe herein described polypeptides. Host cells comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli, yeast, or Baculo virus-infected insect cells. A process for producing any ofthe herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression ofthe desired polypeptide and recovering the desired polypeptide from the cell culture.
In other embodiments, the invention provides chimeric molecules comprising any ofthe herein described polypeptides fused to a heterologous polypeptide or amino acid sequence. Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin.
In yet another embodiment, the invention provides an antibody which specifically binds to any ofthe above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences.
4. Brief Description ofthe Drawings
Figure 1 shows a nucleotide sequence (SEQ ID NO : 1 ) of a native sequence PRO 181 cDNA, wherein SEQ JD NO: 1 is a clone designated herein as "DNA23330-1390".
Figure 2 shows the amino acid sequence (SEQ ID NO:2) derived from the coding sequence of SEQ ID NO:l shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ JD NO:3) of a native sequence PRO 178 cDNA, wherein SEQ ID NO:3 is a clone designated herein as "DNA23339-1130". Figure 4 shows the amino acid sequence (SEQ ID NO:4) derived from the coding sequence of SEQ JD
NO:3 shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ JD NO:5) of a native sequence PR0444 cDNA, wherein SEQ ID NO:5 is a clone designated herein as "DNA26846-1397".
Figure 6 shows the amino acid sequence (SEQ JD NO:6) derived from the coding sequence of SEQ ID NO:5 shown in Figure 5.
Figure 7 shows a nucleotide sequence (SEQ ID NO:7) of a native sequence PRO 195 cDNA, wherein SEQ JD NO:7 is a clone designated herein as "DNA26847-1395".
Figure 8 shows the amino acid sequence (SEQ ID NO: 8) derived from the coding sequence of SEQ ID NO: 7 shown in Figure 7. Figure 9 shows a nucleotide sequence (SEQ JD NO :9) of a native sequence PRO 182 cDNA, wherein SEQ
ID NO:9 is a clone designated herein as "DNA27865-1091".
Figure 10 shows the amino acid sequence (SEQ ID NO:10) derived from the coding sequence of SEQ JD NO:9 shown in Figure 9.
Figure 11 shows a nucleotide sequence (SEQ JD NO: 11) of a native sequence PRO205 cDNA, wherein SEQ ID NO:l 1 is a clone designated herein as "DNA30868-1156".
Figure 12 shows the amino acid sequence (SEQ ID NO:12) derived from the coding sequence of SEQ ID NO : 11 shown in Figure 11.
Figure 13 shows a nucleotide sequence (SEQ ID NO:13) of a native sequence PRO204 cDNA, wherein SEQ ID NO: 13 is a clone designated herein as "DNA30871-1157".
Figure 14 shows the amino acid sequence (SEQ ID NO:14) derived from the coding sequence of SEQ ID NO:13 shown in Figure 13. Figure 15 shows a nucleotide sequence (SEQ ID NO: 15) of a native sequence PR01873 cDNA, wherein
SEQ JD N0:15 is a clone designated herein as "DNA30880".
Figure 16 shows the amino acid sequence (SEQ ID N0:16) derived from the coding sequence of SEQ ID NO: 15 shown in Figure 15.
Figure 17 shows a nucleotide sequence (SEQ ID NO: 17) of a native sequence PR0214 cDNA, wherein SEQ ID NO: 17 is a clone designated herein as "DNA32286-1191".
Figure 18 shows the amino acid sequence (SEQ ID NO: 18) derived from the coding sequence of SEQ JD NO: 17 shown in Figure 17.
Figure 19 shows a nucleotide sequence (SEQ ID NO:19) of a native sequence PR0221 cDNA, wherein SEQ JD NO:19 is a clone designated herein as "DNA33089-1132". Figure 20 shows the amino acid sequence (SEQ JD NO:20) derived from the coding sequence of SEQ JD
NO: 19 shown in Figure 19.
Figure 21 shows a nucleotide sequence (SEQ ID NO:21) of a native sequence PR0228 cDNA, wherein SEQ ID NO:21 is a clone designated herein as "DNA33092-1202".
Figure 22 shows the amino acid sequence (SEQ ID NO:22) derived from the coding sequence of SEQ ID NO:21 shown in Figure 21.
Figure 23 shows a nucleotide sequence (SEQ JD NO:23) of a native sequence PR0229 cDNA, wherein SEQ ID N0:23 is a clone designated herein as "DNA33100-1159".
Figure 24 shows the amino acid sequence (SEQ JD NO:24) derived from the coding sequence of SEQ ID N0:23 shown in Figure 23. Figure 25 shows a nucleotide sequence (SEQ ID NO:25) of a native sequence PRO230 cDNA, wherein
SEQ TD NO:25 is a clone designated herein as "DNA33223-1136".
Figure 26 shows the amino acid sequence (SEQ ID NO:26) derived from the coding sequence of SEQ JD NO:25 shown in Figure 25.
Figure 27 shows a nucleotide sequence (SEQ ID NO:27) of a native sequence PR07223 cDNA, wherein SEQ ID N0:27 is a clone designated herein as "DNA34385".
Figure 28 shows the amino acid sequence (SEQ ID N0:28) derived from the coding sequence of SEQ JD N0:27 shown in Figure 27. Figure 29 shows a nucleotide sequence (SEQ ID NO:29) of a native sequence PR0241 cDNA, wherein SEQ ID N0:29 is a clone designated herein as "DNA34392-1170".
Figure 30 shows the amino acid sequence (SEQ ID NO:30) derived from the coding sequence of SEQ ID NO:29 shown in Figure 29. Figure 31 shows a nucleotide sequence (SEQ ID NO:31) of a native sequence PR0263 cDNA, wherein
SEQ ID NO:31 is a clone designated herein as "DNA34431-1177".
Figure 32 shows the amino acid sequence (SEQ ID NO:32) derived from the coding sequence of SEQ JD N0:31 shown in Figure 31.
Figure 33 shows a nucleotide sequence (SEQ ID NO:33) of a native sequence PR0321 cDNA, wherein SEQ ID N0:33 is a clone designated herein as "DNA34433-1308".
Figure 34 shows the amino acid sequence (SEQ JD N0:34) derived from the coding sequence of SEQ JD N0:33 shown in Figure 33.
Figure 35 shows a nucleotide sequence (SEQ ID NO:35) of a native sequence PR0231 cDNA, wherein SEQ ID N0:35 is a clone designated herein as "DNA34434-1139". Figure 36 shows the amino acid sequence (SEQ ID N0:36) derived from the coding sequence of SEQ JD
NO:35 shown in Figure 35.
Figure 37 shows a nucleotide sequence (SEQ JD NO:37) of a native sequence PR0238 cDNA, wherein SEQ ID NO:37 is a clone designated herein as "DNA35600- 1162".
Figure 38 shows the amino acid sequence (SEQ ID NO:38) derived from the coding sequence of SEQ JD NO:37 shown in Figure 37.
Figure 39 shows a nucleotide sequence (SEQ JD NO: 39) of a native sequence PR0247 cDNA, wherein SEQ JD NO:39 is a clone designated herein as "DNA35673-1201".
Figure 40 shows the amino acid sequence (SEQ ID NO:40) derived from the coding sequence of SEQ JD NO:39 shown in Figure 39. Figure 41 shows a nucleotide sequence (SEQ JD NO:41) of a native sequence PR0256 cDNA, wherein
SEQ ID NO:41 is a clone designated herein as "DNA35880-1160".
Figure 42 shows the amino acid sequence (SEQ ID NO:42) derived from the coding sequence of SEQ JD NO:41 shown in Figure 41.
Figure 43 shows a nucleotide sequence (SEQ JD NO:43) of a native sequence PR0258 cDNA, wherein SEQ JD NO:43 is a clone designated herein as "DNA35918-1174".
Figure 44 shows the amino acid sequence (SEQ JD NO:44) derived from the coding sequence of SEQ ID NO:43 shown in Figure 43.
Figure 45 shows a nucleotide sequence (SEQ JD NO:45) of a native sequence PR0265 cDNA, wherein SEQ ID N0:45 is a clone designated herein as "DNA36350-1158". Figure 46 shows the amino acid sequence (SEQ ID NO :46) derived from the coding sequence of SEQ JD
NO:45 shown in Figure 45.
Figure 47 shows a nucleotide sequence (SEQ ID NO:47) of a native sequence PR021 cDNA, wherein SEQ JD NO:47 is a clone designated herein as "DNA36638-1056".
Figure 48 shows the amino acid sequence (SEQ JD NO:48) derived from the coding sequence of SEQ JD NO:47 shown in Figure 47.
Figure 49 shows a nucleotide sequence (SEQ ID NO:49) of a native sequence PR0295 cDNA, wherein SEQ ID NO:49 is a clone designated herein as "DNA38268-1188".
Figure 50 shows the amino acid sequence (SEQ ID NO:50) derived from the coding sequence of SEQ JD NO:49 shown in Figure 49.
Figure 51 shows a nucleotide sequence (SEQ ID NO:51) of a native sequence PRO302 cDNA, wherein SEQ ID NO:51 is a clone designated herein as "DNA40370-1217". Figure 52 shows the amino acid sequence (SEQ JD NO:52) derived from the coding sequence of SEQ ID
NO:51 shown in Figure 51.
Figure 53 shows a nucleotide sequence (SEQ JD NO:53) of a native sequence PRO301 cDNA, wherein SEQ ID NO:53 is a clone designated herein as "DNA40628-1216".
Figure 54 shows the amino acid sequence (SEQ ID NO:54) derived from the coding sequence of SEQ ID NO:53 shown in Figure 53.
Figure 55 shows a nucleotide sequence (SEQ ID NO:55) of a native sequence PR0337 cDNA, wherein SEQ ID N0:55 is a clone designated herein as "DNA43316-1237".
Figure 56 shows the amino acid sequence (SEQ ID NO:56) derived from the coding sequence of SEQ ID NO:55 shown in Figure 55. Figure 57 shows a nucleotide sequence (SEQ ID NO:57) of a native sequence PR07248 cDNA, wherein
SEQ ID NO:57 is a clone designated herein as "DNA44195".
Figure 58 shows the amino acid sequence (SEQ JD NO:58) derived from the coding sequence of SEQ JD NO:57 shown in Figure 57.
Figure 59 shows a nucleotide sequence (SEQ ID NO:59) of a native sequence PR0846 cDNA, wherein SEQ ID NO:59 is a clone designated herein as "DNA44196-1353".
Figure 60 shows the amino acid sequence (SEQ JD NO:60) derived from the coding sequence of SEQ JD NO:59 shown in Figure 59.
Figure 61 shows a nucleotide sequence (SEQ JD NO: 61 ) of a native sequence PRO 1864 cDNA, wherein SEQ ID NO:61 is a clone designated herein as "DNA45409-2511". Figure 62 shows the amino acid sequence (SEQ ID NO:62) derived from the coding sequence of SEQ ID
NO:61 shown in Figure 61.
Figure 63 shows a nucleotide sequence (SEQ JD NO:63) of a native sequence PR0363 cDNA, wherein SEQ ID N0:63 is a clone designated herein as "DNA45419-1252".
Figure 64 shows the amino acid sequence (SEQ ID NO: 64) derived from the coding sequence of SEQ ID NO:63 shown in Figure 63.
Figure 65 shows a nucleotide sequence (SEQ JD NO:65) of a native sequence PRO730 cDNA, wherein SEQ JD NO:65 is a clone designated herein as "DNA45624-1400". Figure 66 shows the amino acid sequence (SEQ JD NO:66) derived from the coding sequence of SEQ ID NO:65 shown in Figure 65.
Figure 67 shows a nucleotide sequence (SEQ ID NO:67) of a native sequence PR0365 cDNA, wherein SEQ ID NO:67 is a clone designated herein as "DNA46777-1253". Figure 68 shows the amino acid sequence (SEQ ID NO:68) derived from the coding sequence of SEQ JD
NO:67 shown in Figure 67.
Figure 69 shows a nucleotide sequence (SEQ JD NO:69) of a native sequence PR0532 cDNA, wherein SEQ ID NO:69 is a clone designated herein as "DNA48335".
Figure 70 shows the amino acid sequence (SEQ ID NO:70) derived from the coding sequence of SEQ ID NO:69 shown in Figure 69.
Figure 71 shows a nucleotide sequence (SEQ JD NO:71) of a native sequence PR0322 cDNA, wherein SEQ JD NO:71 is a clone designated herein as "DNA48336-1309".
Figure 72 shows tire amino acid sequence (SEQ JD NO:72) derived from the coding sequence of SEQ ID NO:71 shown in Figure 71. Figure 73 shows a nucleotide sequence (SEQ JD NO:73) of a native sequence PROl 120 cDNA, wherein
SEQ JD NO:73 is a clone designated herein as "DNA48606-1479".
Figure 74 shows the amino acid sequence (SEQ ID NO:74) derived from the coding sequence of SEQ ID NO:73 shown in Figure 73.
Figure 75 shows a nucleotide sequence (SEQ JD NO:75) of a native sequence PR07261 cDNA, wherein SEQ ID NO:75 is a clone designated herein as "DNA49149".
Figure 76 shows the amino acid sequence (SEQ ID NO:76) derived from the coding sequence of SEQ JD NO:75 shown in Figure 75.
Figure 77 shows a nucleotide sequence (SEQ ID NO:77) of a native sequence PR0533 cDNA, wherein SEQ TD NO:77 is a clone designated herein as "DNA49435-1219". Figure 78 shows the amino acid sequence (SEQ ID NO:78) derived from the coding sequence of SEQ ID
NO:77 shown in Figure 77.
Figure 79 shows a nucleotide sequence (SEQ ID NO:79) of a native sequence PR0724 cDNA, wherein SEQ ID NO:79 is a clone designated herein as "DNA49631-1328".
Figure 80 shows the amino acid sequence (SEQ JD NO:80) derived from the coding sequence of SEQ JD NO:79 shown in Figure 79.
Figure 81 shows a nucleotide sequence (SEQ JD NO:81) of a native sequence PR0734 cDNA, wherein SEQ ID NO:81 is a clone designated herein as "DNA49817" .
Figure 82 shows the amino acid sequence (SEQ ID NO:82) derived from the coding sequence of SEQ JD NO: 81 shown in Figure 81. Figure 83 shows a nucleotide sequence (SEQ ID NO:83) of a native sequence PR0771 cDNA, wherein
SEQ JD NO:83 is a clone designated herein as "DNA49829-1346".
Figure 84 shows the amino acid sequence (SEQ JD NO:84) derived from the coding sequence of SEQ JD NO: 83 shown in Figure 83.
Figure 85 shows a nucleotide sequence (SEQ JD NO:85) of a native sequence PRO2010 cDNA, wherem SEQ JD NO:85 is a clone designated herein as "DNA50792".
Figure 86 shows the amino acid sequence (SEQ ID NO:86) derived from the coding sequence of SEQ ID NO:85 shown in Figure 85.
Figure 87 shows a nucleotide sequence (SEQ ID NO:87) of a native sequence PR0871 cDNA, wherein SEQ JD NO:87 is a clone designated herein as "DNA50919-1361".
Figure 88 shows the amino acid sequence (SEQ ID NO:88) derived from the coding sequence of SEQ JD NO:87 shown in Figure 87. Figure 89 shows a nucleotide sequence (SEQ JD NO:89) of a native sequence PR0697 cDNA, wherein
SEQ ID NO:89 is a clone designated herein as "DNA50920-1325".
Figure 90 shows the amino acid sequence (SEQ ID NO:90) derived from the coding sequence of SEQ ID NO:89 shown in Figure 89.
Figure 91 shows a nucleotide sequence (SEQ ID NO:91) of a native sequence PRO1083 cDNA, wherein SEQ JD NO:91 is a clone designated herein as "DNA50921-1458".
Figure 92 shows the amino acid sequence (SEQ JD NO:22) derived from the coding sequence of SEQ JD NO:91 shown in Figure 91.
Figure 93 shows a nucleotide sequence (SEQ ID NO:93) of a native sequence PR0725 cDNA, wherein SEQ JD NO:93 is a clone designated herein as "DNA52758-1399". Figure 94 shows the amino acid sequence (SEQ ID NO:94) derived from the coding sequence of SEQ JD
NO:93 shown in Figure 93.
Figure 95 shows a nucleotide sequence (SEQ ID NO:95) of a native sequence PRO720 cDNA, wherein SEQ JD NO:95 is a clone designated herein as "DNA53517-1366-1".
Figure 96 shows the amino acid sequence (SEQ ID NO:96) derived from the coding sequence of SEQ ID NO:95 shown in Figure 95.
Figure 97 shows a nucleotide sequence (SEQ JD NO:97) of a native sequence PR0738 cDNA, wherein SEQ ID NO:97 is a clone designated herein as "DNA53915-1258".
Figure 98 shows the amino acid sequence (SEQ ID NO:98) derived from the coding sequence of SEQ JD NO: 97 shown in Figure 97. Figure 99 shows a nucleotide sequence (SEQ ID NO:99) of a native sequence PR0865 cDNA, wherein
SEQ ID NO:99 is a clone designated herein as "DNA53974-1401".
Figure 100 shows the amino acid sequence (SEQ JD NO: 100) derived from the coding sequence of SEQ JD NO:99 shown in Figure 99.
Figure 101 shows a nucleotide sequence (SEQ ID NO : 101 ) of a native sequence PRO840 cDNA, wherein SEQ JD NO: 101 is a clone designated herein as "DNA53987-1438".
Figure 102 shows the amino acid sequence (SEQ JD NO: 102) derived from the coding sequence of SEQ J NO:101 shown in Figure 101. Figure 103 shows anucleotide sequence (SEQ ID NO: 103) of a native sequence PRO 1080 cDNA, wherein SEQ JD NO:103 is a clone designated herein as "DNA56047-1456".
Figure 104 shows the amino acid sequence (SEQ JD NO: 104) derived from the coding sequence of SEQ ID NO:103 shown in Figure 103. Figure 105 shows a nucleotide sequence (SEQ JD NO: 105) of a native sequence PRO 1079 cDNA, wherein
SEQ ID NO:105 is a clone designated herein as "DNA56050-1455".
Figure 106 shows the amino acid sequence (SEQ ID NO: 106) derived from the coding sequence of SEQ ID NO: 105 shown in Figure 105.
Figure 107 shows a nucleotide sequence (SEQ JD NO : 107) of a native sequence PR0793 cDNA, wherein SEQ ID NO: 107 is a clone designated herein as "DNA56110-1437".
Figure 108 shows the amino acid sequence (SEQ ID NO: 108) derived from the coding sequence of SEQ JD NO: 107 shown in Figure 107.
Figure 109 shows anucleotide sequence (SEQ ID NO:109) of a native sequence PR0788 cDNA, wherein SEQ JD NO:109 is a clone designated herein as "DNA56405-1357". Figure 110 shows the amino acid sequence (SEQ ID NO: 110) derived from the coding sequence of SEQ
ID NO: 109 shown in Figure 109.
Figure 111 shows a nucleotide sequence (SEQ ID NO: 111) of a native sequence PR0938 cDNA, wherein SEQ ID NO:l 11 is a clone designated herein as "DNA56433-1406".
Figure 112 shows the amino acid sequence (SEQ ID NO: 112) derived from the coding sequence of SEQ JD NO:l 11 shown in Figure 111.
Figure 113 shows anucleotide sequence (SEQ ID NO: 113) of a native sequence PRO1012 cDNA, wherein SEQ JD NO: 113 is a clone designated herein as "DNA56439-1376".
Figure 114 shows the amino acid sequence (SEQ JD NO:l 14) derived from the coding sequence of SEQ ID NO:113 shown in Figure 113. Figure 115 shows a nucleotide sequence (SEQ ID NO : 115) of a native sequence PRO 1477 cDNA, wherein
SEQ JD NO:l 15 is a clone designated herein as "DNA56529-1647".
Figure 116 shows the amino acid sequence (SEQ ID NO: 116) derived from the coding sequence of SEQ JD NO: 115 shown in Figure 115.
Figure 117 shows anucleotide sequence (SEQJDNO:l 17) of anative sequence PROl 134 cDNA, wherein SEQ ID NO:l 17 is a clone designated herein as "DNA56865-1491".
Figure 118 shows the amino acid sequence (SEQ ID NO: 118) derived from the coding sequence of SEQ JD NO: 117 shown in Figure 117.
Figure 119 shows a nucleotide sequence (SEQ ID NO: 119) of a native sequence PRO 162 cDNA, wherein SEQ ID NO:119 is a clone designated herein as "DNA56965-1356". Figure 120 shows the amino acid sequence (SEQ JD NO:120) derived from the coding sequence of SEQ
ID NO: 119 shown in Figure 119.
Figure 121 shows a nucleotide sequence (SEQ JD NO: 121) of a native sequence PRO 1114 cDNA, wherein SEQ JD NO:121 is a clone designated herein as "DNA57033-1403-1".
Figure 122 shows the amino acid sequence (SEQ ID NO: 122) derived from the coding sequence of SEQ ID NO-.121 shown in Figure 121.
Figure 123 shows a nucleotide sequence (SEQ ID NO: 123) of a native sequence PR0828 cDNA, wherein SEQ ID NO: 123 is a clone designated herein as "DNA57037-1444".
Figure 124 shows the amino acid sequence (SEQ ID NO: 124) derived from the coding sequence of SEQ ID NO:123 shown in Figure 123.
Figure 125 shows a nucleotide sequence (SEQ ID NO: 125) of a native sequence PR0827 cDNA, wherein SEQ JD NO: 125 is a clone designated herein as "DNA57039-1402". Figure 126 shows the amino acid sequence (SEQ ID NO: 126) derived from the coding sequence of SEQ
ID NO: 125 shown in Figure 125.
Figure 127 shows a nucleotide sequence (SEQ ID NO: 127) of a native sequence PRO 1075 cDNA, wherein SEQ JD NO:127 is a clone designated herein as "DNA57689-1385".
Figure 128 shows the amino acid sequence (SEQ JD NO:128) derived from tire coding sequence of SEQ ID NO: 127 shown in Figure 127.
Figure 129 shows a nucleotide sequence (SEQ ID NO: 129) of a native sequence PRO 1 07 cDNA, wherein SEQ ID NO:129 is a clone designated herein as "DNA57690-1374".
Figure 130 shows the amino acid sequence (SEQ ID NO: 130) derived from the coding sequence of SEQ ID NO: 129 shown in Figure 129. Figure 131 shows a nucleotide sequence (SEQ ID NO: 131) of a native sequence PR0826 cDNA, wherein
SEQ ID NO: 131 is a clone designated herein as "DNA57694-1341".
Figure 132 shows the amino acid sequence (SEQ JD NO: 132) derived from the coding sequence of SEQ ID NO: 131 shown in Figure 131.
Figure 133 shows a nucleotide sequence (SEQ ID NO: 133) of a native sequence PR0819 cDNA, wherein SEQ ID NO: 132 is a clone designated herein as "DNA57695-1340".
Figure 134 shows the amino acid sequence (SEQ ID NO: 134) derived from the coding sequence of SEQ ID NO.T33 shown in Figure 133.
Figure 135 shows anucleotide sequence (SEQ ID NO: 135) of a native sequence PRO 1006 cDNA, wherein SEQ ID NO: 135 is a clone designated herein as "DNA57699-1412". Figure 136 shows the amino acid sequence (SEQ JD NO: 136) derived from the coding sequence of SEQ
ID NO:135 shown in Figure 135.
Figure 137 shows a nucleotide sequence (SEQ JD NO: 137) of a native sequence PR0982 cDNA, wherein SEQ ID NO:137 is a clone designated herein as 'DNA57700-1408".
Figure 138 shows the amino acid sequence (SEQ ID NO:138) derived from the coding sequence of SEQ JD NO:137 shown in Figure 137.
Figure 139 shows a nucleotide sequence (SEQ JD NO: 139) of a native sequence PRO 1005 cDNA, wherein SEQ JD NO: 139 is a clone designated herein as "DNA57708-1411". Figure 140 shows the amino acid sequence (SEQ JD NO: 140) derived from the coding sequence of SEQ ID NO: 139 shown in Figure 139.
Figure 141 shows anucleotide sequence (SEQ JDNO:141) of anative sequence PR0791 cDNA, wherein SEQ ID NO: 141 is a clone designated herein as "DNA57838-1337". Figure 142 shows the amino acid sequence (SEQ ID NO: 142) derived from the coding sequence of SEQ
ID NO:141 shown in Figure 141.
Figure 143 shows a nucleotide sequence (SEQ ID NO : 143) of a native sequence PRO 1071 cDNA, wherein SEQ ID NO: 143 is a clone designated herein as "DNA58847-1383".
Figure 144 shows the amino acid sequence (SEQ ID NO: 144) derived from the coding sequence of SEQ ID NO.T43 shown in Figure 43.
Figure 145 shows a nucleotide sequence (SEQ ID NO : 145) of a native sequence PRO 1415 cDNA, wherein SEQ ID NO:145 is a clone designated herein as "DNA58852-1637".
Figure 146 shows the amino acid sequence (SEQ ID NO: 146) derived from the coding sequence of SEQ ID NO: 145 shown in Figure 145. Figure 147 shows anucleotide sequence (SEQ JD NO: 147) of anative sequence PRO 1054 cDNA, wherein
SEQ ID NO: 147 is a clone designated herein as "DNA58853-1423".
Figure 148 shows the amino acid sequence (SEQ JD NO: 148) derived from the coding sequence of SEQ JD NO: 147 shown in Figure 147.
Figure 149 shows anucleotide sequence (SEQ IDNO:149) of anative sequence PRO 1411 cDNA, wherein SEQ ID NO:149 is a clone designated herein as "DNA59212-1627".
Figure 150 shows the amino acid sequence (SEQ ID NO: 150) derived from the coding sequence of SEQ ID NO: 149 shown in Figure 149.
Figure 151 shows a nucleotide sequence (SEQ JD NO : 151 ) of a native sequence PRO 1184 cDNA, wherein SEQ ID NO:151 is a clone designated herein as "DNA59220-1514". Figure 152 shows the amino acid sequence (SEQ ID NO: 152) derived from the coding sequence of SEQ
ID NO:151 shown in Figure 151.
Figure 153 shows a nucleotide sequence (SEQJDNO:153) of anative sequence PRO 1029 cDNA, wherein SEQ JD NO:153 is a clone designated herein as "DNA59493-1420".
Figure 154 shows the amino acid sequence (SEQ ID NO: 154) derived from the coding sequence of SEQ ID NO:153 shown in Figure 153.
Figure 155 shows anucleotide sequence (SEQ ID NO:155) of anative sequence PROl 139 cDNA, wherein SEQ ID NO: 155 is a clone designated herein as "DNA59497-1496".
Figure 156 shows the amino acid sequence (SEQ JD NO: 156) derived from the coding sequence of SEQ ID NO: 155 shown in Figure 155. Figure 157 shows a nucleotide sequence (SEQ ID NO: 157) of a native sequence PRO 1190 cDNA, wherein
SEQ ID NO: 157 is a clone designated herein as "DNA59586-1520".
Figure 158 shows the amino acid sequence (SEQ ID NO: 158) derived from the coding sequence of SEQ JD NO:157 shown in Figure 157.
Figure 159 shows anucleotide sequence (SEQJDNO:159) of anative sequence PRO1309 cDNA, wherein SEQ ID NO: 159 is a clone designated herein as "DNA59588-1571".
Figure 160 shows the amino acid sequence (SEQ ID NO: 160) derived from the coding sequence of SEQ ID NO: 159 shown in Figure 159.
Figure 161 shows a nucleotide sequence (SEQ JD NO: 161 ) of a native sequence PR0836 cDNA, wherein SEQ ID N0:161 is a clone designated herein as "DNA59620-1463".
Figure 162 shows the amino acid sequence (SEQ ID NO: 162) derived from the coding sequence of SEQ JD NO:161 shown in Figure 161. Figure 163 shows a nucleotide sequence (SEQ JD NO: 163) of a native sequence PRO 1025 cDNA, wherein
SEQ ID NO:163 is a clone designated herein as "DNA59622-1334".
Figure 164 shows the amino acid sequence (SEQ JD NO:164) derived from the coding sequence of SEQ ID NO:163 shown in Figure 163.
Figure 165 shows a nucleotide sequence (SEQ JD NO : 165) of a native sequence PRO 1131 cDNA, wherein SEQ ID NO: 165 is a clone designated herein as "DNA59777-1480".
Figure 166 shows the amino acid sequence (SEQ JD NO: 166) derived from the coding sequence of SEQ ID NO: 165 shown in Figure 165.
Figure 167 shows a nucleotide sequence (SEQ JD NO: 167) of a native sequence PRO 1182 cDNA, wherein SEQ JD NO: 167 is a clone designated herein as "DNA59848-1512". Figure 168 shows the amino acid sequence (SEQ ID NO: 168) derived from the coding sequence of SEQ
JD NO: 167 shown in Figure 167.
Figure 169 shows a nucleotide sequence (SEQ ID NO: 169) of a native sequence PROl 155 cDNA, wherein SEQ JD NO:169 is a clone designated herein as "DNA59849-1504".
Figure 170 shows the amino acid sequence (SEQ JD NO.T70) derived from the coding sequence of SEQ ID NO: 169 shown in Figure 169.
Figure 171 shows a nucleotide sequence (SEQ ID NO: 171) of a native sequence PRO 1186 cDNA, wherein SEQ ID NO:171 is a clone designated herein as "DNA60621-1516".
Figure 172 shows the amino acid sequence (SEQ JD NO: 172) derived from the coding sequence of SEQ ID NO: 171 shown in Figure 171. Figure 173 shows anucleotide sequence (SEQ JDNO: 173) of anative sequence PROl 198 cDNA, wherein
SEQ TD NO-.173 is a clone designated herein as "DNA60622-1525".
Figure 174 shows the amino acid sequence (SEQ JD NO:174) derived from the coding sequence of SEQ ID NO: 173 shown in Figure 173.
Figure 175 shows anucleotide sequence (SEQ J NO: 175) of anative sequence PR01265 cDNA, wherein SEQ JD NO:175 is a clone designated herein as "DNA60764-1533".
Figure 176 shows the amino acid sequence (SEQ JD NO:176) derived from the coding sequence of SEQ J NO: 175 shown in Figure 175. Figure 177 showsanucleotide sequence(SEQ ID NO:177) of anative sequence PR01361 cDNA, wherein SEQ ID NO:177 is a clone designated herein as "DNA60783-1611".
Figure 178 shows the amino acid sequence (SEQ JD NO:178) derived from the coding sequence of SEQ JD NO: 177 shown in Figure 177. Figure 179 shows anucleotide sequence (SEQ IDNO:179) of anative sequence PR01287 cDNA, wherein
SEQ ID NO: 179 is a clone designated herein as "DNA61755- 1554".
Figure 180 shows the amino acid sequence (SEQ ID NO: 180) derived from the coding sequence of SEQ ID NO: 179 shown in Figure 179.
Figure 181 shows anucleotide sequence (SEQ ID NO: 181) of anative sequence PRO 1308 cDNA, wherein SEQ ID NO:181 is a clone designated herein as "DNA62306-1570".
Figure 182 shows the amino acid sequence (SEQ JD NO.T82) derived from the coding sequence of SEQ ID NO: 181 shown in Figure 181.
Figure 183 shows a nucleotide sequence (SEQ ID NO: 183) of a native sequence PR04313 cDNA, wherein SEQ JD NO: 183 is a clone designated herein as "DNA62312-2558". Figure 184 shows the amino acid sequence (SEQ ID NO: 184) derived from the coding sequence of SEQ
ID NO: 183 shown in Figure 183.
Figure 185 shows anucleotide sequence (SEQ JD NO: 185) of anative sequence PROl 192 cDNA, wherein SEQ ID NO:185 is a clone designated herein as "DNA62814-1521".
Figure 186 shows the amino acid sequence (SEQ ID NO:186) derived from the coding sequence of SEQ ID NO: 185 shown in Figure 185.
Figure 187 shows a nucleotide sequence (SEQ ID NO: 187) of a native sequence PRO 1160 cDNA, wherein SEQ ID NO:187 is a clone designated herein as "DNA62872-1509".
Figure 188 shows the amino acid sequence (SEQ ID NO: 188) derived from the coding sequence of SEQ ID NO: 187 shown in Figure 187. Figure 189 shows a nucleotide sequence (SEQ ID NO: 189) of a native sequence PRO 1244 cDNA, wherein
SEQ ID NO:189 is a clone designated herein as "DNA64883-1526".
Figure 190 shows the amino acid sequence (SEQ ID NO: 190) derived from the coding sequence of SEQ ID NO: 189 shown in Figure 189.
Figure 191 shows a nucleotide sequence (SEQ ID NO: 191) of a native sequence PRO 1356 cDNA, wherein SEQ ID NO:191 is a clone designated herein as "DNA64886-1601".
Figure 192 shows the amino acid sequence (SEQ ID NO: 192) derived from the coding sequence of SEQ ID NO:191 shown in Figure 191.
Figure 193 shows a nucleotide sequence (SEQ JD NO: 193) of a native sequence PRO 1274 cDNA, wherein SEQ ID NO:193 is a clone designated herein as "DNA64889-1541". Figure 194 shows the amino acid sequence (SEQ JD NO:194) derived from the coding sequence of SEQ
J NO:193 shown in Figure 193.
Figure 195 shows anucleotide sequence (SEQ JD NO: 195) of anative sequence PRO 1272 cDNA, wherein SEQ JD NO: 195 is a clone designated herein as "DNA64896-1539".
Figure 196 shows the amino acid sequence (SEQ JD NO: 196) derived from the coding sequence of SEQ ID NO: 195 shown in Figure 195.
Figure 197 shows anucleotide sequence (SEQ JD NO: 197) of anative sequence PR01412 cDNA, wherein SEQ ID NO.T97 is a clone designated herein as "DNA64897-1628".
Figure 198 shows the amino acid sequence (SEQ JD NO:198) derived from the coding sequence of SEQ ID NO: 197 shown in Figure 197.
Figure 199 shows anucleotide sequence (SEQ IDNO:199) of anative sequence PROl 286 cDNA, wherein SEQ JD NO: 199 is a clone designated herein as "DNA64903-1553". Figure 200 shows the amino acid sequence (SEQ ID NO:200) derived from the coding sequence of SEQ
JD NO: 199 shown in Figure 199.
Figure 201 shows a nucleotide sequence (SEQ ID NO:201) of a native sequence PRO 1347 cDNA, wherein SEQ JD NO:201 is a clone designated herein as "DNA64950-1590".
Figure 202 shows the amino acid sequence (SEQ ID NO:202) derived from the coding sequence of SEQ ID NO:201 shown in Figure 201.
Figure 203 shows anucleotide sequence (SEQ JD NO:203) of anative sequence PR01273 cDNA, wherein SEQ ID NO:203 is a clone designated herein as "DNA65402-1540".
Figure 204 shows the amino acid sequence (SEQ ID NO:204) derived from the coding sequence of SEQ JD NO:203 shown in Figure 203. Figure 205 shows a nucleotide sequence (SEQ JD NO:205) of anative sequence PR01283 cDNA, wherein
SEQ JD NO:205 is a clone designated herein as "DNA65404-15 1".
Figure 206 shows the amino acid sequence (SEQ JD NO:206) derived from the coding sequence of SEQ ID NO:205 shown in Figure 205.
Figure 207 shows a nucleotide sequence (SEQ ID NO:207) of a native sequence PRO 1279 cDNA, wherein SEQ ID NO:207 is a clone designated herein as "DNA65405-1547".
Figure 208 shows the amino acid sequence (SEQ ID NO:208) derived from the coding sequence of SEQ ID NO:207 shown in Figure 207.
Figure 209 shows anucleotide sequence (SEQ JDNO:209) of anative sequence PRO1306 cDNA, wherein SEQ JD NO:209 is a clone designated herein as "DNA65410-1569". Figure 210 shows the amino acid sequence (SEQ JD NO:210) derived from the coding sequence of SEQ
ID NO:209 shown in Figure 209.
Figure 211 shows anucleotide sequence (SEQIDNO:211) of anative sequence PROl 195 cDNA, wherein SEQ JD NO:211 is a clone designated herein as "DNA65412-1523".
Figure 212 shows the amino acid sequence (SEQ JD NO:212) derived from the coding sequence of SEQ ID NO:211 shown in Figure 211.
Figure 213 shows anucleotide sequence (SEQ IDNO:213) of anative sequence PR04995 cDNA, wherein SEQ JD NO:213 is a clone designated herein as "DNA66307-2661". Figure 214 shows the amino acid sequence (SEQ JD NO:214) derived from the coding sequence of SEQ JD NO:213 shown in Figure 213.
Figure 215 shows a nucleotide sequence (SEQ ID NO:215) of a native sequence PRO 1382 cDNA, wherein SEQ TD NO:215 is a clone designated herein as "DNA66526-1616". Figure 216 shows the amino acid sequence (SEQ ID NO:216) derived from the coding sequence of SEQ
JD NO-.215 shown in Figure 215.
Figure 217 shows a nucleotide sequence (SEQ JD NO:217) of a native sequence PRO 1325 cDNA, wherein SEQ ID NO:217 is a clone designated herein as "DNA66659-1593".
Figure 218 shows the amino acid sequence (SEQ ID NO:218) derived from the coding sequence of SEQ J NO:217 shown in Figure 217.
Figure 219 shows a nucleotide sequence (SEQ ID NO:219) of a native sequence PRO 1329 cDNA, wherein SEQ ID NO.-219 is a clone designated herein as "DNA66660-1585".
Figure 220 shows the amino acid sequence (SEQ ID NO:220) derived from the coding sequence of SEQ ID NO:219 shown in Figure 219. Figure 221 shows a nucleotide sequence (SEQ JD NO:221 ) of a native sequence PRO 1338 cDNA, wherein
SEQ JD NO:221 is a clone designated herein as "DNA66667-1596".
Figure 222 shows the amino acid sequence (SEQ JD NO:222) derived from the coding sequence of SEQ ID NO:221 shown in Figure 221.
Figure 223 shows anucleotide sequence (SEQ IDNO:223) of anative sequence PR01337 cDNA, wherein SEQ JD NO:223 is a clone designated herein as "DNA66672-1586".
Figure 224 shows the amino acid sequence (SEQ JD NO:224) derived from the coding sequence of SEQ JD NO:223 shown in Figure 223.
Figure 225 shows a nucleotide sequence (SEQ ID NO:225) of a native sequence PRO 1343 cDNA, wherein SEQ JD NO:225 is a clone designated herein as "DNA66675-1587". Figure 226 shows the amino acid sequence (SEQ JD NO:226) derived from the coding sequence of SEQ
ID NO:225 shown in Figure 225.
Figure 227 shows anucleotide sequence (SEQ JDNO:227) of anative sequence PR01376 cDNA, wherein SEQ JD NO:227 is a clone designated herein as "DNA67300-1605".
Figure 228 shows the amino acid sequence (SEQ ID NO:228) derived from the coding sequence of SEQ ID NO:227 shown in Figure 227.
Figure 229 shows a nucleotide sequence (SEQ ID NO:229) of a native sequence PR01434 cDNA, wherein SEQ JD N0:229 is a clone designated herein as "DNA68818-2536".
Figure 230 shows the amino acid sequence (SEQ JD NO:230) derived from the coding sequence of SEQ ID NO:229 shown in Figure 229. Figure 231 shows a nucleotide sequence (SEQ ID N0:231) of anative sequence PR03579 cDNA, wherein
SEQ JD NO:231 is a clone designated herein as "DNA68862-2546".
Figure 232 shows the amino acid sequence (SEQ ID NO:232) derived from the coding sequence of SEQ ID NO:231 shown in Figure 231.
Figure 233 shows anucleotide sequence (SEQ JDNO-.233) of anative sequence PR01387 cDNA, wherein SEQ ID NO:233 is a clone designated herein as "DNA68872-1620".
Figure 234 shows the amino acid sequence (SEQ ID NO:234) derived from the coding sequence of SEQ JD NO:233 shown in Figure 233.
Figure 235 shows a nucleotide sequence (SEQ JD NO:235) of a native sequence PRO 1419 cDNA, wherein SEQ ID NO:235 is a clone designated herein as "DNA71290-1630".
Figure 236 shows the amino acid sequence (SEQ ID NO:236) derived from the coding sequence of SEQ ID NO:235 shown in Figure 235. Figure 237 shows anucleotide sequence (SEQ IDNO:237) of anative sequence PRO 1488 cDNA, wherein
SEQ ID NO:237 is a clone designated herein as "DNA73736-1657".
Figure 238 shows the amino acid sequence (SEQ ID NO:238) derived from the coding sequence of SEQ ID NO:237 shown in Figure 237.
Figure 239 shows a nucleotide sequence (SEQ ID NO:239) of a native sequence PR01474 cDNA, wherein SEQ JD NO:239 is a clone designated herein as "DNA73739-1645".
Figure 240 shows the amino acid sequence (SEQ JD NO:240) derived from the coding sequence of SEQ ID NO:239 shown in Figure 239.
Figure 241 shows a nucleotide sequence (SEQ JD NO:241 ) of a native sequence PRO 1508 cDNA, wherein SEQ ID NO:241 is a clone designated herein as "DNA73742-1662". Figure 242 shows the amino acid sequence (SEQ ID NO:242) derived from the coding sequence of SEQ
ID NO:241 shown in Figure 241.
Figure 243 shows a nucleotide sequence (SEQ ID NO:243) of a native sequence PRO 1754 cDNA, wherein SEQ ID NO:243 is a clone designated herein as "DNA76385-1692".
Figure 244 shows the ammo acid sequence (SEQ ID NO:244) derived from the coding sequence of SEQ JD NO:243 shown in Figure 243.
Figure 245 shows a nucleotide sequence (SEQ JD NO :245) of a native sequence PRO 1550 cDNA, wherein SEQ ID NO:245 is a clone designated herein as "DNA76393-1664".
Figure 246 shows the amino acid sequence (SEQ JD NO:246) derived from the coding sequence of SEQ JD NO:245 shown in Figure 245. Figure 247 shows a nucleotide sequence (SEQ ID NO:247) of a native sequence PRO 1758 cDNA, wherein
SEQ ID NO:247 is a clone designated herein as "DNA76399-1700".
Figure 248 shows the amino acid sequence (SEQ JD NO:248) derived from the coding sequence of SEQ ID NO:247 shown in Figure 247.
Figure 249 shows a nucleotide sequence (SEQ ID NO:249) of a native sequence PRO 1917 cDNA, wherein SEQ JD NO:249 is a clone designated herein as "DNA76400-2528".
Figure 250 shows the amino acid sequence (SEQ JD NO:250) derived from the coding sequence of SEQ JD NO:249 shown in Figure 249. Figure 251 shows a nucleotide sequence (SEQ ID NO:251) of a native sequence PRO 1787 cDNA, wherein SEQ ID NO:251 is a clone designated herein as "DNA76510-2504".
Figure 252 shows the amino acid sequence (SEQ ID NO:252) derived from the coding sequence of SEQ ID NO-.251 shown in Figure 251. Figure 253 shows a nucleotide sequence (SEQ JD NO:253) of a native sequence PROl 556 cDNA, wherein
SEQ ID NO:253 is a clone designated herein as "DNA76529-1666".
Figure 254 shows the amino acid sequence (SEQ ID NO:254) derived from the coding sequence of SEQ ID NO:253 shown in Figure 253.
Figure 255 shows anucleotide sequence (SEQ JD NO:255) of anative sequence PRO 1760 cDNA, wherein SEQ ID NO:255 is a clone designated herein as "DNA76532-1702".
Figure 256 shows the amino acid sequence (SEQ ID NO:256) derived from the coding sequence of SEQ ID NO:255 shown in Figure 255.
Figure 257 shows a nucleotide sequence (SEQ ID NO:257) of a native sequence PR01567 cDNA, wherein SEQ ID NO:257 is a clone designated herein as "DNA76541-1675". Figure 258 shows the amino acid sequence (SEQ ID NO:258) derived from the coding sequence of SEQ
ID NO:257 shown in Figure 257.
Figure 259 shows a nucleotide sequence (SEQ JD NO:259) of a native sequence PRO 1600 cDNA, wherein SEQ JD NO:259 is a clone designated herein as "DNA77503-1686".
Figure 260 shows the amino acid sequence (SEQ ID NO:260) derived from the coding sequence of SEQ JD NO:259 shown in Figure 259.
Figure 261 shows anucleotide sequence (SEQ ID NO:261) of anative sequence PR01868 cDNA, wherein SEQ JD NO:261 is a clone designated herein as "DNA77624-2515".
Figure 262 shows the amino acid sequence (SEQ ID NO:262) derived from the coding sequence of SEQ JD NO.-261 shown in Figure 261. Figure 263 shows a nucleotide sequence (SEQ JD NO:263) of a native sequence PROl 890 cDNA, wherein
SEQ ID NO:263 is a clone designated herein as "DNA79230-2525".
Figure 264 shows the amino acid sequence (SEQ ID NO:264) derived from the coding sequence of SEQ ID NO:263 shown in Figure 263.
Fi ure 265 shows anucleotide sequence (SEQ ID O:265) of anative sequence PROl 887 cDNA, wherein SEQ JD NO:265 is a clone designated herein as "DNA79862-2522".
Figure 266 shows the amino acid sequence (SEQ ID NO:265) derived from the coding sequence of SEQ JD NO:265 shown in Figure 265.
Figure 267 shows a nucleotide sequence (SEQ JD NO:267) of anative sequence PR04353 cDNA, wherein SEQ ID NO:267 is a clone designated herein as "DNA80145-2594". Figure 268 shows the amino acid sequence (SEQ ID NO:268) derived from the coding sequence of SEQ
TD NO:267 shown in Figure 267.
Figure269 shows anucleotide sequence (SEQ IDNO:269) of anative sequence PRO 1782 cDNA, wherein SEQ ED NO-.269 is a clone designated herein as "DNA80899-2501".
Figure 270 shows the amino acid sequence (SEQ ID NO:270) derived from the coding sequence of SEQ ID NO:269 shown in Figure 269.
Figure 271 shows anucleotide sequence (SEQ ID O:271) of anative sequence PR01928 cDNA, wherein SEQ ID NO:271 is a clone designated herein as "DNA81754-2532".
Figure 272 shows the amino acid sequence (SEQ ID NO:272) derived from the coding sequence of SEQ ID NO-.271 shown in Figure 271.
Figure 273 shows a nucleotide sequence (SEQ ID NO:273) of a native sequence PROl 865 cDNA, wherein SEQ JD NO:273 is a clone designated herein as "DNA81757-2512". Figure 274 shows the amino acid sequence (SEQ JD NO:274) derived from the coding sequence of SEQ
JD NO:273 shown in Figure 273.
Figure 275 shows a nucleotide sequence (SEQ JD NO:275) of a native sequence PR04341 cDNA, wherein SEQ JD NO:275 is a clone designated herein as "DNA81761-2583".
Figure 276 shows the amino acid sequence (SEQ ID NO:276) derived from the coding sequence of SEQ JD NO:275 shown in Figure 275.
Figure 277 shows anucleotide sequence (SEQ IDNO:277) of anative sequence PR06714 cDNA, wherein SEQ JD NO:277 is a clone designated herein as "DNA82358-2738".
Figure 278 shows the amino acid sequence (SEQ ID NO:278) derived from the coding sequence of SEQ ID NO:277 shown in Figure 277. Figure 279 shows a nucleotide sequence (SEQ JD NO:279) of anative sequence PR05723 cDNA, wherein
SEQ ID NO:279 is a clone designated herein as "DNA82361".
Figure 280 shows the amino acid sequence (SEQ JD NO:280) derived from the coding sequence of SEQ JD NO.-279 shown in Figure 279.
Figure 281 shows anucleotide sequence (SEQ JD NO:281) of anative sequence PR03438 cDNA, wherein SEQ ID NO:281 is a clone designated herein as "DNA82364-2538".
Figure 282 shows the amino acid sequence (SEQ JD NO:282) derived from the coding sequence of SEQ JD NO:281 shown in Figure 281.
Figure 283 shows a nucleotide sequence (SEQ ID NO:283) of anative sequence PRO6071 cDNA, wherein SEQ JD NO:283 is a clone designated herein as "DNA82403-2959". Figure 284 shows the amino acid sequence (SEQ ID NO:284) derived from the coding sequence of SEQ
JD NO:283 shown in Figure 283.
Figure 285 shows a nucleotide sequence (SEQ ID NO:285) of a native sequence PROl 801 cDNA, wherein SEQ JD NO:285 is a clone designated herein as "DNA83500-2506".
Figure 286 shows the amino acid sequence (SEQ JD NO:286) derived from the coding sequence of SEQ JD NO-.285 shown in Figure 285.
Figure287 shows a nucleotide sequence (SEQ JD NO:287) ofa native sequence PR04324cDNA, wherein SEQ JD NO:287 is a clone designated herein as "DNA83560-2569". Figure 288 shows the amino acid sequence (SEQ ID NO:288) derived from the coding sequence of SEQ JD NO:287 shown in Figure 287.
Figure 289 shows anucleotide sequence (SEQ IDNO:289) of anative sequence PR04333 cDNA, wherein SEQ ID NO-.289 is a clone designated herein as "DNA84210-2576". Figure 290 shows the amino acid sequence (SEQ ID NO:290) derived from the coding sequence of SEQ
JD NO:289 shown in Figure 289.
Figure 291 shows anucleotide sequence (SEQ JDNO:291) of anative sequence PRO4405 cDNA, wherein SEQ ID NO:291 is a clone designated herein as "DNA84920-2614".
Figure 292 shows the amino acid sequence (SEQ ID NO:292) derived from the coding sequence of SEQ JD NO:291 shown in Figure 291.
Figure 293 shows a nucleotide sequence (SEQ ID NO:293) of a native sequence PR04356 cDNA, wherein SEQ ID N0:293 is a clone designated herein as "DNA86576-2595".
Figure 294 shows the amino acid sequence (SEQ ID NO:294) derived from the coding sequence of SEQ ID NO:293 shown in Figure 293. Figure 295 shows a nucleotide sequence (SEQ ID NO:295) of a native sequence PR03444 cDNA, wherein
SEQ ID NO:295 is a clone designated herein as "DNA87997".
Figure 296 shows the amino acid sequence (SEQ ID NO:296) derived from the coding sequence of SEQ ID NO:295 shown in Figure 295.
Figure 297 shows anucleotide sequence (SEQ JD NO:297) of anative sequence PRO4302 cDNA, wherein SEQ ID NO:297 is a clone designated herein as "DNA92218-2554". •
Figure 298 shows the amino acid sequence (SEQ ID NO:298) derived from the coding sequence of SEQ ID NO:297 shown in Figure 297.
Figure 299 shows a nucleotide sequence (SEQ ID NO:299) of a native sequence PR04371 cDNA, wherein SEQ JD N0:299 is a clone designated herein as "DNA92233-2599". Figure 300 shows the amino acid sequence (SEQ ID NO:300) derived from the coding sequence of SEQ
JD NO:299 shown in Figure 299.
Figure 301 shows anucleotide sequence (SEQIDNO:301) of anative sequence PR04354 cDNA, wherein SEQ ID NO:301 is a clone designated herein as "DNA92256-2596".
Figure 302 shows the amino acid sequence (SEQ ID NO:302) derived from the coding sequence of SEQ ID NO:301 shown in Figure 301.
Figure 303 shows a nucleotide sequence (SEQ ID NO:303) of a native sequence PR05725 cDNA, wherein SEQ JD NO:303 is a clone designated herein as "DNA92265-2669".
Figure 304 shows the amino acid sequence (SEQ ID NO:304) derived from the coding sequence of SEQ JD NO.-303 shown in Figure 303. Figure 305 shows anucleotide sequence (SEQ JD NO: 305) of anative sequence PRO4408 cDNA, wherein
SEQ ID NO:305 is a clone designated herein as "DNA92274-2617".
Figure 306 shows the amino acid sequence (SEQ JD NO:306) derived from the coding sequence of SEQ JD NO:305 shown in Figure 305.
Figure 307 shows a nucleotide sequence (SEQ JD NO:307) of a native sequence PRO9940 cDNA, wherein SEQ ID NO:307 is a clone designated herein as "DNA92282".
Figure 308 shows the amino acid sequence (SEQ ID NO:308) derived from the coding sequence of SEQ JD NO:307 shown in Figure 307.
Figure 309 shows anucleotide sequence (SEQ JD NO:309) of a native sequence PR05737 cDNA, wherein SEQ ID NO:309 is a clone designated herein as "DNA92929-2534-1".
Figure 310 shows the amino acid sequence (SEQ ID NO:310) derived from the coding sequence of SEQ ID NO:309 shown in Figure 309. Figure 311 shows a nucleotide sequence (SEQ ID NO:311) of a native sequence PR04425 cDNA, wherein
SEQ ID NO:311 is a clone designated herein as "DNA93011-2637".
Figure 312 shows the amino acid sequence (SEQ ID NO:312) derived from the coding sequence of SEQ JD NO:311 shown in Figure 311.
Figure313 shows anucleotide sequence (SEQ JDNO:313) of anative sequence PR04345 cDNA, wherein SEQ JD NO:313 is a clone designated herein as "DNA94854-2586".
Figure 314 shows the amino acid sequence (SEQ ID NO:314) derived from the coding sequence of SEQ JD NO.-313 shown in Figure 313.
Figure 315 shows a nucleotide sequence (SEQ ID NO:315) of a native sequence PR04342 cDNA, wherein SEQ JD NO:315 is a clone designated herein as "DNA96787-2534-1". Figure 316 shows the amino acid sequence (SEQ JD NO:316) derived from the coding sequence of SEQ
ID NO-.315 shown in Figure 315.
Figure 317 shows a nucleotide sequence (SEQ JD NO:317) of a native sequence PR03562 cDNA, wherein SEQ ID NO:317 is a clone designated herein as "DNA96791".
Figure 318 shows the amino acid sequence (SEQ ID NO:318) derived from the coding sequence of SEQ ID NO:317 shown in Figure 317.
Figure 319 shows a nucleotide sequence (SEQ JD NO:319) of a native sequence PR04422 cDNA, wherein SEQ ID NO-.319 is a clone designated herein as "DNA96867-2620".
Figure 320 shows the amino acid sequence (SEQ ID NO:320) derived from the coding sequence of SEQ ID NO:319 shown in Figure 319. Figure 321 shows a nucleotide sequence (SEQ JD NO:321 ) of a native sequence PR05776 cDNA, wherein
SEQ JD N0:321 is a clone designated herein as "DNA96872-2674".
Figure 322 shows the amino acid sequence (SEQ ID NO:322) derived from the coding sequence of SEQ JD NO:321 shown in Figure 321.
Figure 323 shows a nucleotide sequence (SEQ JD NO:323) of a native sequence PRO4430 cDNA, wherein SEQ JD NO:323 is a clone designated herein as "DNA96878-2626".
Figure 324 shows the amino acid sequence (SEQ ID NO:324) derived from the coding sequence of SEQ JD NO:323 shown in Figure 323. Figure 325 shows anucleotide sequence (SEQ J NO:325) of anative sequence PR04499 cDNA, wherein SEQ JD NO:325 is a clone designated herein as "DNA96889-2641".
Figure 326 shows the amino acid sequence (SEQ JD NO:326) derived from the coding sequence of SEQ ID NO:325 shown in Figure 325. Figure 327 shows a nucleotide sequence (SEQ ID NO:327) of a native sequence PRO4503 cDNA, wherein
SEQ ID NO:327 is a clone designated herein as "DNA100312-2645".
Figure 328 shows the amino acid sequence (SEQ ID NO:328) derived from the coding sequence of SEQ ID NO:327 shown in Figure 327.
Figure 329 shows a nucleotide sequence (SEQ ID NO:329) of a native sequence PRO10008 cDNA, wherein SEQ ID NO:329 is a clone designated herein as "DNA101921".
Figure 330 shows the amino acid sequence (SEQ JD NO:330) derived from the coding sequence of SEQ JD NO:329 shown in Figure 329.
Figure 331 shows anucleotide sequence (SEQ JD NO:331) of a native sequence PRO5730 cDNA, wherein SEQ JD NO:331 is a clone designated herein as "DNA101926". Figure 332 shows the amino acid sequence (SEQ ID NO:332) derived from the coding sequence of SEQ
ID NO:331 shown in Figure 331.
Figure 333 shows anucleotide sequence (SEQ JD NO:333) of anative sequence PRO6008 cDNA, wherein SEQ JD NO:333 is a clone designated herein as "DNA102844".
Figure 334 shows the amino acid sequence (SEQ JD NO:334) derived from the coding sequence of SEQ ID NO:333 shown in Figure 333.
Figure 335 shows a nucleotide sequence (SEQ ID NO : 335) of a native sequence PR04527 cDNA, wherein SEQ ID NO:335 is a clone designated herein as "DNA103197".
Figure 336 shows the amino acid sequence (SEQ ID NO:336) derived from the coding sequence of SEQ ID NO:335 shown in Figure 335. Figure 337 shows a nucleotide sequence (SEQ JD NO:337) of anative sequence PR04538 cDNA, wherein
SEQ ID NO:337 is a clone designated herein as "DNA103208".
Figure 338 shows the amino acid sequence (SEQ JD NO:338) derived from the coding sequence of SEQ JD NO:337 shown in Figure 337.
Figure 339 shows a nucleotide sequence (SEQ JD NO:339) of a native sequence PR04553 cDNA, wherein SEQ ID NO:339 is a clone designated herein as "DNA103223".
Figure 340 shows the amino acid sequence (SEQ ID NO:340) derived from the coding sequence of SEQ ID NO:339 shown in Figure 339.
Figure 341 shows a nucleotide sequence (SEQ JD NO:341 ) of a native sequence PRO6006 cDNA, wherein SEQ JD NO:341 is a clone designated herein as "DNA105782-2693". Figure 342 shows the amino acid sequence (SEQ ID NO:342) derived from the coding sequence of SEQ
ID NO:341 shown in Figure 341.
Figure 343 shows a nucleotide sequence (SEQ JD NO:343) of a native sequence PRO6029 cDNA, wherein SEQ ID NO:343 is a clone designated herein as "DNA105849-2704".
Figure 344 shows the amino acid sequence (SEQ ID NO:344) derived from the coding sequence of SEQ JD NO:343 shown in Figure 343.
Figure 345 shows a nucleotide sequence (SEQ JD NO:345) of a native sequence PR09821 cDNA, wherein SEQ ID NO:345 is a clone designated herein as "DNA108725-2766".
Figure 346 shows the amino acid sequence (SEQ ID NO:346) derived from the coding sequence of SEQ ID NO:345 shown in Figure 345.
Figure 347 shows a nucleotide sequence (SEQ ID NO:347) of a native sequence PRO9820 cDNA, wherein SEQ ID NO:347 is a clone designated herein as "DNA108769-2765". Figure 348 shows the amino acid sequence (SEQ JD NO:348) derived from the coding sequence of SEQ
ID NO:347 shown in Figure 347.
Figure 349 shows a nucleotide sequence (SEQ JD NO:349) of anative sequence PR09771 cDNA, wherein SEQ ID N0:349 is a clone designated herein as "DNA119498-2965".
Figure 350 shows the amino acid sequence (SEQ JD NO:350) derived from the coding sequence of SEQ ID NO:349 shown in Figure 349.
Figure 351 shows anucleotide sequence (SEQ ID NO:351) of anative sequence PR07436 cDNA, wherein SEQ ID NO:351 is a clone designated herein as "DNA119535-2756".
Figure 352 shows the amino acid sequence (SEQ ID NO:352) derived from the coding sequence of SEQ ID NO:351 shown in Figure 351. Figure 353 shows a nucleotide sequence (SEQ ID NO:353) of a native sequence PRO10096 cDNA, wherein SEQ ID NO:353 is a clone designated herein as "DNA125185-2806".
Fi ure 354 shows the amino acid sequence (SEQ ID NO:354) derived from the coding sequence of SEQ JD NO:353 shown in Figure 353.
Figure 355 shows a nucleotide sequence (SEQ JD NO:355) of a native sequence PRO19670 cDNA, wherein SEQ ID NO:355 is a clone designated herein as "DNA131639-2874".
Figure 356 shows the amino acid sequence (SEQ ID NO:356) derived from the coding sequence of SEQ ID NO:355 shown in Figure 355.
Figure 357 shows a nucleotide sequence (SEQ ID NO:357) of a native sequence PRO20044 cDNA, wherein SEQ ED NO:357 is a clone designated herein as "DNA139623-2893". Figure 358 shows the amino acid sequence (SEQ ID NO:358) derived from the coding sequence of SEQ
JD NO:357 shown in Figure 357.
Figure 359 shows anucleotide sequence (SEQ JD NO:359) of anative sequence PR09873 cDNA, wherein SEQ ID NO:359 is a clone designated herein as "DNA143076-2787".
Figure 360 shows the amino acid sequence (SEQ JD NO:360) derived from the coding sequence of SEQ ID NO:359 shown in Figure 359.
Figure 361 shows a nucleotide sequence (SEQ ID NO:361) of a native sequence PR021366 cDNA, wherein SEQ ID NO:361 is a clone designated herein as "DNA143276-2975". Figure 362 shows the amino acid sequence (SEQ JD NO:362) derived from the coding sequence of SEQ ID NO:361 shown in Figure 361.
Figure 363 shows a nucleotide sequence (SEQ ID NO:363) of a native sequence PRO20040 cDNA, wherein SEQ ID NO:363 is a clone designated herein as "DNA164625-2890". Figure 364 shows the amino acid sequence (SEQ ID NO:364) derived from the coding sequence of SEQ
ID NO:363 shown in Figure 363.
Figure 365 shows a nucleotide sequence (SEQ ID NO:365) of a native sequence PR021184 cDNA, wherein SEQ ID NO:365 is a clone designated herein as "DNA167678-2963".
Figure 366 shows the amino acid sequence (SEQ ID NO:366) derived from the coding sequence of SEQ ID NO:365 shown in Figure 365.
Figure 367 shows a nucleotide sequence (SEQ ID NO:367) of a native sequence PRO21055 cDNA, wherein SEQ ID NO:367 is a clone designated herein as "DNA170021-2923".
Figure 368 shows the amino acid sequence (SEQ ID NO:368) derived from the coding sequence of SEQ ID NO:367 shown in Figure 367. Figure 369 shows a nucleotide sequence (SEQ ID NO:369) of a native sequence PR028631 cDNA, wherein SEQ ID NO:369 is a clone designated herein as "DNA170212-3000".
Figure 370 shows the amino acid sequence (SEQ ID NO:370) derived from the coding sequence of SEQ ID NO:369 shown in Figure 369.
Figure 371 shows a nucleotide sequence (SEQ ID NO:371) of a native sequence PR021384 cDNA, wherein SEQ JD NO:371 is a clone designated herein as "DNA177313-2982".
Figure 372 shows the amino acid sequence (SEQ JD NO:372) derived from the coding sequence of SEQ ED NO:371 shown in Figure 371.
Figure 373 shows a nucleotide sequence (SEQ ID NO:373) of a native sequence PRO 1449 cDNA, wherein SEQ ID NO:373 is a clone designated herein as "DNA64908-1163-1". Figure 374 shows the amino acid sequence (SEQ ID NO:374) derived from the coding sequence of SEQ
ID NO:373 shown in Figure 373.
Figure 375 shows wholemount in situ hybridization results on mouse embryos using a mouse orthologue of PR01449 whichhas about 78% amino acid identity with PRO 1449. The results show thatPR01449 orthologue is expressed in the developing vasculature. The cross-section further shows expression in endothelial cells and progenitors of endothelial cells.
Figure 376 shows that a PR01449 orthologue having about 78% amino acid identity with PR01449 is expressed in vasculature of many inflamed and diseased tissues, but is very low, or lacking, in normal adult vessels.
Figure 377 shows that a PR01449 orthologue having about 78% amino acid identity with PR01449 induces ectopic vessels in the eyes of chicken embryos. 5. Detailed Description ofthe Invention
5.1. Definitions
The phrases "cardiovascular, endothelial and angiogenic disorder", "cardiovascular, endothelial and angiogenic dysfunction", "cardiovascular, endothelial or angiogenic disorder" and "cardiovascular, endothelial or angiogenic dysfunction" are used interchangeably and refer in part to systemic disorders that affect vessels, such as diabetes mellitus, as well as diseases ofthe vessels themselves, such as ofthe arteries, capillaries, veins, and/or lymphatics. This would include indications that stimulate angiogenesis and or cardiovascularization, and those that inhibit angiogenesis and/or cardiovascularization. Such disorders include, for example, arterial disease, such as atherosclerosis, hypertension, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, and arterial restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, cancer such as vascular tumors, e.g. , hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and lymphangiosarcoma, tumor angiogenesis, trauma such as wounds, bu s, and other injured tissue, implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. This would also include angina, myocardial infarctions such as acute myocardial infarctions, cardiac hypertrophy, and heart failure such as CHF.
"Hypertrophy", as used herein, is defined as an increase in mass of an organ or structure independent of natural growth that does not involve tumor formation. Hypertrophy of an organ or tissue is due either to an increase in the mass ofthe individual cells (true hypertrophy), or to an increase in the number of cells making up the tissue
(hyperplasia), or both. Certain organs, such as the heart, lose the ability to divide shortly after birth. Accordingly, "cardiac hypertrophy" is defined as an increase in mass of the heart, which, in adults, is characterized by an increase in myocyte cell size and contractile protein content without concomitant cell division. The character ofthe stress responsible for inciting the hypertrophy, (e.g., increased preload, increased afterload, loss of myocytes, as in myocardial infarction, or primary depression of contractility), appears to play a critical role in determining the nature ofthe response. The early stage of cardiac hypertrophy is usually characterized morphologically by increases in the size of myofibrils and mitochondria, as well as by enlargement of mitochondria and nuclei. At this stage, while muscle cells are larger than normal, cellular organization is largely preserved. At a more advanced stage of cardiac hypertrophy, there are preferential increases in the size or number of specific organelles, such as mitochondria, and new contractile elements are added in localized areas ofthe cells, in an irregular manner. Cells subjected to long-standing hypertrophy show more obvious disruptions in cellular organization, including markedly enlarged nuclei with highly lobulated membranes, which displace adjacent myofibrils and cause breakdown of normal Z-band registration. The phrase "cardiac hypertrophy" is used to include all stages ofthe progression of this condition, characterized by various degrees of structural damage ofthe heart muscle, regardless ofthe underlying cardiac disorder. Hence, the term also includes physiological conditions instrumental 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 where the heart does not pump blood at the rate needed for the requirements of metabolizing tissues. The heart failure can be caused by a number of factors, including ischemic, congenital, rheumatic, or idiopathic forms.
"Congestive heart failure" (CHF) is a progressive pathologic state where the heart is increasingly unable to supply adequate cardiac output (the volume of blood pumped by the heart over time) to deliver the oxygenated blood to peripheral tissues. As CHF progresses, structural and hemodynamic damages occur. While these damages have a variety of manifestations, one characteristic symptom is ventricular hypertrophy. CHF is a common end result of a number of various cardiac disorders.
"Myocardial infarction" generally results from atherosclerosis of the coronary arteries, often with superimposed coronary thrombosis. It may be divided into two major types: transmural infarcts, in which myocardial necrosis involves the full thickness ofthe ventricular wall, and subendocardial (nontransmural) infarcts, in which the necrosis involves the subendocardium, the intramural myocardium, or both, without extending all the way through the ventricular wall to the epicardium. Myocardial infarction is known to cause both a change in hemodynamic effects and an alteration in structure in the damaged and healthy zones of the heart. Thus, for example, myocardial infarction reduces the maximum cardiac output and the stroke volume of the heart. Also associated with myocardial infarction is a stimulation of the DNA synthesis occurring in the interstice as well as an increase in the formation of collagen in the areas of the heart not affected.
As a result ofthe increased stress or strain placed on the heart in prolonged hypertension due, for example, to the increased total peripheral resistance, cardiac hypertrophy has long been associated with "hypertension". A characteristic ofthe ventricle that becomes hypertrophic as a result of chronic pressure overload is an impaired diastolic performance. Fouad e/α/., J. Am. Coll. Cardiol..4: 1500-1506 (1984); Smith etal, J. Am. Coll. Cardiol.,
5: 869-874 (1985). Aprolonged left ventricular relaxation has been detected in early essential hypertension, in spite of normal or supranormal systolic function. Hartford et al, Hypertension, 6: 329-338 (1984). However, there is no close parallelism between blood pressure levels and cardiac hypertrophy. Altliough improvement in left ventricular function in response to antihypertensive therapy has been reported in humans, patients variously treated with a diuretic (hydrochlorothiazide), a β-blocker (propranolol), or a calcium channel blocker (diltiazem), have shown reversal of left ventricular hypertrophy, without improvement in diastolic function. Inouye et al, Am. J. Cardiol., 53: 1583-7 (1984).
Another complex cardiac disease associated with cardiac hypertrophy is "hypertrophic cardiomyopathy". This condition is characterized by a great diversity of morphologic, functional, and clinical features (Maron et al, N. Engl. J. Med.. 316: 780-789 (1987); Spirito et al, N. Engl. J. Med.. 320: 749-755 (1989); Louie and Edwards,
Prog. Cardiovasc. Pis.. 36: 275-308 (1994); Wigle et al, Circulation, 92: 1680-1692 (1995)), the heterogeneity of which is accentuated by the fact that it afflicts patients of all ages. Spirito et al, N. Engl. J. Med.. 336: 775-785 (1997). The causative factors of hypertrophic cardiomyopathy are also diverse and little understood. In general, mutations in genes encoding sarcomeric proteins are associated with hypertrophic cardiomyopathy. Recent data suggest that β-myosin heavy chain mutations may account for approximately 30 to 40 percent of cases of familial hypertrophic cardiomyopathy. Watkins etal, N. Engl. J. Med..326: 1108-1114 (1992); Schwartz etal, Circulation, 91 : 532-540 (1995); Marian and Roberts, Circulation. 92: 1336-1347 (1995); Thierfelder et al, Cell,'77: 701-712 (1994); Watkins et al, Nat. Gen.. JJ,: 434-437 (1995). Besides β-myosin heavy chain, other locations of genetic mutations include cardiac froponin T, alpha topomyosin, cardiac myosin binding protein C, essential myosin light chain, and regulatory myosin light chain. See, Malik and Watkins, Curr. Opin. Cardiol.. 12: 295-302 (1997).
Supravalvular "aortic stenosis" is an inherited vascular disorder characterized by narrowing of the ascending aorta, but other arteries, including the pulmonary arteries, may also be affected. Untreated aortic stenosis may lead to increased intracardiac pressure resulting in myocardial hypertrophy and eventually heart failure and death. The pathogenesis of this disorder is not fully understood, but hypertrophy and possibly hyperplasia of medial smooth muscle are prominent features of this disorder. It has been reported that molecular variants ofthe elastin gene are involved in the development and pathogenesis of aortic stenosis. U.S. Patent No. 5,650,282 issued July 22, 1997.
"Valvular regurgitation" occurs as a result of heart diseases resulting in disorders ofthe cardiac valves. Various diseases, like rheumatic fever, can cause the shrinking or pulling apart of the valve orifice, while otlier diseases may result in endocarditis, an inflammation ofthe endocardium or lining membrane ofthe atrioventricular orifices and operation ofthe heart. Pefects such as the narrowing ofthe valve stenosis or the defective closing of the valve result in an accumulation of blood in the heart cavity or regurgitation of blood past the valve. If uncorrected, prolonged valvular stenosis or insufficiency may result in cardiac hypertrophy and associated damage to the heart muscle, which may eventually necessitate valve replacement.
The treatment of all these, and other cardiovascular, endothelial and angiogenic disorders, which may or may not be accompanied by cardiac hypertrophy, is encompassed by the present invention. The terms "cancer", "cancerous", and "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's andnon-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer. The preferred cancers for treatment herein are breast, colon, lung, melanoma, ovarian, and others involving vascular tumors as noted above. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., 1311, 1251, 90Y, and 186Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, folic acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil, cisplatin, purine nucleosides, amines, amino acids, triazol nucleosides, or corticosteroids. Specific examples include Adriamycin, Poxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Oaunomycin, Carminomycin, Aminopterin, Pactinomycin, Mitomycins, Esperamicins (see U.S. Pat. No.4,675, 187), Melphalan, and other related nitrogen mustards. Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors, such as tamoxifen and onapristone.
A "growtli-inhibitory agent" when used herein refers to a compound or composition that inhibits growth of a cell, such as an Wnt-overexpressing cancer cell, either in vitro or in vivo. Thus, the growm-inhibitory agent is one which significantly reduces the percentage of malignant cells in S phase. Examples of growth-inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce
GI arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest GI also spill over into S-phase arrest, for example, PNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds., Chapter 1 , entitled "Cell cycle regulation, oncogenes, and antineoplastic drags" by Murakami etal. (WB Saunders: Philadelphia, 1995), especially p. 13. Additional examples include tumor necrosis factor (TNF), an antibody capable of inliibiting or neutralizing the angiogenic activity of acidic or basic FGF or hepatocyte growth factor (HGF), an antibody capable of inhibiting or neutralizing the coagulant activities of tissue factor, protein C, or protein S (see, WO 91/01753, published 21 February 1991), or an antibody capable of binding to HER2 receptor (WO 89/06692), such as the 4P5 antibody (and functional equivalents thereof) (e.g., WO 92/22653).
"Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a cardiovascular, endothelial, and angiogenic disorder. The concept of treatment is used in the broadest sense, and specifically includes the prevention (prophylaxis), moderation, reduction, and curing of cardiovascular, endothelial, and angiogenic disorders of any stage. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) or ameliorate a cardiovascular, endothelial, and angiogenic disorder such as hypertrophy. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. The disorder may result from any cause, including idiopathic, cardiotrophic, or myotrophic causes, or ischemia or ischemic insults, such as myocardial infarction.
"Chronic" administration refers to administration ofthe agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial effect, such as an anti-hypertrophic effect, for an extended period of time.
"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, pigs, etc. Preferably, the mammal is human.
Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. The phrase "cardiovascular, endothelial or angiogenic agents" refers generically to any drag that acts in treating cardiovascular, endothelial, and angiogenic disorders. Examples of cardiovascular agents are those that promote vascular homeostasis by modulating blood pressure, heart rate, heart contractility, and endothelial and smooth muscle biology, all of which factors have a role in cardiovascular disease. Specific examples of these include angiotensin-II receptor antagonists; endothelin receptor antagonists such as, for example, BOSENTAN™ and MOXONOPIN™; interferon-gamma (IFN-γ); des-aspartate-angiotensin I; thrombolytic agents, e.g., streptokinase, urokinase, t-PA, and a t-PA variant specifically designed to have longer half-life and very high fibrin specificity, TNK t-PA (a Tl 03N, NI 17Q, KHRR(296-299)AAAA t-PA variant, Keyt et al., Proc. Natl. Acad. Sci. USA. 91: 3670-3674 (1994)); inotropic or hypertensive agents such as digoxigenin and β-adrenergic receptor blocking agents, e.g., propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, and carvedilol; angiotensin converting enzyme (ACE) inhibitors, e.g., quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, and lisinopril; diuretics, e.g., clilorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, and indapamide; and calcium channel blockers, e.g., diltiazem, nifedipine, verapamil, nicardipine. One preferred category of this type is a therapeutic agent used for the treatment of cardiac hypertrophy or of a physiological condition instrumental in the development 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, if applicable, vasculogenesis. This would include factors that accelerate wound healing, such as growth hormone, insulin-like growth factor-I (IGF-I), VEGF, VIGF, PPGF, epidermal growth factor (EGF), CTGF and members of its family, FGF, and TGF- and TGF-β.
"Angiostatic agents" are active agents that inhibit angiogenesis or vasculogenesis or otherwise inhibit or prevent growth of cancer cells. Examples include antibodies or other antagonists to angiogenic agents as defined above, such as antibodies to VEGF. They additionally include cytotherapeutic agents such as cytotoxic agents, chemotherapeutic agents, growth-inhibitory agents, apoptotic agents, and other agents to treat cancer, such as anti- HER-2, anti-CP20, and other bioactive and organic chemical agents.
In a pharmacological sense, in the context ofthe present invention, a "therapeutically effective amount" of an active agent such as a PRO polypeptide or agonist or antagonist thereto or an anti-PRO antibody, refers to an amount effective in the treatment of a cardiovascular, endothelial or angiogenic disorder in a mammal and can be determined empirically. As used herein, an "effective amount" of an active agent such as a PRO polypeptide or agonist or antagonist thereto or an anti-PRO antibody, refers to an amount effective for carrying out a stated purpose, wherein such amounts may be determined empirically for the desired effect.
The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (i.e., PRO/number) refers to specific polypeptide sequences as described herein. The terms "PRO/number polypeptide" and "PRO/number" wherein the term "number" is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein). The PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence PRO polypeptide" specifically encompasses naturally-occurring truncated or secreted forms ofthe specific PRO polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In various embodiments ofthe invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons are shown in bold font and underlined in the figures. However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the PRO polypeptides. The PRO polypeptide "extracellular domain" or "ECP" refers to a form ofthe PRO polypeptide which is essentially free ofthe transmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have less than 1% of such transmembrane and or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the PRO polypeptides ofthe present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end ofthe domain as initially identified herein. Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are comtemplated by the present invention. The approximate location ofthe "signal peptides" ofthe various PRO polypeptides disclosed herein are shown in the present specification and or the accompanying figures. It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side ofthe signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al, Prot Eng„ 10:1-6 (1997) and von Heinje et al, Nucl. Acids Res.,
14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side ofthe C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
"PRO polypeptide variant" means an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more ammo acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a PRO polypeptide variant will have at least about 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, PRO variant polypeptides are at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 amino acids in length and alternatively at least about 300 amino acids in length, or more.
"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Table 1 has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under
U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
. 100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations, Tables 2-3 demonstrate how to calculate the % amino acid sequence identity ofthe amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "PRO".
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al, Nucleic Acids Res..25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih. gov. or otherwise obtained from the National Institute of Health, Bethesda, MD. NCBI- BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multipass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 tunes the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al, Methods in Enzymology. 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i. e. , the adjustable parameters, are set with 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 determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence ofthe PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (i. e. , the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues ofthe PRO polypeptide of interest. For example, in the statement "a polypeptide comprising an amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B", the amino acid sequence A is the comparison ammo acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, a PRO variant polynucleotide will have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97% or 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence.
Ordinarily, PRO variant polynucleotides are at least about 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 450, or 600 nucleotides in length and alternatively at least about 900 nucleotides in length, or more.
"Percent (%) nucleic acid sequence identity" with respect to the PRO polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN- 2 program is provided in Table 1. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Table 1 has been filed with user documentation in the U.S. Copyright
Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Tables 4-5 demonstrate how to calculate the % nucleic acid sequence identity ofthe nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO- DNA".
Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al. ,
Nucleic Acids Res., 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. or otherwise obtained from the National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence P) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI- BLAST2 in that program's alignment of C and P, and where Z is the total number of nucleotides in P. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence P, the % nucleic acid sequence identity of C to P will not equal the % nucleic acid sequence identity of P to C.
In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST-
2 computer program (Altschul et al, Methods in Enzymology, 266:460-480 (1996)). Most ofthe WU-BLAST-2 search parameters are set to the default values. Those not set to default values, . e. , the adjustable parameters, are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. For purposes herein, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence ofthe PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (i.e., the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides ofthe PRO polypeptide- encoding nucleic acid molecule of interest. For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence ofthe PRO polypeptide-encoding nucleic acid molecule of interest. In other embodiments, PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO polypeptide as shown in the specification herein and accompanying figures. PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide.
"Isolated", when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and or recovered from a component of its natural environment. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SPS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component ofthe PRO natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step. An "isolated" nucleic acid molecule encoding a PRO polypeptide or an "isolated" nucleic acid molecule encoding an anti-PRO antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source ofthe PRO-encoding nucleic acid or the natural source ofthe anti-PRO-encoding nucleic acid. Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated. An isolated PRO-encoding nucleic acid molecule or an isolated anti-PRO-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the PRO-encoding nucleic acid molecule or from the anti-PRO-encoding nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule encoding a PRO polypeptide or an isolated nucleic acid molecule encoding an anti- PRO antibody includes PRO-nucleic acid molecules or anti-PRO-nucleic acid molecules contained in cells that ordinarily express PRO polypeptides or anti-PRO antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize, for example, promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a PRO polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an enviromnent below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see, Ausubel et al, Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995).
"Stringent conditions" or "high-stringency conditions", as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll 0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.
"Moderately-stringent conditions" may be identified as describedby Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Press, 1989), and include the use of wasliing solution and hybridization conditions (e.g., temperature, ionic strength, and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mMNaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
The modifier "epitope-tagged" when used herein refers to a chimeric polypeptide comprising a PRO polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity ofthe polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
"Active" or "activity" in the context of PRO variants refers to form(s) of PRO proteins that retain the biologic and or immunologic activities of a native or naturally-occurring PRO polypeptide.
"Biological activity" in the context of a molecule that antagonizes a PRO polypeptide that can be identified by the screening assays disclosed herein (e.g. , an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to bind or complex with the PRO polypeptide identified herein, or otherwise interfere with the interaction of the PRO polypeptide with other cellular proteins or otherwise inhibits the transcription or translation of the PRO polypeptide. Particularly preferred biological activity includes cardiac hypertrophy, activity that acts on systemic disorders that affect vessels, such as diabetes mellitus, as well as diseases ofthe arteries, capillaries, veins, and/or lymphatics, and cancer.
The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes one or more ofthe biological activities of a native PRO polypeptide disclosed herein, for example, if applicable, its mitogenic or angiogenic activity. Antagonists of a PRO polypeptide may act by interfering with the binding of a PRO polypeptide to a cellular receptor, by incapacitating or killing cells that have been activated by a PRO polypeptide, or by interfering with vascular endothelial cell activation after binding of a PRO polypeptide to a cellular receptor. All such points of intervention by a PRO polypeptide antagonist shall be considered equivalent for purposes of this invention. The antagonists inhibit the mitogenic, angiogenic, or other biological activity of PRO polypeptides, and thus are useful for the treatment of diseases or disorders characterized by undesirable excessive neovascularization, including by way of example tumors, and especially solid malignant tumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and otlier retinopathies, retrolental fibroplasia, age- related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation. The antagonists also are useful for the treatment of diseases or disorders characterized by undesirable excessive vascular permeability, such as edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, lung inflammation, neplrrotic syndrome, pericardial effusion (such as that associated with pericarditis), andpleural effusion. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments, or amino acid sequence variants of native PRO polypeptides, peptides, small organic molecules, etc.
A "small molecule" is defined herein to have a molecular weight below about 500 daltons. The term "PRO polypeptide receptor" as used herein refers to a cellular receptor for a PRO polypeptide, ordinarily a cell-surface receptor found on vascular endothelial cells, as well as variants thereof that retain the ability to bind a PRO polypeptide.
"Antibodies" (Abs) and "immunoglobulms" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulms include both antibodies and otlier antibody-like molecules that lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas. The term "antibody" is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.
"Native antibodies" and "native immunoglobulms" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and hght chain also has regularly spaced intrachain disulfide bridges. 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 at one end (VL) and a constant domain at its other end; the constant domain ofthe light chain is aligned with the first constant domain ofthe heavy chain, and the light-chain variable domain is aligned with the variable domain ofthe heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains. The term "variable" refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody to and for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. See, Kabat et al, NHI Publ, No.91-3242. Vol. I, pages 647-669 (1991). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation ofthe antibody in antibody-dependent cellular toxicity.
"Antibody fragments" comprise 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 Fv fragments; diabodies; linear antibodies (Zapata et al. , Protein Eng..8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. 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. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) ofthe constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The "light chains" of antibodies (immunoglobulms) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ) and lambda (λ), based on the amino acid sequences of their constant domains.
Depending on the amino acid sequence ofthe constant domain of their heavy chains, hiimunoglobulins can be assigned to different classes. There are five major classes of immunoglobulms: IgA, IgD, IgE, IgG, and IgM; and several of these maybe further divided into subclasses (isotypes), e.g., IgGl , IgG2, IgG3, IgG4, IgA, and IgA2.
The heavy-chain constant domains that correspond to the different classes of immunoglobulms are called , δ, e, γ, and μ, respectively. The subunit stractures and three-dimensional configurations of different classes of immunoglobulms are well known.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i. e. , the individual antibodies comprising the population are identical except forpossible naturally-occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulms. The modifier "monoclonal" indicates the character ofthe antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, Nature.256: 495 ( 1975), or may be made by recombinant DNA methods (see, e.g., U.S. PatentNo.4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 581-597 (1991), for example. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulms) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder ofthe chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. U.S. PatentNo.4,816,567; Morrison etal, Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulms, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2j or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulms (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. The humanized antibody preferably also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
For further details, see Jones et al, Nature.321: 522-525 (1986); Reichmann et al, Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992). The humanized antibody includes a PRIMATIZED™ antibody wherein the antigen-binding region ofthe antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv see, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113. Rosenburg and Moore, eds. (Springer- Verlag: New York, 1994), pp. 269-315. The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VjJ connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; 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 diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody 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.
An antibody that "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
The word "label" when used herein refers to a detectable compound or other composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label may be detectable by itself (e.g. , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable. Radionuclides that can serve as detectable labels include, for example, 1-131, 1-123, 1-125, Y-90, Re-188, At-211, Cu-67, Bi-212, and Pd-109. The label may also be a non- detectable entity such as a toxin.
By "solid phase" is meant a non-aqueous matrix to which an antibody ofthe present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.
A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant that is useful for delivery of a drag (such as the PRO polypeptide or antibodies thereto disclosed herein) to a mammal.
The components ofthe liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
As used herein, the term "immunoadhesin" designates antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity that is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG- 1 , IgG- 2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.
As shown below, Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program. This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program.
In addition, Tables 2-5 show hypothetical exemplifications for using the below described method to deteimine % amino acid sequence identity (Tables 2-3) and % nucleic acid sequence identity (Tables 4-5) using the ALIGN-2 sequence comparison computer program, wherein "PRO" represents the amino acid sequence of a hypothetical PRO polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared, "PRO-DNA" represents a hypothetical PRO-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule of interest is being compared, "X", "Y", and "Z" each represent different hypothetical amino acid residues and "N", "L" and "V" each represent different hypothetical nucleotides.
Table 1
/* *
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is _ ; stop-stop = 0; J (joker) match : */
#define M /* value of a match with a stop */ int day[26][26] = {
/* A' B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/
/*A*/ 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0},
/*B*/ 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1},
/*C*/ -2,-4,15 -5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5},
/*D*/ 0, 3,-5 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2},
/*E*/ 0, 2,-5 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3},
/* p */ ■4,-5,-4. -6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5},
/*G*/ 1, 0,-3. 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-l,-l,-3, 1, 0, 0,-1,-7, 0,-5, 0},
/*H*/ ■1, 1,-3 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0, 2},
1*1*1 •1,-2,-2. -2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2,_M,-2,-2,-2,-l, 0, 0, 4,-5, 0,-1,-2},
/* */ 0,0,0 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/*K*/ -1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-4, 0},
/*L*/ -2,-3,-6 -4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-l, 0, 2,-2, 0,-1,-2},
/*M*/ ■1,-2,-5 -3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-l, 0,-2,-1, 0, 2,-4, 0,-2,-1},
/*N*/ 0, 2,-4 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2, 1},
/*o*/ M, M. _M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M, 0,_M,_M,_M, M,_M,_M,_M,_M,_M,_M,_M},
/* p */ 1,-1,-3 -1,-1,-5,-1, 0,-2, 0,-l,-3,-2,-l,_M, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5, 0},
/*Q*/ 0, 1,-5 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 1,-1,-1, 0,-2,-5, 0,-4, 3},
/*R*/ -2, 0,-4 -1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4, 0},
/*S */ 1, 0, 0 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1,_M, 1,-1, 0, 2, 1, 0,-1,-2, 0,-3, 0}, /* Ύ */ 1,0,-2.0, 0,-3, 0,-1, 0, 0, 0,-1,-1, 0,_M, 0,-1,-1, 1, 3, 0, 0,-5, 0,-3, 0},
/*U*/ 0, 0, 0 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* v */ 0,-2,-2 -2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2,_M,-l, -2,-2,-1, 0, 0, 4,-6, 0,-2,-2},
/*w*/ -6,-5,-8 -7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4,_M,-6,-5, 2,-2,-5, 0,-6,17, 0, 0,-6}, l*X*I 0, 0, 0 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, j* Y */ -3,-3, 0 -4,-4, 7,-5, 0,-1, 0,-4,-l,-2,-2,_M,-5,-4,-4,-3,-3, 0,-2, 0, 0,10,-4},
/*z*/ 0, 1,-5 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1,_M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4} };
Table 1 (conf)
/* */
^include <stdio.h> ^include <ctype.h>
#define MAXJMP 16 /* max jumps in a diag */
#define MAXGAP 24 /* don't continue to penalize gaps larger than this */
#defme JMPS 1024 /* maxjmps in an path */
#defme MX 4 /* save if there' s at least MX-1 bases since last jmp */
#define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty for mismatched bases */
#defme DINSO 8 /* penalty for a gap */
#define DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap */
#defme PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. of jmp in seq x */
}; /* limits seq to 2A16 -1 */ struct diag { int score; /* score at last jmp */ long offset; /* offset of prev block */ short ijmp; /* current jmp index */ struct jmp jp; /* list of jmps */
}; struct path { int spc; /* number of leading spaces */ short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (last elem before gap) */
}; char *ofile; /* output file name */ char *namex[2]; /* seq names: getseqs( ) */ char *prog; /* prog name for err msgs */ char *seqx[2]; /* seqs: getseqs( ) */ int dmax; /* best diag: nw( ) */ int dmaxO; /* final diag */ int dna; /* set if dna: main( ) */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int lenO, lenl; /* seq lens */ int ngapx, ngapy; /* total size of gaps */ int smax; /* max score: nw( ) */ int *xbm; /* bitmap for matching */ long offset; /* current offset in jmp file */ struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds path for seqs */ char *calloc( ), *malloc( ), *index( ), *strcpy( ); char *getseq( ), *g_calloc( ); Table 1 (conf)
/* Needleman-Wunsch alignment program
*
* usage: progs filel file2 where filel and file2 are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ';','>' or ' < ' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align. out" *
* The program may create a imp file in /imp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650 */
#include "nw.h" #include "day.h" static _dbval[26] = { 1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
}; static _pbval[26] = {
1, 2|(1< <('D'-'A'))|(1< <('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1< < 10, 1< < 11, 1< < 12, 1< < 13, 1< < 14,
1< < 15, 1< < 16, 1< < 17, 1< < 18, 1< < 19, 1< <20, 1< <21, 1< <22,
1<<23, 1<<24, 1<<25|(1<<(Ε'-Α'))|(1<<('Q'-Α'))
}; main(ac, av) mam int ac; char *av[]; prog = av[0]; if(ac!=3){ fprintf(stderr, "usage: s filel fϊle2\n", prog); fprintf(stderr, "where filel and fϊle2 are two dna or two protein sequences. \n"); frintf(stderr, "The sequences can be in upper- or lower-case\n"); fprintf(stderr, " Any lines beginning with ';' or ' < ' are ignored\n"); fprintf(stderr, "Output is in the file \"align.out\"\n"); exit(l);
} namex[0] = av[l]; namexfl] = av[2]; seqx[0] = getseq(namex[0], &len0); seqx[l] = getseq(namex[l], &lenl); xbm = (dna)? _dbval : pbval; endgaps = 0; /* 1 to penalize endgaps */ ofile = "align.out" /* output file */ nw(); /* fill in the matrix, get the possible jmps */ readjmps( ); /* get the actual jmps */ print( ); /* print stats, alignment */ cleanup(0); /* unlink any tmp files */ Table 1 (cont^
/* do the alignment, return best score: main( )
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y. nw( ) n
{ char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /* keep track of dely */ int ndelx, delx; /* keep track of delx */ int *tmρ; /* for swapping rowO, rowl */ int mis; /* score for each type */ int insO, insl; /* insertion penalties */ register id; /* diagonal index */ register ϋ; /* jmp index */ register *col0, *coll; /* score for curr, last row */ register xx, yy; /* index into seqs */ dx = (struct diag *)g_calloc("to get diags", lenO+lenl + 1, sizeof(struct diag)); ndely = (int *)g_calloc("to get ndely", lenl + 1, sizeof(int)); dely = (int *)g_calloc("to get dely", lenl 4-1, sizeof(int)); colO = (int *)g_calloc("to get colO", lenl + 1, sizeof(int)); coll = (int *)g_calloc("to get coll", lenl + 1, sizeof(int)); insO = (dna)? DINSO : PINSO; insl = (dna)? DINS1 : PINS1; smax = -10000; if (endgaps) { for (col0[0] = dely[0] = -insO, yy = 1; yy < = lenl; yy++) { col0[yy] = delyfyy] = col0[yy-l] - insl; ndelyfyyj = yy;
} col0[0] = 0; /* Waterman Bull Math Biol 84 */
} else for (yy = 1; yy < = lenl; yy+ +) delyfyy] = -insO;
/* fill in match matrix */ for (px = seqx[0], xx = 1; xx < = lenO; px+ + , xx+ +) { /* initialize first entry in col */ if (endgaps) { if (xx == 1) coll[0] = delx = -(insO+insl); else collIO] = delx = col0[0] - insl; ndelx = xx;
} else { coll[0] = 0; delx = -insO: ndelx = 0; Table 1 (conf)
...n or (py = seqx[l], yy = 1; yy < = lenl; py+ +, yy+ +) { mis = col0[yy-l]; if (dna) mis + = (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS; else mis + = _day[*px-'A'][*py-'A'];
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps 1 1 ndely[yy] < MAXGAP) { if (col0[yy] - insO > = delyfyy]) { dely[yy] = col0[yy] - (insO+insl); ' ndely[yy] = 1;
} else { dely[yy] -= insl; ndely[yy] + + ; }
} else { if (col0[yy] - (insO+insl) > = dely[yy]) { dely[yy] = col0[yy] - (insO+insl); ndely[yy] = 1;
} else ndely[yy] + + ;
}
/* update penalty for del in y seq; * favor new del over ongong del */ if (endgaps 1 1 ndelx < MAXGAP) { if (coll[yy-l] - insO > = delx) { delx = coll[yy-l] - (insO+insl); ndelx = 1; } else { delx -= insl; ndelx+ + ; }
} else { if (coll[yy-l] - (insO+insl) > = delx) { delx = coll[yy-l] - (insO+insl); ndelx = 1;
} else ndelx+ +;
}
/* pick the maximum score; we're favoring * mis over any del and delx over dely
*/
Table 1 (conf)
...n id = x - yy + lenl - 1; if (mis > = delx && mis > = delyfyy]) collfyy] = mis; else if (delx > = delyfyy]) { collfyy] = delx; ij = dx[id].ijmp; if (dx[id].jp.n[0] && (!dna 1 1 (ndelx > = MAXJMP && xx > dx[id].jp.x[ij]+MX) 1 1 mis > dx[id].score+DINS0)) { dx[id].ijmp+ + ; if (+ +ij > = MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id]. offset = offset; offset + = sizeof(struct jmp) + sizeof(offset);
} } dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id]. score = delx;
} else { collfyy] = dely[yy]; ij = dx[id].ijmρ; if (dx[id].jp.n[0] && (!dna 1 1 (ndely[yy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) | [ mis > dx[id]. score +DINS0)) { dx[id].ijmp+ + ; if (+ +ij > = MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id]. offset = offset; offset + = sizeof(struct jmp) + sizeof(offsef); } } dx[ιd].jp.n[ij] = -ndely[yy]; dx[id].jp.x[ij] = xx; dx[id]. score = dely[yy];
} if (xx = = lenO && yy < lenl) { /* last col */ if (endgaps) collfyy] -= insO+insl*(lenl-yy); if (coll [yy] > smax) { smax = coll[yy]; dmax = id; }
, J if (endgaps && xx < lenO) coll[yy-l] -= insO+insl*(lenO-xx); if (collfyy- 1] > smax) { smax = coll[yy-l]; dmax = id;
} tmp = colO; colO = coll; coll = imp;
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)co!0);
(void) free((char *)coll); } Table 1 (conf)
/*
* print( ) — only routine visible outside this module
*
* static:
* getmat( ) — trace back best path, count matches: print( )
* pr_align( ) — print alignment of described in array p[]: print( )
* dumpblock( ) — dump a block of lines with numbers, stars: pr_align( )
* nums( ) — put out a number line: dumpblock( )
* putline( ) — put out a line (name, [num], seq, [num]): dumpblock( )
* stars( ) - -put a line of stars: dumpblock( )
* stripname( ) — strip any path and prefix from a seqname */
#include "nw.h"
#define SPC 3 #defme P LINE 256 /* maximum output line */ #define P_SPC 3 /* space between name or num and seq */ extern _day[26][26]; int olen; /* set output line length */
FILE *fx; /* output file */ print( ) print { int Ix, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, "w")) == 0) { fprintf(stderr,"%s: can't write %s\n", prog, ofile); cleanup(l);
} φrintf(fx, " < first sequence: %s (length = %d)\n", namex[0], lenO); fprintf(fx, " < second sequence: %s (length = %d)\n", namexfl], lenl); olen = 60; lx = lenO; ly = lenl; firstgap = lastgap = 0; if (dmax < lenl - 1) { /* leading gap in x */ pp[0].spc = firstgap = lenl - dmax - 1; ly -= pp[0].spc;
} else if (dmax > lenl - 1) { /* leading gap in y */ pp[l].spc = firstgap = dmax - (lenl - 1); lx -= pp[l].spc;
} if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = lenO - dmaxO -1; lx -= lastgap;
} else if (dmaxO > lenO - 1) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1); ly -= lastgap;
} getmat(lx, ly, firstgap, lastgap); pr_align( ); Table 1 (conf)
/*
* trace back the best path, count matches
*/ static getmat(lx, ly, firstgap, lastgap) getmat int lx, ly; /* "core" (minus endgaps) */ int firstgap, lastgap; /* leading trailing overlap */ int nm, iO, il, si: zO, sizl; char outx[32]; double pet; register nO, nl; register char *pO, *pl;
/* get total matches, score */ iO = il = sizO = sizl = 0; pO = seqx[0] + pp[l].spc; pi = seqx[l] + pp[0].spc; nO = pp[l].spc + 1; nl = ρp[0].spc + 1; nm = 0; while ( *p0 && *pl ) { if (sizO) { pl + + ; nl + + ; sizO— ;
} else if (sizl) { p0+ + ; n0+ + ; sizl-
} else { if (xbm[*pO-'A']&xbm[*pl-'A']) nm+ + ; if (nO+ + = = pp[0].x[iO]) sizO = pp[0].n[iO+ +]; if (nl + + = -= pp[l].x[il]) sizl = pp[l].n[il + +]; p0+ + ; pl + +;
}
/* pet homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core */ if (endgaps) lx = (lenO < lenl)? lenO : lenl; else lx = (lx < ly)? lx : ly; pet = 100.*(double)nm/(double)lx; φrintf(fx, "\n"); f rintf(fx, " < %d match%s in an overlap of %d: %.2f percent similarity\n", nm, (nm = = 1)? "" : "es", lx, pet); . Table 1 (conf) φrintf(fx, " <gaps in first sequence: %d", gapx); ...getmat if (gapx) {
(void) sprintf(outx, " (%ά %s%s)", ngapx, (dna)? "base":"residue", (ngapx = = 1)? "":"s"); φrintf(fx,"%s", outx); φrintf(fx, ", gaps in second sequence: %d", gapy); if (gapy) {
(void) sprintf(outx, " (%d %s%s)", ngapy, (dna)? "base": "residue", (ngapy == 1)? "":"s"); φrintf(fx," %s", outx);
} if (dna) φrintf(fx,
"\n< score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n", smax, DMAT, DMIS, DINSO, DINS1); else φrintf(fx,
"\n<score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per residue)\n", smax, PINSO, PINS1); if (endgaps) φrintf(fx,
" <endgaps penalized, left endgap: %d %s%s, right endgap: %d %s s\n", firstgap, (dna)? "base" : "residue", (firstgap = = 1)? "" : "s", lastgap, (dna)? "base" : "residue", (lastgap == 1)? "" : "s"); else φrintf(fx, " < endgaps not penalized\n");
static nm; /* matches in core — for checking */ static Imax; /* lengths of stripped file names */ static ij[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[2]; /* current elem number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next ouφut char slot */ static char out[2][P LINE]; /* output line */ static char star[P LINE]; /* set by stars( ) */
/*
* print alignment of described in struct path pp[]
*/ static pr_align( ) pr align { int nn; /* char count : int more; register i; for (i = 0, lmax = 0; i < 2; i+ +) { nn = stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; Table 1 (conf) for (nn = nm = 0, more = 1; more; ) { ...pr_align for (i = more = 0; i < 2; i++) { /*
* do we have more of this sequence? */ if(!*ρs[i]) continue; more++; if (pp[i]-spc) { /* leading space */ *po[i] + + = ' '; pp[i].spc~;
} else if (siz[ij) { /* in a gap */
*po[i] + + = '-'; siz[i]-;
} else { /* we're putting a seq element
*/ *po[i] = *ps[i]; if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]); po[i] + + ; ps[i] + + ;
/* * are we at next gap for this seq? if(ni[i]==pp[i].x[ij[i]]){ /*
* we need to merge all gaps
* at this location */ siz[i] =pp[i].n[ij[i] + +]; while (ni[i] == pp[i].x[ij[i]]) siz[i]+=pp[i].n[ij[i] + +];
} ni[i] + +;
} if (+ +nn = = olen 11 !more && nn) { dumpblock( ); for(i = 0;i < 2;i++) po[i] = out[i]; nn = 0;
}
/*
* dump a block of lines, including numbers, stars: pr_align( )
*/ static dumpbiock( ) dumpblock
{ register i; for(i = 0;i < 2; i++) *po[i]~ = '\0'; Table 1 (conf)
.dumpblock
(void)putc('\n', fx); for(i = 0; i < 2;i++){ if (*out[i] && (*out[i] != ' ' 11 *(po[i]) !='')){ if(i==0) nums(i); if(i==0&&*out[l]) stars( ); putline(i); if(i ==0&&*out[l]) φrintf(fx, star); if(i==l) nums(i);
}
/*
* put out a number line: dumpblock( ) */ static nums(ix) nums int ix; /* index in out[] holding seq line */ char nline[P_LINE]; register i, j; register char *pn, *px, *py; for (pn = nline, i = 0; i < lmax+P_SPC; i+ + , pn++)
*pn = ' '; for (i = nc[ix], py = out[ix]; *py; py+ + , pn++) { if(*py==" || *py -=-='-') else if (i%10 == 011 (i == 1 &&nc[ix] != 1)) { j = (i < 0)? -i : i; for (px -= pn; j; j /= 10, px-)
*px = j%10 + '0'; if (i < 0)
*px = '-'; else i+ + ;
} nc[ix] = i; for (pn = nline; *pn; pn+ +) (void) putc(*pn, fx); (void)putc('\n', fx);
}
/*
* put out a line (name, [num], seq, [num]): dumpblock( )
*/ static putiine(ix) putline int ix; { Table 1 (conf)
.putline int 1; register char *px; for (px = namexøx], i = 0; *px && *px != ':'; px+ + , i++)
(void) putc(*px, fx); for (; i < lmax+P_SPC; i+ +)
(void) putc(' ', fx);
/* these count from 1:
* ni[] is current element (from 1)
* nc[] is number at start of current line */ for (px = out[ix]; *px; px++)
(void) putc(*ρx&0x7F, fe); (void)ρutc('\n', fx);
/*
* put a line of stars (seqs always in out[0], out[l]): dumpblock( )
*/ static stars( ) stars int i; register char *p0, *pl, ex, *px; if (!*out[0] 11 (*out[0] ==''&& *(po[0]) == ' ') 11 !*out[l] 11 (*out[l] ==''&& *(po[l]) =='')) return; px = star; for (i = lmax+P_SPC; i; i-)
*px++ = ' '; for (pO = out[0], pi = out[l]; *p0 && *pl; p0++, pl + +) { if (isalpha(*pO) && isalpha(*pl)) { if (xbm[*pO-'A']&xbm[*pl-'A']) { ex = '*'; nm+ + ; ,
} elseif(!dna&&_day[*pO-'A'][*pl-'A'] > 0) ex = '.'; else ex = ' ';
} else ex = ' '; *px++ = ex;
}
*px++ = '\n'; *px= '\0'; Table 1 (conf)
/*
* strip path or prefix from pn, retarn len: pr_align( ) */ static stripname(pn) Stripname char *pn; /* file name (may be path) */
{ register char *px, *py; py = 0; for (px = pn; *px; px+ +) if (*px == V) py = px + 1; if (ρy)
(void) strcpy(pn, py); return(strlen(pn)) ;
Table 1 (conf)
/*
* cleanup( ) — cleanup any tmp file
* getseq( ) — read in seq, set dna, len, maxlen
* g__calloc( ) — calloc( ) with error checkin
* readjmps( ) — get the good jmps, from tmp file if necessary
* writejmps( ) — write a filled array of jmps to a tmp file: nw( ) */
^include "πw.h" #include <sys/file.h> char *jname = "/tmp/homgXXXXXX"; /* tmp file for jmps */ FILE *fj; int cleanup( ); /* cleanup tmp file */ long lseek( );
/* * remove any tmp file if we blow
*/ cleanup® cleanup int { if Cβ)
(void) unlink(jname); exit(i);
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with ';', ' < ', or ' > '
* seq in upper or lower case */ char * getseq(file, len) getseq char *file; /* file name */ int *len; /* seq len */
{ char line[1024J, *ρseq; register char *px, *py; int natgc, tlen;
FILE *φ; if ((fp = fopen(file,"r")) = = 0) { φrintf(stderr,"%s: can't read %s\n", prog, file); exit(l);
} tlen = natgc = 0; while (fgets(line, 1024, fp)) { if (*line = = ';' | | *line = = ' < ' | | *line = = ' > ') continue; for (px = line; *px != '\n'; px++) if (isupper(*px) 1 1 islower(*px)) tlen++;
} if ((pseq = malloc((unsigned)(tlen+6))) = = 0) { φrintf(stderr," s: malloc( ) failed to get %d bytes for %s\n", prog, flen+6, file); exit(l);
} pseq[0] = pseq[l] = pseq[2] = ρseq[3] = '\0'; Table 1 (conf)
..getseq py = pseq + 4; *len = tlen; rewind(φ); while (fgets(line, 1024, φ)) { if (*line = = ';' | | *line == ' < ' | | *line = = ' > ') continue; for (px = line; *px != '\n'; px+ +) { if (isuρper(*px))
*py+ + = *px; else if (islower(*px))
*py+ + = toupper(*px); if (index("ATGCU",*(py-l))) natgc+ + ;
> >
*py++ = '\0';
*py = '\0';
(void) fclose(φ); dna = natgc > (tlen/3); return(pseq+4);
char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, calling routine */ int nx, sz; /* number and size of elements */
{ char *px, *calloc( ); if ((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) { φrintf(stderr, "%s: g_calloc( ) failed %s (n= %d, sz= %d)\n", prog, msg, nx, sz); exit(l); } } return(px);
}
/* * get final jmps from dx[] or tmp file, set pp[], reset dmax: main( )
*/ readjmps( ) readjmps
{ int fd = -1; int siz, iO, il; register i, j, xx; if (fj) {
(void) fclose(fj); if ((fd = open(jname, 0_RDONLY, 0)) < 0) { fprintf(stderr, "%s: can't open( ) %s\n", prog, jname); cleanup(l); }
} for (i = iO = il = 0, dmaxO = dmax, xx = lenO; ; i+ +) { while (1) { for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j-) Table 1 (conf)
...readjmps if (j < 0 && dx[dmax]. offset && fj) {
(void) lseek(fd, dx[dmax]. offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax]. offset, sizeof(dx[dmax]. offset)); dx[dmax].ijmp = MAXJMP-1;
} else break;
} if (i > = JMPS) { φrintf(stderr, " %s: too many gaps in alignment\n", prog); cleanup(l);
} if (j > = 0) { siz = dx[dmax].jp.njr ]; xx = dx[dmax].jp.x[j]; dmax + = siz; if (siz < 0) { /* gap in second seq */ pp[l].n[il] = -siz; xx + = siz;
/* id = xx - yy + lenl - 1 */ pp[l].x[il] = xx - dmax + lenl - 1; gapy+ + ; ngapy -= siz; /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP 1 1 endgaps)? -siz : MAXGAP; il + + ;
> else if (siz > 0) { /* gap in first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx+ + ; ngapx + = siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP 1 1 endgaps)? siz : MAXGAP; i0+ + ; } } else break; }
/* reverse the order of jmps */ for 0 = 0, i0~; j < iO; j + + , i0~) { i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = PP[O].x 1; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = i;
} for (j = 0, il— ; j < il; j+ + , il-) { i = pp[l].n[j]; pp[l].n[j] = pp[l].n[il]; pp[l].n[il] = i; i = pp[l].xD]; pp[l].x[j] = pp[l].x[il]; pp[l].x[il] = i;
} if (fd > = 0)
(void) close(fd); if (® {
(void) unlink(jname); fj = 0; offset = 0; } } Table 1 (conf)
/*
* write a filled jmp struct offset ofthe prev one (if any): nw( ) */ writejmps(iχ) writejmps int ix;
{ char *mkιemp( ); if (!fj) { if (mktemp(jname) < 0) { φrintf(stderr, "%s: can't mktemp( ) %s\n", prog, jname); cleanup(l);
} if ((fj = fopen(jname, "w")) = = 0) { φrintf(stderr, " %s: can't write s\n", prog, jname); exit(l); } }
(void) fwrite((char *)&dx[ix].jp, sizeof (struct jmp), 1, fj); (void) f rite((char *)&dx[ix]. offset, sizeof(dx[ix]. offset), 1, fj);
Table 2
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by
ALIGN-2) divided by (the total number of amino acid residues o the PRO polypeptide) =
5 divided by 15 = 33.3%
O Table 3
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by
ALIGN-2) divided by (the total number of amino acid residues ofthe PRO polypeptide) =
5 divided by 10 = 50%
Table 4
:o PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-
,5 2) divided by (the total number of nucleotides ofthe PRO-DNA nucleic acid sequence) =
6 divided by 14 = 42.9%
Table 5
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLW (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-
2) divided by (the total number of nucleotides ofthe PRO-DNA nucleic acid sequence) =
4 divided by 12 = 33.3% 5.2. Compositions and Methods ofthe Invention
5.2.1. PRO Variants
In addition to the full-length native sequence PRO polypeptides described herein, it is contemplated that
PRO variants can be prepared. PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA, and/or by synthesis ofthe desired PRO polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes ofthe PRO polypeptide such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PRO polypeptide or in various domains of the PRO polypeptide described herein, can be made, for example, using any of tlie techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding tlie PRO polypeptide that results in a change in tlie amino acid sequence ofthe PRO polypeptide as compared with the native sequence PRO polypetide. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more ofthe domains ofthe PRO polypeptide. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing tlie sequence ofthe PRO polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i. e. , conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.
Table 6
Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gin; lys; arg arg He (I) leu; val; met; ala; phe; norleucine leu
Leu (L) norleucine; ile; val; met; ala; phe ile
Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr tlir Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) tip; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu
Substantial modifications in function or immunological identity ofthe PRO polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk ofthe side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: tip, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as oligonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al, Nucl. Acids Res.. 13:4331 (1986); Zoller et al, Nucl. Acids Res.. 10:6487 (1987)], cassette mutagenesis [Wells et al, Gene, 34:315 (1985)], restriction selection mutagenesis [Wells etal, Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation ofthe variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150: 1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
5.2.2. Modifications of PRO Polypeptides
Covalent modifications of PRO polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a PRO polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues ofthe PRO polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking the PRO polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO antibodies, and vice- versa. Commonly used crosslinking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N- maleimido-l,8-octane and agents such as methyl-3-[(ρ-azidoρhenyl)dithio]propioimidate. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation ofthe α-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties. W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation ofthe N-terminal amine, and amidation of any C-terminal carboxyl group. Another type of covalent modification ofthe PRO polypeptide included within the scope of this invention comprises altering the native glycosylation pattern ofthe polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in the native sequence PRO polypeptide (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means) , and or adding one or more glycosylation sites that are not present in the native sequence PRO polypeptide. In addition, tlie phrase includes qualitative changes in tlie glycosylation ofthe native proteins, involving a change in the nature and proportions ofthe various carbohydrate moieties present.
Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO polypeptide (for O-linked glycosylation sites). The PRO amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
Another means of increasing tlie number of carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp.259-306 (1981). Removal of carbohydrate moieties present on tlie PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al, Arch. Biochem. Biophvs., 259:52 (1987) and by Edge et al., Anal. Biochem.. 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al, Metli. Enzymol..138:350 (1987).
Another type of covalent modification ofthe PRO polypeptide comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The PRO polypeptide ofthe present invention may also be modified in a way to form a chimeric molecule comprising the PRO polypeptide fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion ofthe PRO polypeptide with a protein transduction domain which targets the PRO polypeptide for delivery to various tissues and more particularly across the brain blood barrier, using, for example, the protein transduction domain of human immunodeficiency virus TAT protein (Schwarze et al, 1999, Science 285: 1569-72).
In another embodiment, such a chimeric molecule comprises a fusion ofthe PRO polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus ofthe PRO polypeptide. The presence of such epitope- tagged forms ofthe PRO polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-His) or poly-histidine-glycine (poly- His-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al, Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al, Molecular and Cellular Biology. 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody
[Paborsky et al. , Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp etal, BioTechnology, 6:1204-1210 (1988)]: the KT3 epitope peptide [Martin etal. Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinner et al, J. Biol. Chem..266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth etal, Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)]. In an alternative embodiment, the chimeric molecule may comprise a fusion ofthe PRO polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form ofthe chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI , CH2 and CH3 regions of an IgGl molecule. For the production of immunoglobulin fusions see also, U.S. Patent No.5,428, 130 issued June 27, 1995.
5.2.3. Preparation ofthe PRO Polypeptide
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides. In particular, cDNAs encoding PRO polypeptides have been identified and isolated, as disclosed in further detail in the Examples below. It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed. However, for sake of simplicity, in the present specification the protein encoded by the PRO DNA as well as all further native homologues and variants included in the foregoing definition of PRO polypeptides, will be referred to as "PRO" regardless of their origin or mode of preparation.
Tlie description below relates primarily to production of PRO polypeptides by culturing cells transformed or transfected with a vector containing nucleic acid encoding PRO polypeptides. It is, of course, contemplated that alternative methods that are well known in the art may be employed to prepare the PRO polypeptide. For instance, the PRO polypeptide sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques. See, e.g., Stewart etal. Solid-Phase Peptide Synthesis (W.H. Freeman Co.: San Francisco, CA, 1969); Merrifϊeld, J. Am. Chem. Soc. 85: 2149-2154 (1963). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, with an Applied Biosystems
Peptide Synthesizer (Foster City, CA) using manufacturer's instructions. Various portions ofthe PRO polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full- length PRO polypeptide.
5.2.3.1. Isolation of DNA Encoding PRO Polypeptides DNA encoding tlie PRO polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA encoding the PRO polypeptide and to express it at a detectable level. Accordingly, DNAs encoding the human PRO polypeptide can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in the Examples. The gene encoding the PRO polypeptide may also be obtained from a genomic library or by oligonucleotide synthesis. Libraries can be screened with probes (such as antibodies to the PRO polypeptide or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al, supra. An alternative means to isolate the gene encoding the PRO polypeptide is to use PCR methodology. Sambrook et al, supra; Dieffenbach et al, PCR Primer: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1995).
The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radio labels like 32P-labeled ATP, biotinylation, or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al, supra.
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of tlie molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as
ALIGN, DNAstar, and INHERIT, which employ various algorithms to measure homology.
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al, supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
5.2.3.2. Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described herein for PRO polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformanfs, or amplifying the genes encoding tlie desired sequences. The culture conditions, such as media, temperature, pH, and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach. M. Butler, ed. (IRL Press, 1991) and Sambrook et al, supra. Methods of transfection are known to the ordinarily skilled artisan, for example, CaP04 treatment and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al, supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection •withAgrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al. , Gene. 23: 315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 ( 1978) can be employed. General aspects ofmammalian cell host system transformations have been described in U.S. PatentNo. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al, J. Bact, 130: 946 (1977) and Hsiao et al, Proc. Natl. Acad. Sci. (USA). 76: 3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene or polyoπuthine, may also be used. For various techniques for transforming mammalian cells, see, Keown et al, Methods in Enzymology. 185: 527-537 (1990) and Mansour et al, Nature. 336: 348-352 (1988).
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coliYΛl strain MM294 (ATCC 31,446); £. co/ X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325); and K5 772 (ATCC 53,635). Otlier suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g.,B. licheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as
P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to tlie host, with examples of such hosts including E. coli W3110 strain 1 A2, which has the complete genotype tonA ; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonAptr3phoA E15 (argF-lac)169 degP ompTkan'; E. coli W3110 strain 37D6, which has the complete genotype tonAptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kaif; E. coliW3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No.4,946,783 issued 7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding the PRO polypeptide. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al,
Bio/Technology. 9: 968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al, J. BacterioL. 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178),K. waltii (ATCC 56,500),K. drosophilarum (ATCC 36.906: Van den Berg etal. Bio/Technology, 8: 135 (1990)), K. thermotolerans, and K. maj-χianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al, J. Basic Microbiol. 28: 265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa
(Case et al, Proc. Natl. Acad. Sci. USA. 76: 5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al, Biochem. Biophvs. Res. Commun.. 112: 284-289 [1983]; Tilburn et al, Gene. 26: 205-221 [1983]; Yelton et al, Proc. Natl. Acad. Sci. USA. 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4: 475-479
[1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
Suitable host cells for the expression of nucleic acid encoding glycosylated PRO polypeptides are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary
(CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryomc kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al. . Gen. Virol, 36: 59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA. 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor
(MMT 060562, ATCC CCL51). The selection ofthe appropriate host cell is deemed to be within tlie skill in the art.
5.2.3.3. Selection and Use of a Replicable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding the PRO polypeptide may be inserted into a replicable vector for cloning (amplification ofthe DNA) or for expression. Various vectors are publicly available.
The vector may, for example, be in tlie form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence if the sequence is to be secreted, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
Construction of suitable vectors containing one or more of these components employs standard ligation techniques that are known to the skilled artisan.
The PRO polypeptide may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at tlie N-terminus of tlie mature protein or polypeptide. In general, the signal sequence may be a component ofthe vector, or it may be a part ofthe DNA encoding the PRO polypeptide that is inserted into tlie vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a- factor leaders, the latter described in U.S. PatentNo.5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362, 179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990.
In mammalian cell expression, mammalian signal sequences may be used to direct secretion ofthe protein, such as signal sequences from secreted polypeptides ofthe same or related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker.
Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillim neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the nucleic acid encoding the PRO polypeptide such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is tlie CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). A suitable selection gene for use in yeast is the trpl gene present in the yeastplasmid YRp7. Stinchcomb et al. , Nature.282: 39 (1979);
Kingsman et al. , Gene. 7: 141 (1979); Tschemper et al. Gene, 10: 157 (1980). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or
PEP4-1. Jones, Genetics. 85: 12 (1977). Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding the PRO polypeptide to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al, Nature, 275: 615 (1978); Goeddel et al, Nature, 281: 544 (1979)), alkaline phosphatase, a tryptophan (tip) promoter system (Goeddel, Nucleic Acids Res.. 8 : 4057 (1980); EP 36,776), and hybrid promoters such as the tac promoter (deBoer et al, Proc. Natl. Acad. Sci. USA. 80: 21-25 (1983)). Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S .D.) sequence operably linked to the DNA encoding the PRO polypeptide.
Examples of suitable promoting sequences for use with yeast hosts include tlie promoters for 3- phosphoglycerate kinase QHitzeman et al, J. Biol. Chem..255: 2073 (1980)) or other glycolytic enzymes (Hess et al. J. Adv. Enzyme Reg..7: 149 (1968); Holland, Biochemistry.17: 4900 (1978)), such as enolase, glyceraldehyde-
3-phosρhate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphoffuctokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters that are inducible promoters having tlie additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phos- phate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
PRO nucleic acid transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published
5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, and Simian Virus 40 (SV40); by heterologous mammalian promoters, e.g. , the actin promoter or an immunoglobulin promoter; and by heat-shock promoters, provided such promoters are compatible with the host cell systems.
Transcription of a DNA encoding the PRO polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side ofthe replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the sequence coding for PRO polypeptides, but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the PRO polypeptide.
Still other methods, vectors, and host cells suitable for adaptation to the synthesis ofthe PRO polypeptide in recombinant vertebrate cell culture are described in Gething et al, Nature, 293: 620-625 (1981); Mantei et al, Nature.281: 40-46 (1979); EP 117,060; and EP 117,058.
5.2.3.4. Detecting Gene Amplification/Expression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional
Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on tlie sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such as ihimunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, tlie antibodies may be prepared against a native-sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA encoding the PRO polypeptide and encoding a specific antibody epitope.
5.2.3.5. Purification of PRO Polypeptides Forms of PRO polypeptides may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., TRITON-X™ 100) or by enzymatic cleavage. Cells employed in expression of nucleic acid encoding the PRO polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell-lysing agents. It may be desired to purify the PRO polypeptide from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by f actionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms ofthe PRO polypeptide. Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Deutscher, Methods in
Enzymology. 182 (1990^: Scopes. Protein Purification: Principles and Practice (Springer- Verlag: New York, 1982). The purification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO polypeptide produced.
5.2.4. Uses of PRO Polypeptides 5.2.4.1. Assays for Cardiovascular. Endothelial. and Angiogenic Activity
Various assays can be used to test the polypeptide herein for cardiovascular, endothelial, and angiogenic activity. Such assays include those provided in the Examples below.
Assays for testing for endothelin antagonist activity, as disclosed in U.S. Pat. No. 5,773,414, include a rat heart ventricle binding assay where the polypeptide is tested for its ability to inhibit iodinized endothelin- 1 binding in a receptor assay, an endothelin receptor binding assay testing for intact cell binding of radiolabeled endothelin- 1 using rabbit renal artery vascular smooth muscle cells, an inositol phosphate accumulation assay where functional activity is determined in Rat-1 cells by measuring infra-cellular levels of second messengers, an arachidonic acid release assay that measures tlie ability of added compounds to reduce endothelin-stimulated arachidonic acid release in cultured vascular smooth muscles, in vitro (isolated vessel) studies using endothelium from male New Zealand rabbits, and in vivo studies using male Sprague-Dawley rats.
Assays for tissue generation activity include, without limitation, those described in WO 95/16035 (bone, cartilage, tendon); WO 95/05846 (nerve, neuronal), and WO 91/07491 (skin, endothelium).
Assays for wound-healing activity include, for example, those described in Winter, Epidermal Wound Healing. Maibach, HI and Rovee, DT, eds. (Year Book Medical Publishers, Inc., Chicago), pp.71- 112, as modified by the article of Eaglstein and Mertz, J. Invest. Dermatol.. 71: 382-384 (1978).
An assay to screen for a test molecule relating to a PRO polypeptide that binds an endothelin B! (ETBj) receptor polypeptide and modulates signal transduction activity involves providing a host cell transformed with a DNA encoding endothelin : receptor polypeptide, exposing the cells to the test candidate, and measuring endothelin B, receptor signal transduction activity, as described, e.g., in U.S. Pat. No. 5,773,223. There are several cardiac hypertrophy assays. In vitro assays include induction of spreading of adult rat cardiac myocytes. In this assay, ventricular myocytes are isolated from a single (male Sprague-Dawley) rat, essentially following a modification ofthe procedure described in detail by Piper et al. , "Adult ventricular rat heart muscle cells" in Cell Culture Techniques in Heart and Vessel Research. H.M. Piper, ed. (Berlin: Springer- Verlag, 1990), pp. 36-60. This procedure permits tlie isolation of adult ventricular myocytes and the long-term culture of these cells in the rod-shaped phenotype. Phenylephrine and Prostaglandin F2α (PGF2«) have been shown to induce a spreading response in these adult cells. The inhibition of myocyte spreading induced by PGF2« or PGF2„ analogs
(e.g., fluprostenol) and phenylephrine by various potential inhibitors of cardiac hypertrophy is then tested.
One example of an in vivo assay is a test for inhibiting cardiac hypertrophy induced by fluprostenol in vivo. This pharmacological model tests the ability ofthe PRO polypeptide to inhibit cardiac hypertrophy induced in rats (e.g., male Wistar or Sprague-Dawley) by subcutaneous injection of fluprostenol (an agonist analog of PGF2„). It is known that rats with pathologic cardiac hypertrophy induced by myocardial infarction have chronically elevated levels of extractable PGF2o in their myocardium. Lai et al, Am. J. Phvsiol. (Heart Circ. PhvsioL). 271: H2197- H2208 (1996). Accordingly, factors that can inhibit the effects of fluprostenol on myocardial growth in vivo are potentially useful for treating cardiac hypertrophy. The effects ofthe PRO polypeptide on cardiac hypertrophy are determined by measuring tlie weight of heart, ventricles, and left ventricle (normalized by body weight) relative to fluprostenol-treated rats not receiving the PRO polypeptide. -
Another example of an in vivo assay is the pressure-overload cardiac hypertrophy assay. For in vivo testing it is common to induce pressure-overload cardiac hypertrophy by constriction ofthe abdominal aorta of test animals. In a typical protocol, rats (e.g., male Wistar or Sprague-Dawley) are treated under anesthesia, and the abdominal aorta of each rat is narrowed down just below the diaphragm. Beznak M., Can. J. Biochem. Phvsiol., 33: 985-94 (1955). The aorta is exposed through a surgical incision, and a blunted needle is placed next to the vessel. The aorta is constricted with a ligature of silk thread around the needle, which is immediately removed and which reduces the lumen ofthe aorta to the diameter ofthe needle. This approach is described, for example, in Rossi et al, Am. Heart J.. 124: 700-709 (1992) and O'Rourke and Reibel, P.S.E.M.B.. 200: 95-100 (1992).
In yet another in vivo assay, the effect on cardiac hypertrophy following experimentally induced myocardial infarction (MI) is measured. Acute MI is induced in rats by left coronary artery ligation and confirmed by electrocardiographic examination. A sham-operated group of animals is also prepared as control animals. Earlier data have shown that cardiac hypertrophy is present in the group of animals with MI, as evidenced by an 18% increase in heart weight-to-body weight ratio. Lai et al, supra. Treatment of these animals with candidate blockers of cardiac hypertrophy, e.g., the PRO polypeptide, provides valuable information about the therapeutic potential ofthe candidates tested. One further such assay test for induction of cardiac hypertrophy is disclosed in
U.S. Pat. No. 5,773,415, using Sprague-Dawley rats.
For cancer, a variety of well-known animal models can be used to further understand the role ofthe genes identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies and other antagonists of native PRO polypeptides, such as small-molecule antagonists. The in vivo nature of such models makes them particularly predictive of responses in human patients. Animal models of tumors and cancers (e.g., breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g. , murine models. Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e.g., subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e.g., colon cancer cells implanted in colonic tissue. See, e.g., PCT publication No. WO 97/33551, published September 18, 1997. Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice. The observation that the nude mouse with thymic hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose. The autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII, and S JL. In addition, a wide variety of other animals with inherited immunological defects other than tlie nude mouse have been bred and used as recipients of tumor xenografts. For further details see, e.g., The Nude Mouse in Oncology Research, E. Boven and B. Winograd, eds. (CRC Press, Inc., 1991).
The cells introduced into such animals can be derived from known tumor/cancer cell lines, such as any ofthe above-listed tumor cell lines, and, for example, the B104-1-1 cell line (stable NTH-3T3 cell line transfected with the neu protooncogene); røs-transfected NTH-3T3 cells; Caco-2 (ATCC HTB-37); or a moderately well- differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38); or from tumors and cancers. Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions involving freezing and storing in liquid nitrogen. Karmali et al, Br. J. Cancer. 48: 689-696 (1983).
Tumor cells can be introduced into animals such as nude mice by a variety of procedures. The subcutaneous (s.c.) space in mice is very suitable for tumor implantation. Tumors can be transplanted s.c. as solid blocks, as needle biopsies by use of a frochar, or as cell suspensions. For solid-block or frochar implantation, tumor tissue fragments of suitable size are introduced into the s.c. space. Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously. Tumor cells can also be injected as subdermal implants. In this location, the inoculum is deposited between the lower part ofthe dermal connective tissue and the s.c. tissue.
Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogene was initially isolated), or new-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al. Proc. Nat. Acad. Sci. USA. 83: 9129-9133 (1986).
Similarly, animal models of colon cancer can be generated by passaging colon cancer cells in animals, e.g. , nude mice, leading to the appearance of tumors in these animals. An or hotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang et al, Cancer Research.54: 4726-4728 (1994) and Too et al, Cancer Research, 55: 681-684 (1995). This model is based on the so-called "METAMOUSE™" sold by AntiCancer, Inc., (San Diego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from the in vitro cultures can then be passaged to animals. Such tumors can serve as targets for further testing or drug screening. Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest. Such passaging techniques can be performed with any known tumor or cancer cell lines.
For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al, J. Exp. Med.. 146: 720 (1977)), which provide a highly controllable model system for studying tlie anti-tumor activities of various agents. Palladino et al, J. Immunol., 138: 4023-4032 (1987). Briefly, tumor cells are propagated in vitro in cell culture. Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about lOxlO6 to 10x10' cells/ml. The animals are then infected subcutaneously with 10 to 100 μl of the cell suspension, allowing one to three weeks for a tumor to appear. In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most thoroughly studied experimental tumors, can be used as an investigational rumor model. Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small-cell carcinoma of tlie lung
(SCCL). This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture. Zupi etal, Br. J. Cancer, 41: suppl.4, 30 (1980). Evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive. For further information about this tumor model see, Zacharski, Haemostasis, 16: 300-320 (1986). One way of evaluating the efficacy of a test compound in an animal model with an implanted tumor is to measure the size of the tumor before and after treatment. Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions. The measure limited to two dimensions does not accurately reflect the size ofthe tumor; therefore, it is usually converted into the corresponding volume by using a mathematical formula. However, the measurement of tumor size is very inaccurate. The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay. Another important variable in the description of tumor growth is the tumor volume doubling time. Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen. Proc.6th Int. Workshop on Immune-Deficient Animals, WuandShengeds. (Basel, 1989),ρ.301. It is noted, however, that necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially. Therefore, these changes need to be carefully monitored, by a combination of a morphometric method and flow cytometric analysis.
Further, recombinant (transgenic) animal models can be engineered by introducing tlie coding portion of the PRO gene identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals. Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees and monkeys. Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (U.S. Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g., Van der Putten et al, Proc. Natl. Acad. Sci. USA. 82: 6148-615 (1985)); gene targeting in embryonic stem cells (Thompson et al, Cell, 56: 313-321 (1989)); electroporation of embryos (Lo, Mol. Cell. Biol, 3: 1803-1814 (1983)); and sperm-mediated gene transfer. Lavitrano et al, Cell. 57: 717-73 (1989). For a review, see for example, U.S. Patent No. 4,736,866.
For the purpose ofthe present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals"). The transgene can be integrated either as a single transgene, or in concatamers, e.g. , head-to-head or head-to-tail tandems. Selective introduction of a transgene into a particular cell type is also possible by following, for example, tlie technique of Lasko et al, Proc. Natl. Acad. Sci. USA.89: 6232- 636 (1992). The expression ofthe transgene in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification can be used to verify the integration ofthe transgene. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry. The animals are further examined for signs of tumor or cancer development.
Alternatively, "knock-out" animals can be constructed that have a defective or altered gene encoding a PRO polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the PRO polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell ofthe animal. For example, cDNA encoding a particular PRO polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques. A portion ofthe genomic DNA encoding a particular PRO polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector. See, e.g. , Thomas and Capecchi, Cell, 51: 503 (1987) for a description of homologous recombination vectors. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected. See, e.g., Li et al, Cell. 69: 915 (1992). The selected cells are then injected into a blastocyst of an animal (e.g. , a mouse or rat) to form aggregation chimeras. See, e.g. , Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL: Oxford, 1987), pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock-out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized, for instance, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to absence ofthe PRO polypeptide.
The efficacy of antibodies specifically binding the PRO polypeptides identified herein, and other drug candidates, can be tested also in tlie treatment of spontaneous animal tumors. A suitable target for such studies is the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% ofthe oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor. These tumors are usually not amenable to surgery, primarily because ofthe anatomy ofthe feline oral cavity. At present, there is no effective treatment for this tumor. Prior to entry into the study, each cat undergoes complete clinical examination and biopsy, and is scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves. Each cat is treated repeatedly, over a longer period of time. Photographs ofthe tumors will be taken daily during the treatment period, and at each subsequent recheck. After treatment, each cat undergoes another CT scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter. The data are evaluated for differences in survival, response, and toxicity as compared to control groups. Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span.
In addition, other spontaneous animal tumors, such as fibrosarcoma, adenocarcinoma, lymphoma, chondroma, or leiomyosarcoma of dogs, cats, and baboons can also be tested. Of these, mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans. However, the use of this model is limited by the rare occurrence of this .type of tumor in animals.
Other in vitro and in vivo cardiovascular, endothelial, and angiogenic tests known in tlie art are also suitable herein.
5.2.4.2. Tissue Distribution
The results of tlie cardiovascular, endothelial, and angiogenic assays herein can be verified by further studies, such as by determining mRNA expression in various human tissues.
As noted before, gene amplification and/or gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA.77:5201-5205 (1980)), dot blotting QDNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
Gene expression in various tissues, alternatively, may be measured by immunological methods, such as immmiohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly tlie expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native-sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO DNA and encoding a specific antibody epitope. General techniques for generating antibodies, and special protocols for in situ hybridization are provided hereinbelow.
5.2.4.3. Antibody Binding Studies
The results ofthe cardiovascular, endothelial, and angiogenic study can be further verified by antibody binding studies, in which the ability of anti-PRO antibodies to inhibit the effect of the PRO polypeptides on endothelial cells or other cells used in the cardiovascular, endothelial, and angiogenic assays is tested. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow.
Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques (CRC Press, Inc., 1987), pp.147-158. Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of target protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte that remain unbound.
Sandwich assays involve the use of two antibodies, each capable of binding to a different mimunogenic portion, or epitope, ofthe protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay) . For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
For immunohistochemistry, the tissue sample may be fresh or frozen or may be embedded hi paraffin and fixed with a preservative such as formalin, for example.
5.2.4.4. Cell-Based Tumor Assays Cell-based assays and animal models for cardiovascular, endothelial, and angiogenic disorders, such as tumors, can be used to verify the findings of a cardiovascular, endothelial, and angiogenic assay herein, and further to understand the relationship between the genes identified herein and the development and pathogenesis of undesirable cardiovascular, endothelial, and angiogenic cell growth. The role of gene products identified herein in the development and pathology of undesirable cardiovascular, endothelial, and angiogenic cell growth, e.g., tumor cells, can be tested by using cells or cells lines that have been identified as being stimulated or inhibited by the PRO polypeptide herein. Such cells include, for example, those set forth in the Examples below.
In a different approach, cells of a cell type known to be involved in a particular cardiovascular, endothelial, and angiogenic disorder are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessive growth or inhibit growth is analyzed. If the cardiovascular, endothelial, and angiogenic disorder is cancer, suitable tumor cells include, for example, stable tumor cell lines such as the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and rαs-transfected NTH-3T3 cells, which can be transfected with the desired gene and monitored for tumorigenic growth. Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumorigenic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC). Cells transfected with the coding sequences ofthe genes identified herein can further be used to identify drug candidates for the treatment of cardiovascular, endothelial, and angiogenic disorders such as cancer.
In addition, primary cultures derived from tumors in transgenic animals (as described above) can be used in tlie cell-based assays herein, although stable cell lines are preferred. Techniques to derive continuous cell lines from transgenic animals are well known in the art. See, e.g., Small et al, Mol. Cell. Biol., 5: 642-648 (1985). 5.2.4.5. Gene Therapy Described below are methods and compositions whereby disease symptoms may be ameliorated. Certain diseases are brought about, at least in part, by an excessive level of gene product, or by tlie presence of a gene product exhibiting an abnormal or excessive activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of such disease symptoms.
Alternatively, certain other diseases are brought about, at least in part, by the absence or reduction ofthe level of gene expression, or a reduction in the level of a gene product's activity. As such, an increase in the level of gene expression and/or tlie activity of such gene products would bring about the amelioration of such disease symptoms. In some cases, tlie up-regulation of a gene in a disease state reflects a protective role for that gene product in responding to the disease condition. Enhancement of such a target gene's expression, or the activity ofthe target gene product, will reinforce the protective effect it exerts. Some disease states may result from an abnormally low level of activity of such a protective gene. In these cases also, an increase in the level of gene expression and/or the activity of such gene products would bring about the amelioration of such disease symptoms. The PRO polypeptides described herein and polypeptidyl agonists and antagonists may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as gene therapy.
There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells: in vivo and ex vivo. For in vivo delivery tlie nucleic acid is injected directly into the patient, usually at tlie sites where the PRO polypeptide is required, i.e., the site of synthesis of tlie PRO polypeptide, if known, and the site (e.g., wound) where biological activity of the PRO polypeptide is needed. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated withinporous membranes that are implanted into the patient (see, e.g., U.S. Pat. Nos.4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or transferred in vivo in the cells ofthe intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. Transduction involves the association of a replication-defective, recombinant viral (preferably retroviral) particle with a cellular receptor, followed by introduction ofthe nucleic acids contained by the particle into the cell. A commonly used vector for ex vivo delivery ofthe gene is a retrovirus.
The currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non- viral vectors (such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) and lipid-based systems (useful lipids for lipid-mediated transfer ofthe gene are, for example, DOTMA, DOPE, and DC-Choi; see, e.g., Tonkinson et al, Cancer Investigation. 14(1): 54-65 (1996)). The most preferred vectors for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral vector such as a retroviral vector includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. In addition, a viral vector such as a retroviral vector includes a nucleic acid molecule that, when transcribed in the presence of a gene encoding the PRO polypeptide, is operably linked thereto and acts as a translation initiation sequence. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to tlie virus used (if these are not already present in the viral vector). In addition, such vector typically includes a signal sequence for secretion of tlie PRO polypeptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence, most preferably the native signal sequence for the PRO polypeptide. Optionally, the vector construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such vectors will typically include a 5' LTR, a fRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
In some situations, it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on tlie target cell, etc. Where liposomes are employed, proteins that bind to a cell-surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al. , J. Biol. Chem..262: 4429-4432 (1987); and Wagner et al , Proc. Natl. Acad.
Sci. USA. 87: 3410-3414 (1990). For a review ofthe currently known gene marking and gene therapy protocols, see, Anderson et al, Science, 256: 808-813 (1992). See also WO 93/25673 and the references cited therein.
Suitable gene therapy and methods for making retroviral particles and structural proteins can be found in, e.g., U.S. Pat. No. 5,681,746.
5.2.4.6. Use of Gene as a Diagnostic
This invention is also related to the use of the gene encoding the PRO polypeptide as a diagnostic. Detection of a mutated fonn ofthe PRO polypeptide will allow a diagnosis of a cardiovascular, endothelial, and angiogenic disease or a susceptibility to a cardiovascular, endothelial, and angiogenic disease, such as a tumor, since mutations in the PRO polypeptide may cause tumors. Individuals carrying mutations in the genes encoding a human PRO polypeptide may be detected at the
' DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy, and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki etal, Nature, 324: 163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding the PRO polypeptide can be used to identify and analyze the PRO polypeptide mutations. For example, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA encoding the PRO polypeptide, or alternatively, radiolabeled antisense DNA sequences encoding the PRO polypeptide. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamidine gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures. See, e.g., Myers et al, Science. 230: 1242 (1985).
Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method, for example, Cotton et al, Proc. Natl. Acad. Sci. USA. 85: 4397-4401 (1985).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or tlie use of restriction enzymes, e.g., restriction fragment length polymorphisms (RFLP), and Southern blotting of genomic DNA.
5.2.4.7. Use to Detect PRO Polypeptide Levels In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis. Expression of nucleic acid encoding the PRO polypeptide may be linked to vascular disease or neovascularization associated with tumor formation. If the PRO polypeptide has a signal sequence and the mRNA is highly expressed in endothelial cells and to a lesser extent in smooth muscle cells, this indicates that the PRO polypeptide is present in serum. Accordingly, an anti-PRO polypeptide antibody could be used to diagnose vascular disease or neovascularization associated with tumor formation, since an altered level of this PRO polypeptide may be indicative of such disorders.
A competition assay may be employed wherein antibodies specific to the PRO polypeptide are attached to a solid support and the labeled PRO polypeptide and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity ofthe PRO polypeptide in the sample.
5.2.4.8. Chromosome Mapping
The sequences of tlie present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis for the 3 '- untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to tlie primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sub localization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome- specific cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use ofthe clones from which tlie gene encoding the PRO polypeptide was derived, and the longer the better. For example, 2,000 bp is good, 4,000 bp is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage ofthe time. For a review of this technique, see, Verma et al, Human Chromosomes: a Manual of Basic Techniques (Pergamon Press, New York, 1988).
Once a sequence has been mapped to aprecise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick,
Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region is then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all ofthe affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent ofthe disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes.
(This assumes 1 megabase mapping resolution and one gene per 20 kb).
5.2.4.9. Screening Assays for Drug Candidates
This invention encompasses methods of screening compounds to identify those that mimic the PRO polypeptide (agonists) or prevent the effect ofthe PRO polypeptide (antagonists). Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO polypeptide encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art. All assays for antagonists are common in that they call for contacting the drug candidate with a PRO polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.
In binding assays, the interaction is binding and the complex formed can be isolated or detected in tlie reaction mixture. In a particular embodiment, the PRO polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g. , on a microtiter plate, by covalent or non-covalent attachments.
Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the PRO polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the PRO polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When tlie originally non- immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex. If the candidate compound interacts with but does not bind to a particular PRO polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co- immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein- protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London).340: 245-246 (1989); Chien et al. Proc. Natl. Acad. Sci. USA, 88: 9578-9582
(1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA. 89: 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech.
This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions. Compounds that interfere with the interaction of a gene encoding a PRO polypeptide identified herein and other infra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product ofthe gene and the infra- or extracellular component under conditions and for a time allowing for the interaction and binding ofthe two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in tlie presence of tlie test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the infra- or extracellular component present in tlie mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction ofthe test compound and its reaction partner. If the PRO polypeptide has the ability to stimulate the proliferation of endothelial cells in the presence of the co-mitogen ConA, then one example of a screening method takes advantage of this ability. Specifically, in tlie proliferation assay, human umbilical vein endothelial cells are obtained and cultured hi 96-well flat-bottomed culture plates (Costar, Cambridge, MA) and supplemented with a reaction mixture appropriate for facilitating proliferation ofthe cells, the mixture containing Con-A (Calbiochem, La Jolla, CA). Con-A and the compound to be screened are added and after incubation at 37°C, cultures are pulsed with 3Η-thymidine and harvested onto glass fiber filters (phD; Cambridge Technology, Watertown, MA). Mean3Η- thymidine incorporation (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA). Significant 3"03)- thymidine incorporation indicates stimulation of endothelial cell proliferation.
To assay for antagonists, the assay described above is performed; however, in this assay the PRO polypeptide is added along with the compound to be screened and the ability ofthe compound to inhibit
3"(H)tlιymidine incorporation in the presence ofthe PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide. Alternatively, antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of tlie potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those ofskillintheart, for example, ligand panning and FACS sorting. Coligan et al.. Current Protocols in Irnmun.. 1 (2) : Chapter 5 (1991). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the PRO polypeptide. Transfected cells that are grown on glass slides are exposed to the labeled PRO polypeptide. The PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, tlie slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled PRO polypeptide can be photoaffinity- linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The am o acid sequence obtained from micro-sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor. In another assay for antagonists, mammalian cells or a membrane preparation expressing tlie receptor would be incubated with the labeled PRO polypeptide in the presence ofthe candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
The compositions useful in the treatment of cardiovascular, endothelial, and angiogenic disorders include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple-helix molecules, etc., that inhibit the expression and/or activity of the target gene product.
More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with a PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the PRO polypeptide that recognizes the receptor but imparts no effect, thereby competitively hiliibiting the action ofthe PRO polypeptide.
Another potential PRO polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein franslation. 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 binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion ofthe polynucleotide sequence, which encodes the mature PRO polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region ofthe gene involved in transcription (triple helix - see, Lee et al, Nucl. Acids Res.. 6:3073 (1979); Cooney et al, Science. 241: 456 (1988);' Dervan et al, Science, 251:1360 (1991)), thereby preventing transcription and the production of the PRO polypeptide. A sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand ofthe duplex DNA may thus be tested, or triplex helix formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO polypeptide (antisense - Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988). The antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide canbe modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g. , for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989, Proc. Natl. Acad. Sci.
U.S.A. 86:6553-6556; Lemaitre, et al, 1987, Proc. Natl. Acad. Sci. U.S.A. 84:648-652; PCT Publication No. WO88/09810, published December 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published April 25, 1988), hybridization-triggered cleavage agents (see, e.g., Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g. , a peptide, hybridization triggered cross-linking agent, transport agent, hybridization- triggered cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymetlιylaminometlιyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-mexhyladenine, 2-methylguanine, 3-metlιylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-memoxyaininomethyl-2-thioxiracil, beta-D-mannosylqueosfne, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-mexhylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxos ie, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-
2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphofriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An α- anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gavάieτ, etal, 1987 ', Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2'-0-methylribonucleotide (Inoue, et al, 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue, et al, 1987, FEBSLett. 215:327-330).
Oligonucleotides ofthe invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988, Nucl.
Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin, et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc. The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions ofthe target gene nucleotide sequence, are preferred. Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases hi length, or more.
Potential antagonists further include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site ofthe PRO polypeptide, thereby blocking the normal biological activity ofthe PRO polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds. Additionalpotential antagonists are ribozymes, which are enzymatic RNAmolecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to tlie complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biology, 4: 469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions which form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known hi the art and is described more fully in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, (see especially Figure 4, page
833) and in Haseloff and Gerlach, 1988, Nature, 334:585-591, which is incorporated herein by reference in its entirety.
Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5' end ofthe target gene mRNA, i. e. , to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al, 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al, 1986, Nature, 324:429-433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-
216). The Cech-type ribozymes have an eight base pair active site that hybridizes to a target RNA sequence whereafter cleavage ofthe target RNA takes place. The invention encompasses those Cech-type ribozymes that target eight base-pair active site sequences that are present in the target gene.
As hi the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo. A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities ofthe ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of pur nes or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551, supra.
These small molecules can be identified by any one or more ofthe screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.
5.2.4.10. Types of Cardiovascular. Endothelial, and Angiogenic Disorders to be Treated
The PRO polypeptides, or agonists or antagonists thereto, that have activity in the cardiovascular, angiogenic, and endothelial assays described herein, and or whose gene product has been found to be localized to the cardiovascular system, are likely to have therapeutic uses in a variety of cardiovascular, endothelial, and angiogenic disorders, including systemic disorders that affect vessels, such as diabetes mellitus. Their therapeutic utility could include diseases ofthe arteries, capillaries, veins, and or lymphatics. Examples of treatments hereunder include treating muscle wasting disease, treating osteoporosis, aiding in implant fixation to stimulate the growth of cells around the implant and therefore facilitate its attachment to its intended site, increasing IGF stability in tissues or in serum, if applicable, and increasing binding to the IGF receptor (since IGF has been shown in vitro to enhance human marrow erythroid and granulocytic progenitor cell growth). The PRO polypeptides or agonists or antagonists thereto may also be employed to stimulate erythropoiesis or granulopoiesis, to stimulate wound healing or tissue regeneration and associated therapies concerned with re- growth of tissue, such as connective tissue, skin, bone, cartilage, muscle, lung, or kidney, to promote angiogenesis, to stimulate or inhibit migration of endothelial cells, and to proliferate the growth of vascular smooth muscle and endothelial cell production. The increase in angiogenesis mediated by the PRO polypeptide or agonist would be beneficial to ischemic tissues and to collateral coronary development in the heart subsequent to coronary stenosis.
Antagonists are used to inhibit the action of such polypeptides, for example, to limit the production of excess connective tissue during wound healing or pulmonary fibrosis if the PRO polypeptide promotes such production. This would include treatment of acute myocardial infarction and heart failure.
Moreover, the present invention provides the treatment of cardiac hypertrophy, regardless ofthe xmderlying cause, by administering a therapeutically effective dose ofthe PRO polypeptide, or agonist or antagonist thereto.
If the objective is the treatment of human patients, the PRO polypeptide preferably is recombinant human PRO polypeptide (rhPRO polypeptide). The treatment for cardiac hypertrophy can be performed at any of its various stages, which may result from a variety of diverse pathologic conditions, including myocardial infarction, hypertension, hypertrophic cardiomyopathy, and valvular regurgitation. The treatment extends to all stages ofthe progression of cardiac hypertrophy, with of without structural damage of tlie heart muscle, regardless of the underlying cardiac disorder.
The decision of whether to use the molecule itself or an agonist thereof for any particular indication, as opposed to an antagonist to the molecule, would depend mainly on whether the molecule herein promotes cardiovascularization, genesis of endothelial cells, or angiogenesis or inhibits these conditions. For example, if the molecule promotes angiogenesis, an antagonist thereof would be useful for treatment of disorders where it is desired to limit or prevent angiogenesis. Examples of such disorders include vascular tumors such as haemangioma, tumor angiogenesis, neovascularization in the retina, choroid, or cornea, associated with diabetic retinopathy or premature infant retinopathy or macular degeneration and proliferative vitreoretinopathy, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hypersthnulation, psoriasis, endometriosis associated with neovascularization, restenosis subsequent to balloon angioplasty, scar tissue overproduction, for example, that seen in a keloid that forms after surgery, fibrosis after myocardial infarction, or fϊbrotic lesions associated with pulmonary fibrosis.
If, however, the molecule inhibits angiogenesis, it would be expected to be used directly for freatment of the above conditions.
On the other hand, if the molecule stimulates angiogenesis it would be used itself (or an agonist thereof) for indications where angiogenesis is desired such as peripheral vascular disease, hypertension, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, arterial restenosis, thrombophlebitis, lymphangitis, lymphedema, wound healing and tissue repair, ischemia reperfusion injury, angina, myocardial infarctions such as acute myocardial infarctions, chronic heart conditions, heart failure such as congestive heart failure, and osteoporosis.
If, however, the molecule inhibits angiogenesis, an antagonist thereof would be used for treatment of those conditions where angiogenesis is desired.
Specific types of diseases are described below, where the PRO polypeptide herein or agonists or antagonists thereof may serve as useful for vascular-related drug targeting or as therapeutic targets for the treatment or prevention ofthe disorders. Atherosclerosis is a disease characterized by accumulation of plaques of intimal thickening in arteries, due to accumulation of lipids, proliferation of smooth muscle cells, and formation of fibrous tissue within the arterial wall. The disease can affect large, medium, and small arteries in any organ. Changes in endothelial and vascular smooth muscle cell function are known to play an important role in modulating the accumulation and regression of these plaques.
Hypertension is characterized by raised vascular pressure in the systemic arterial, pulmonary arterial, or portal venous systems. Elevated pressure may result from or result in impaired endothelial function and/or vascular disease.
Inflammatory vasculitides include giant cell arteritis, Takayasu's arteritis, polyarteritis nodosa (including the microangiopathic form), Kawasaki's disease, microscopic polyangiitis, Wegener's granulomatosis, and a variety of infectious-related vascular disorders (including Henoch-Schonlein prupura). Altered endothelial cell function has been shown to be important in these diseases.
Reynaud's disease and Reynaud's phenomenon are characterized by intermittent abnormal impairment of tlie circulation through the extremities on exposure to cold. Altered endothelial cell function has been shown to be important in this disease.
Aneurysms are saccular or fusiform dilatations ofthe arterial or venous tree that are associated with altered endothelial cell and/or vascular smooth muscle cells.
Arterial restenosis (restenosis ofthe arterial wall) may occur following angioplasty as a result of alteration in the function and proliferation of endothelial and vascular smooth muscle cells. Thrombophlebitis and lymphangitis are inflammatory disorders of veins and lymphatics, respectively, that may result from, and/or in, altered endothelial cell function. Similarly, lymphedema is a condition involving impaired lymphatic vessels resulting from endothelial cell function.
The family of benign and malignant vascular tumors are characterized by abnormal proliferation and growth of cellular elements of the vascular system. For example, lymphangiomas are benign tumors of the lymphatic system that are congenital, often cystic, malformations ofthe lymphatics that usually occur in newborns.
Cystic tumors tend to grow into the adjacent tissue. Cystic tumors usually occur in tlie cervical and axillary region. They can also occur in the soft tissue ofthe extremities. The main symptoms are dilated, sometimes reticular, structured lymphatics and lymphocysts surrounded by connective tissue. Lymphangiomas are assumed to be caused by improperly connected embryonic lymphatics or their deficiency. The result is impaired local lymph drainage. Griener et al, Lvmphology. 4: 140-144 (1971).
Another use for the PRO polypeptides herein or agonists or antagonists thereto is in the prevention of tumor angiogenesis, which involves vascularization of a tumor to enable it to growth and/or metastasize. This process is dependent on the growth of new blood vessels. Examples of neoplasms and related conditions that involve tumor angiogenesis include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, tliecomas, arrhenoblastomas, cervical carcinomas, endometrial carcinoma, endomefrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma, abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
Age-related macular degeneration (AMD) is a leading cause of severe visual loss in the elderly population. The exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment. Because choroidal neovascularization is associated with a dramatic worsening in prognosis, the PRO polypeptide or agonist or antagonist thereto is expected to be useful in reducing the severity of AMD. Healing of trauma such as wound healing and tissue repair is also a targeted use for the PRO polypeptides herein or their agonists or antagonists. Formation and regression of new blood vessels is essential for tissue healing and repair. This category includes bone, cartilage, tendon, ligament, and/or nerve tissue growth or regeneration, as well as wound healing and tissue repair and replacement, and in the freatment of burns, incisions, and ulcers. A PRO polypeptide or agonist or antagonist thereof that induces cartilage and/or bone growth in circumstances where bone is not normally formed has application in tlie healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a PRO polypeptide or agonist or antagonist thereof may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma- induced, or oncologic, resection-induced craniofacial defects, and also is useful in cosmetic plastic surgery.
PRO polypeptides or agonists or antagonists thereto may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a PRO polypeptide or agonist or antagonist thereto may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, or endothelium), muscle (smooth, skeletal, or cardiac), and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part ofthe desired effects may be by inhibition or modulation of fϊbrotic scarring to allow normal tissue to regenerate.
A PRO polypeptide herein or agonist or antagonist thereto may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage. Also, the PRO polypeptide or agonist or antagonist thereto may be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells, or for inhibiting the growth of tissues described above.
A PRO polypeptide or agonist or antagonist thereto may also be used in the treatment of periodontal diseases and in other tooth-repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells, or induce differentiation of progenitors of bone-forming cells. A PRO polypeptide herein or an agonist or an antagonist thereto may also be useful in the treatment of osteoporosis or osteoarfhritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes, since blood vessels play an important role in the regulation of bone turnover and growth.
Another category of tissue regeneration activity that may be attributable to tlie PRO polypeptide herein or agonist or antagonist thereto is tendon/ligament formation. A protein that induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed has application in the healing of tendon or ligament tears, deformities, and other tendon or ligament defects in humans and other animals. Such a preparation may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition ofthe PRO polypeptide herein or agonist or antagonist thereto contributes to the repair of congenital, trauma-induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions herein may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament- forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions herein may also be useful in the freatment of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
The PRO polypeptide or its agonist or antagonist may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i. e. , for the treatment of central and peripheral nervous system disease and neuropathies, as well as mechanical and traumatic disorders, that involve degeneration, death, or trauma to neural cells or nerve tissue. More specifically, a PRO polypeptide or its agonist or antagonist may be used in the treatment of diseases ofthe peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma, and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a PRO polypeptide herein or agonist or antagonist thereto.
Ischemia-reperfusion injury is another indication. Endothelial cell dysfunction may be important in both the initiation of, and in regulation ofthe sequelae of events that occur following ischemia-reperfusion injury.
Rheumatoid arthritis is a further indication. Blood vessel growth and targeting of inflammatory cells through the vasculature is an important component in the pathogenesis of rheumatoid and sero-negative forms of arthritis.
A PRO polypeptide or its agonist or antagonist may also be administered prophylactically to patients with cardiac hypertrophy, to prevent the progression of the condition, and avoid sudden death, including death of asymptomatic patients. Such preventative therapy is particularly warranted in the case of patients diagnosed with massive left ventricular cardiac hypertrophy (a maximal wall thickness of 35 mm or more in adults, or a comparable value in children), or in instances when the hemodynamic burden on the heart is particularly strong.
A PRO polypeptide or its agonist or antagonist may also be useful in the management of afrial fibrillation, which develops in a substantial portion of patients diagnosed with hypertrophic cardiomyopathy.
Further indications include angina, myocardial infarctions such as acute myocardial infarctions, and heart failure such as congestive heart failure. Additional non-neoplastic conditions include psoriasis, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion. In view ofthe above, the PRO polypeptides or agonists or antagonists thereof described herein, which are shown to alter or impact endothelial cell function, proliferation, and/or form, are likely to play an important role in the etiology and pathogenesis of many or all of tlie disorders noted above, and as such can serve as therapeutic targets to augment or inhibit these processes or for vascular-related drug targeting in these disorders.
5.2.4.11. Administration Protocols, Schedules. Doses, and Formulations
The molecules herein and agonists and antagonists thereto are pharmaceutically useful as a prophylactic and therapeutic agent for various disorders and diseases as set forth above.
Therapeutic compositions ofthe PRO polypeptides or agonists or antagonists are prepared for storage by mixing the desired molecule having tlie appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences. 16th edition, Osol, A. ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or hnmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non- ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol Q°EG).
Additional examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodiurn hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium frisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol. Carriers for topical or gel-based forms of agonist or antagonist include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols. For all administrations, conventional depot forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and sustained-release preparations. The PRO polypeptides or agonists or antagonists will typically be formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml.
Another formulation comprises incorporating a PRO polypeptide or agonist or antagonist thereof into formed articles. Such articles can be used in modulating endothelial cell growth and angiogenesis. In addition, tumor invasion and metastasis may be modulated with these articles. PRO polypeptides or agonists or antagonists to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. PRO polypeptides ordinarily will be stored in lyophilized form or in solution if admmistered systemically. If in lyophilized form, the PRO polypeptide or agonist or antagonist thereto is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at tlie time for use. An example of a liquid formulation of a PRO polypeptide or agonist or antagonist is a sterile, clear, colorless unpreserved solution filled in a single-dose vial for subcutaneous injection. Preserved pharmaceutical compositions suitable for repeated use may contain, for example, depending mainly on the indication and type of polypeptide: a) PRO polypeptide or agonist or antagonist thereto; b) a buffer capable of maintaining the pH in a range of maximum stability of the polypeptide or other molecule in solution, preferably about 4-8; c) a detergent/surfactant primarily to stabilize thepolypeptide or molecule against agitation-induced aggregation; d) an isotonifier; e) a preservative selected from the group of phenol, benzyl alcohol and a benzethonium halide, e.g. , chloride; and f) water.
If the detergent employed is non-ionic, it may, for example, be polysorbates (e.g., POLYSORBATE™
(TWEEN™) 20, 80, etc.) or poloxa ers (e.g. , POLOXAMER™ 188). The use of non-ionic surfactants permits the formulation to be exposed to shear surface stresses without causing denaturation ofthe polypeptide. Further, such surfactant-containing formulations may be employed in aerosol devices such as those used in a pulmonary dosing, and needleless jet injector guns (see, e.g., EP 257,956).
An isotonifier may be present to ensure isotonicity of a liquid composition ofthe PRO polypeptide or agonist or antagonist thereto, and includes polyhydric sugar alcohols, preferably frihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. These sugar alcohols can be used alone or in combination. Alternatively, sodium chloride or other appropriate inorganic salts may be used to render the solutions isotonic.
The buffer may, for example, be an acetate, citrate, succinate, or phosphate buffer depending on the pH desired. The pH of one type of liquid formulation of this invention is buffered in the range of about 4 to 8, preferably about physiological pH.
The preservatives phenol, benzyl alcohol and benzethonium halides, e.g. , chloride, are known antimicrobial agents that may be employed.
Therapeutic PRO polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The formulations are preferably administered as repeated intravenous (i.v.), subcutaneous (s.c), or intramuscular
(i.m.) injections, or as aerosol formulations suitable for intranasal or intrapulmonary delivery (for infrapulmonary delivery see, e.g., EP 257,956). PRO polypeptides can also be administered in the form of sustained-released preparations. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in tlie form of shaped articles, e.g. , films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g. , poly(2-hydroxyethyl-methacrylate) as described by Langer et al, J. Biomed. Mater. Res.. 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma etliyl-L-glutamate (Sidman etal. , Biopolymers, 22: 547-556 (1983)), non-degradable ethylene- vinyl acetate (Langer et al, supra), degradable lactic acid-glycolic acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-)-3-hydroxybutyric acid (EP 133,988).
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 encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for protein stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
Sustained-release PRO polypeptide compositions also include liposomally entrapped PRO polypeptides. Liposomes containing the PRO polypeptide are prepared by methods no-fmper se: DE 3,218,121; Eρstein e/α/.,
Proc. Natl. Acad. Sci. USA. 82: 3688-3692 (1985); Hwang et al, Pro 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 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are ofthe small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal therapy.
The therapeutically effective dose of a PRO polypeptide or agonist or antagonist thereto will, of course, vary depending on such factors as the pathological condition to be treated (including prevention), the method of administration, the type of compound being used for treatment, any co-therapy involved, the patient's age, weight, general medical condition, medical history, etc., and its determination is well within the skill of a practicing physician. Accordhigly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the maximal therapeutic effect. If the PRO polypeptide has a narrow host range, for the treatment of human patients formulations comprising human PRO polypeptide, more preferably native- sequence human PRO polypeptide, are preferred. The clinician will administer the PRO polypeptide until a dosage is reached that achieves the desired effect for treatment ofthe condition in question. For example, if the objective is the treatment of CHF, the amount would be one that inhibits the progressive cardiac hypertrophy associated with this condition. The progress of this therapy is easily monitored by echo cardiography. Similarly, in patients with hypertrophic cardiomyopathy, the PRO polypeptide can be administered on an empirical basis. With the above guidelines, the effective dose generally is within the range of from about 0.001 to about 1.0 mg/kg, more preferably about 0.01-1.0 mg/kg, most preferably about 0.01-0.1 mg/kg.
For non-oral use in treating human adult hypertension, it is advantageous to administer the PRO polypeptide in the form of an injection at about 0.01 to 50 mg, preferably about 0.05 to 20 mg, most preferably 1 to 20 mg, per kg body weight, 1 to 3 times daily by intravenous injection. For oral administration, a molecule based on the PRO polypeptide is preferably administered at about 5 mg to 1 g, preferably about 10 to 100 mg, per kg body weight, 1 to 3 times daily. It should be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng/mg protein. Moreover, for human admimstration, the formulations preferably meet sterility, pyrogenicity, general safety, and purity as required by FDA Office and Biologies standards. The dosage regimen of a pharmaceutical composition containing the PRO polypeptide to be used in tissue regeneration will be determined by the attending physician considering various factors that modify the action of the polypeptides, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g. , bone), the patient's age, sex, and diet, die severity of any infection, time of administration, and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF-I, to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations, and tetracycline labeling.
The route of PRO polypeptide or antagonist or agonist administration is in accord with known methods, e.g., by injection or infusion by intravenous, intramuscular, infracerebral, intraperitoneal, intracerobrospinal, subcutaneous, intraocular, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes, or by sustained-release systems as noted below. The PRO polypeptide or agonist or antagonists thereof also are suitably administered by infratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. The intraperitoneal route is expected to be particularly useful, for example, in the freatment of ovarian tumors.
If a peptide or small molecule is employed as an antagonist or agonist, it is preferably administered orally or non-orally in the form of a liquid or solid to mammals.
Examples of pharmacologically acceptable salts of molecules that form salts and are useful hereunder include alkali metal salts (e.g. , sodium salt, potassium salt), alkaline earth metal salts (e.g. , calcium salt, magnesium salt), ammonium salts, organic base salts (e.g., pyridine salt, triethylamine salt), inorganic acid salts (e.g., hydrochloride, sulfate, nitrate), and salts of organic acid (e.g., acetate, oxalate, p-toluenesulfonate).
For compositions herein that are useful for bone, cartilage, tendon, or ligament regeneration, the tlierapeutic method includes administering the composition topically, systemically, or locally as an implant or device. When administered, the therapeutic composition for use is in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage, or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Preferably, for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing composition to tlie site of bone and/or cartilage damage, providing a structure for tlie developing bone and cartilage and preferably capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance, and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid, and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.
Matrices may be comprised of combinations of any ofthe above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. One specific embodiment is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the polypeptide compositions from disassociating from the matrix.
One suitable family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydoxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, one preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt%, based on total formulation weight, which represents the amount necessary to prevent desorption ofthe polypeptide (or its antagonist) from the polymer matrix and to provide appropriate handling ofthe composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide (or its antagonist) the opportunity to assist the osteogenic activity ofthe progenitor cells.
5.2.4.12. Combination Therapies The effectiveness ofthe PRO polypeptide or an agonist or antagonist thereof in preventing or treating the disorder in question may be improved by administering the active agent serially or in combination with another agent that is effective for those purposes, either in the same composition or as separate compositions.
For example, for treatment of cardiac hypertrophy, PRO polypeptide therapy can be combined with the administration of inhibitors of known cardiac myocyte hypertrophy factors, e.g. , inhibitors of α-adrenergic agonists such as phenylephrine; endothelin-1 inhibitors such as BOSENTAN™ and MOXONODTN™; inhibitors to CT-1 (U.S. Pat. No. 5,679,545); inhibitors to LIF; ACE inhibitors; des-aspartate-angiotensin I inhibitors (U.S. Pat. No. 5,773,415), and angiotensin II inhibitors.
For treatment of cardiac hypertrophy associated with hypertension, the PRO polypeptide can be administered in combination with β-adrenergic receptor blocking agents, e.g., propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol; ACE inhibitors, e.g., quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, or lisinopril; diuretics, e.g., chlorotliiazide, hydrochlorothiazide, hydroflumethazide, metliylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, or indapamide; and/or calcium channel blockers, e.g. , diltiazem, nifedipine, verapamil, or nicardipine. Pharmaceutical compositions comprising the therapeutic agents identified herein by their generic names are commercially available, and are to be administered following the manufacturers' instructions for dosage, administration, adverse effects, contraindications, etc. See, e.g. , Physicians' Desk Reference (Medical Economics Data Production Co. : Montvale, N.J., 1997), 51th Edition.
Preferred candidates for combination therapy in the treatment of hypertrophic cardiomyopathy are β- adrenergic-blocking drugs (e.g., propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol), verapamil, difedipine, or diltiazem. Treatment of hypertrophy associated with high blood pressure may require the use of antihypertensive drug therapy, using calcium channel blockers, e.g., diltiazem, nifedipine, verapamil, or nicardipine; β-adrenergic blocking agents; diuretics, e.g., chlorotliiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, or indapamide; and/or ACE-inhibitors, e.g., quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, or lisinopril.
For other indications, PRO polypeptides or their agonists or antagonists may be combined with other agents beneficial to the treatment ofthe bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as EGF, PDGF, TGF-α or TGF-β, IGF, FGF, and CTGF.
In addition, PRO polypeptides or their agonists or antagonists used to treat cancer may be combined with cytotoxic, chemotherapeutic, or growth-inhibitory agents as identified above. Also, for cancer treatment, the PRO polypeptide or agonist or antagonist thereof is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances.
The effective amounts of tlie therapeutic agents administered in combination with the PRO polypeptide or agonist or antagonist thereof will be at the physician's or veterinarian's discretion. Dosage admhiisfration and adjustment is done to achieve maximal management of the conditions to be treated. For example, for treating hypertension, these amounts ideally take into account use of diuretics or digitalis, and conditions such as hyper- or hypotension, renal impairment, etc. The dose will additionally depend on such factors as the type ofthe therapeutic agent to be used and the specific patient being treated. Typically, the amount employed will be the same dose as that used, if the given therapeutic agent is administered without tlie PRO polypeptide. 5.2.4.13, Articles of Manufacture
An article of manufacture such as a kit containing the PRO polypeptide or agonists or antagonists thereof useful for the diagnosis or treatment ofthe disorders described above comprises at least a container and a label.
Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for diagnosing or freating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is the PRO polypeptide or an agonist or antagonist thereto. The label on, or associated with, the container indicates that the composition is used for diagnosing or freating the condition of choice. The article of manufacture may further comprise a second contamer comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. The article of manufacture may also comprise a second or third container with another active agent as described above.
5.2.5. Antibodies
Some ofthe mostpromising drug candidates according to the present invention are antibodies and antibody fragments that may inhibit tlie production or the gene product of the genes identified herein and/or reduce the activity of tlie gene products.
5.2.5.1. Polyclonal Antibodies Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in amammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the PRO polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A or synthetic frehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
5.2.5.2. Monoclonal Antibodies
The anti-PRO antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma metiiods, such as those described by Kohler and Milstein, Nature. 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the PRO polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice (New York: Academic Press. 1986),pp.59-103. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), tlie culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. Kozbor, J. Immunol.. 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Inc.: New York, 1987) pp. 51-63.
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the PRO polypeptide. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) . Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the
Scatchard analysis of Munson and Pollard, Anal. Biochem..107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard metiiods. Goding, supra. Suitable cultare media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures suchas, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. PatentNo. 4,816,567. DNA encoding the monoclonal antibodies ofthe invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce irnmunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Patent No.
4,816,567; Morrison et al, supra) or by covalently joining to tlie immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy- chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art.
5.2.5.3. Human and Humanized Antibodies The anti-PRO antibodies may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g. , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2> or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulms (recipient antibody) in which residues from a CDR ofthe recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues ofthe human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody preferably also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al. , Nature, 321: 522-
525 (1986); Riechmann et al, Nature. 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol.. 2:593-596 (1992).
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. ,
Nature.321: 522-525 (1986); Riechmann etal. Nature, 332: 323-327 (1988); Verhoeven etal. Science, 239: 1534- 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. PatentNo.4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol.. 227: 381 (1991); Marks et al, J. Mol. Biol.. 222: 581 (1991). The techniques of Cole et al. and Boerner et al are also available for the preparation of human monoclonal antibodies. Cole et al. , Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, ρ. 77 (1985) and Boerner etal, J. Immunol., 147(1): 86-95 (1991). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g. , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661 ,016, and in the following scientific publications: Marks et al, Bio/Technology. 10: 779-783 (1992); Lonberg etal, Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild era/., Nature Biotechnology. 14: 845-851 (1996); Neuberger, Nature Biotechnology. 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol.. 13: 65-93 (1995).
5.2.5.4. Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one ofthe binding specificities is for the PRO polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities. Milstein and Cuello, Nature. 305: 537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al.. EMBO J.. 10: 3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant-domain sequences. The fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have tlie first heavy-chain constant region (CHI) containing the site necessary for light-chain bindhig present in at least one of the fusions. DNAs encoding the hnmunoglobulin heavy-chain fusions and, if desired, tlie immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies, see, for example, Suresh et al, Methods in Enzymology. 121: 210 (1986).
5.2.5.5. Heteroconjugate Antibodies
Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune-system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection. WO 91/00360; WO 92/200373; EP 03089. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminofhiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
5.2.5.6. Effector Function Engineering
It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al, J. Exp. Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enlianced anti- tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al, Cancer Research. 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See, Stevenson et al, Anti-Cancer Drug Design.
3: 219-230 (1989).
5.2.5.7. Immunoconiugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianfhin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tticotiiecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, I311, 131In, 90Y, and 18δRe.
Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succh imidyl-3-(2-pyridylditlιiol) propionate (SPDP), iminofhiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disucciriimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science. 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- ethyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, WO94/11026.
In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand"
(e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
5.2.5.8. Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al, Proc Natl. Acad. Sci. USA. TP. 4030 (1980); and U.S. Pat. Nos. 4,485,045 and
4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments ofthe antibody ofthe present invention can be conjugated to the liposomes as described in Martin et al,
J. Biol. Chem.. 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorabicin) is optionally contained within the liposome. See, Gabizon et al, J. National Cancer Inst. 81(19): 1484 (1989).
5.2.5.9. Pharmaceutical Compositions of Antibodies Antibodies specifically binding a PRO polypeptide identified herein, as well as other molecules identified by tlie screening assays disclosed hereinbefore, can be administered for the freatment of various disorders as noted above and below in the form of pharmaceutical compositions.
If the PRO polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain ofthe target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA. 90: 7889-7893 (1993). The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in tlie form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, ρoly(2-hydroxyefhyl-mefhacrylate), or ρoly(vinylalcohol)), polylactides (U.S. Pat. No.
3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and 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 encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
5.2.5.10. Methods of Treatment using the Antibody It is contemplated that tlie antibodies to a PRO polypeptide may be used to treat various cardiovascular, endothelial, and angiogenic conditions as noted above.
The antibodies are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, infracerobrospinal, subcutaneous, infra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration ofthe antibody is preferred. Other therapeutic regimens may be combined with the administration of the antibodies of the instant invention as noted above. For example, if the antibodies are to treat cancer, the patient to be treated with such antibodies may also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by tlie skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, Ed., M.C. Perry (Williams & Wilkins: Baltimore, MD, 1992). The chemotherapeutic agent may precede, or follow administration of the antibody, or may be given simultaneously therewith. The antibody may be combined with an anti-estrogen compound such as tamoxifen or EVISTA™ or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
If tlie antibodies are used for treating cancer, it may be desirable also to administer antibodies against other tumor-associated antigens, such as antibodies that bind to one or more ofthe ErbB2, EGFR, ErbB3, ErbB4, or
VEGF receptor(s). These also include the agents set forth above. Also, the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances. Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to the patient. Sometimes, it may be beneficial also to administer one or more cytokines to the patient. In a preferred embodiment, the antibodies herein are co- administered with a growth-inliibitory agent. For example, tlie growth-inhibitory agent may be administered first, followed by an antibody ofthe present invention. However, simultaneous administration or adminisfration ofthe antibody of tlie present invention first is also contemplated. Suitable dosages for tlie growth-inhibitory agent are those presently used and may be lowered due to the combined action (synergy) ofthe growth-inhibitory agent and the antibody herein.
In one embodiment, vascularization of tumors is attacked in combination therapy. The anti-PRO polypeptide antibody and another antibody (e.g., anti- VEGF) are administered to tumor-bearing patients at therapeutically effective doses as determined, for example, by observing necrosis ofthe tumor or its metastatic foci, if any. This therapy is continued until such time as no further beneficial effect is observed or clinical examination shows no trace ofthe tumor or any metastatic foci. Then TNF is administered, alone or in combination with an auxiliary agent such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin, anti-heregulin antibody, D-factor, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), or agents that promote microvascular coagulation in tumors, such as anti-protein C antibody, anti-protein S antibody, or C4b binding protein (see, WO 91/01753, published 21 February 1991), or heat or radiation. Since the auxiliary agents will vary in their effectiveness, it is desirable to compare their impact on the tumor by matrix screening in conventional fashion. The administration of anti-PRO polypeptide antibody and TNF is repeated until the desired clinical effect is achieved. Alternatively, the anti-PRO polypeptide antibody is administered together with TNF and, optionally, auxiliary agent(s). In instances where solid tumors are found in the limbs or in other locations susceptible to isolation from the general circulation, tlie tlierapeutic agents described herein are administered to the isolated tumor or organ. In other embodiments, a FGF or PDGF antagonist, such as an anti-FGF or an anti-PDGF neutralizing antibody, is administered to the patient in conjunction with the anti-PRO polypeptide antibody. Treatment with anti-PRO polypeptide antibodies preferably may be suspended during periods of wound healing or desirable neovascularization.
For the prevention or treatment of cardiovascular, endothelial, and angiogenic disorder, tlie appropriate dosage of an antibody herein will depend on the type of disorder to be treated, as defined above, the severity and course ofthe disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to tlie patient at one time or over a series of treatments.
For example, depending on the type and severity ofthe disorder, about 1 μg/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for adminisfration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily or weekly dosage might range from about
1 μg/kg to 100 mg kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated or sustained until a desired suppression of disorder symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging.
5.2.5.11. Articles of Manufacture with Antibodies
An article of manufacture containing a container with the antibody and a label is also provided. Such articles are described above, wherein the active agent is an anti-PRO antibody.
5.2.5.12. Diagnosis and Prognosis of Tumors using Antibodies If the indication for which the antibodies are used is cancer, while cell-surface proteins, such as growth receptors over expressed in certain tumors, are excellent targets for drug candidates or tumor (e.g., cancer) treatment, the same proteins along with PRO polypeptides find additional use in the diagnosis and prognosis of tumors. For example, antibodies directed against the PRO polypeptides may be used as tumor diagnostics or prognostics. For example, antibodies, including antibody fragments, can be used qualitatively or quantitatively to detect the expression of genes including the gene encoding the PRO polypeptide. The antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, ftuorimetry, or otlier techniques known in the art. Such binding assays are performed essentially as described above. In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of tlie marker gene product in the tissue examined. It will be apparent to those skilled in the art that a wide variety of histological methods are readily available for in situ detection. The following Examples are offered for illustrative purposes only, and are not intended to limit the scope ofthe present invention in any way.
The disclosures of all patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
6. EXAMPLES
Commercially available reagents referred to in the Examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following Examples, and throughout the specification, by ATCC accession numbers is tlie American Type Culture Collection, Manassas, VA. Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks: Sambrook et al, supra; Ausubel et al, Current
Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience. N. Y.. 1989) : Innis et al. , PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.: N.Y., 1990); Harlow et al, Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford, 1984); Freshney. Animal Cell Culture. 1987; Coligauetal, Current Protocols in Immunology. 1991.
6.1. EXAMPLE 1: Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNA Encoding Therefor The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public databases (e.g., Dayhoff, GenBank), and proprietary databases (e.g. LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST-2
(Altschul et al, Methods hi Enzymology. 266:460-480 (1996)) as a comparison ofthe ECD protein sequences to a 6 frame translation ofthe EST sequences. Those comparisons with a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, WA). Using this extracellular domain homology screen, consensus DNA sequences were assembled relative to the other identified EST sequences using phrap. In addition, the consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST or BLAST-2 and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.
Based upon the consensus sequences obtained as described above, oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone ofthe full-length coding sequence for a PRO polypeptide. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al, Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one ofthe primer pairs.
The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al, Science. 253:1278-1280 (1991)) in the unique Xhol and Notl sites.
6.2. EXAMPLE 2: Isolation of cDNA Clones by Amylase Screening 6.2.1. Preparation of oligo dT primed cDNA library mRNA was isolated from a human tissue of interest using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2). This RNA was used to generate an oligo dT primed cDNA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System). In this procedure, the double sfranded cDNA was sized to greater than 1000 bp and the Sall/Notl linkered cDNA was cloned into Xhol/Notl cleaved vector. pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restriction enzyme site preceding the Xhol/Notl cDNA cloning sites.
6.2.2. Preparation of random primed cDNA library
A secondary cDNA library was generated in order to preferentially represent the 5' ends of tlie primary cDNA clones. Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY.O using reagents and protocols from Life
Technologies (Super Script Plasmid System, referenced above). In this procedure the double stranded cDNA was sized to 500-1000 bp, linkered with blunt to Notl adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector. pSST-AMY.O is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites. Thus, cDNAs cloned into this vector that are fused in frame with amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies.
6.2.3. Transformation and Detection
DNA from the library described in paragraph 2 above was chilled on ice to which was added elecfrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was then electroporated as recommended by the manufacturer. Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37°C for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37°C). Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet using standard protocols, e.g. , CsCl-gradient. The purified DNA was then carried on to the yeast protocols below. The yeast methods were divided into three categories: (1) Transformation of yeast with theplasmid/cDNA combined vector; (2) Detection and isolation of yeast clones secreting amylase; and (3) PCR amplification ofthe insert directly from the yeast colony and purification ofthe DNA for sequencing and further analysis.
The yeast strain used was HD56-5A (ATCC-90785). This strain has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-l 12, his3-l 1, his3-15, MAL+, SUC+, GAL+. Preferably, yeast mutants can be employed that have deficient post-translational pathways. Such mutants may have franslocation deficient alleles in seel 1 , secl2, sec62, with truncated seclλ being most preferred. Alternatively, antagonists (including antisense nucleotides and/or ligands) which interfere with the normal operation of these genes, otlier proteins implicated in this post translation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJlp or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast.
Transformation was performed based on the protocol outlined by Gietz et al, Nucl. Acid. Res.. 20:1425 (1992). Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and grown overnight at 30°C. The YEPD broth was prepared as described in Kaiser et al, Methods in Yeast Genetics. Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). The overnight cultare was then diluted to about 2 x IO6 cells/ml (approx. OD600=0.1) into fresh YEPD broth (500 ml) and regrown to 1 x IO7 cells/ml (approx.
OD600=0.4-0.5).
The cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval
GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tabes at 3,500 rpm in a Beckman GS-6KR centrifuge. The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5,
100 mM Li2OOCCH3), and resuspended into LiAc/TE (2.5 ml).
Transformation took place by mixing the prepared cells (100 μl) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 μg, vol. < 10 μl) in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li2OOCCH3, pH 7.5) was added. This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes. The cells were then heat shocked at 42°C for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTApH 7.5) followed by recentrifugation. The cells were then diluted into TE (1 ml) and aliquots (200 μl) were spread onto the selective media previously prepared in 150 mm growth plates (VWR).
Alternatively, instead of multiple small reactions, the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly.
The selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al, Methods in Yeast Genetics. Cold Spring Harbor Press, Cold Spring Harbor, NY, p.208- 210 (1994). Transformants were grown at 30°C for 2-3 days.
The detection of colonies secreting amylase was performed by including red starch in the selective growth media. Starch was coupled to the red dye (Reactive Red- 120, Sigma) as per the procedure described by Biely et al, Anal. Biochem., 172:176-179 (1988). The coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0 (50-100 mM final concentration).
The positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies. Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar. Positive colonies were determined by their ability to break down starch resulting in a clear halo around tlie positive colony visualized directly.
6.2.4. Isolation of DNA by PCR Amplification When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile water (30 μl) in a 96 well plate. At this time, the positive colonies were either frozen and stored for subsequent analysis or immediately amplified. An aliquot of cells (5 μl) was used as a template for the PCR reaction in a 25 μl volume containing: 0.5 μl Klentaq (Clontech, Palo Alto, CA); 4.0 μl 10 mM dNTP's (Perkin Ehner-Cetas); 2.5 μl Kentaq buffer (Clontech); 0.25 μl forward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. The sequence ofthe forward oligonucleotide 1 was: 5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID NO:382)
The sequence of reverse oligonucleotide 2 was:
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID NO:383)
PCR was then performed as follows: a. Denature 92°C, 5 minutes b. 3 cycles of: Denature 92°C, 30 seconds
Anneal 59°C, 30 seconds Extend 72°C, 60 seconds c. 3 cycles of: Denature 92°C, 30 seconds
Anneal 57°C, 30 seconds Extend 72°C, 60 seconds d. 25 cycles of: .Denature 92°C, 30 seconds
Anneal 55°C, 30 seconds Extend 72°C, 60 seconds e. Hold 4°C
The underlined regions ofthe oligonucleotides annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY.O when no insert was present. Typically, the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers. Thus, the total product ofthe PCR reaction from an empty vector was 343 bp. However, signal sequence-fused cDNA resulted in considerably longer nucleotide sequences. Following the PCR, an aliquot ofthe reaction (5 μl) was examined by agarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA (TBE) buffering system as described by Sambrook et al, supra. Clones resulting in a single strong PCR product larger than 400 bp were further analyzed by DNA sequencing after purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc., Chatsworfh, CA).
6.3. EXAMPLE 3: Isolation of cDNA Clones Using Signal Algorithm Analysis
Various polypeptide-encoding nucleic acid sequences were identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc., (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e.g., GenBank) and/or private Q IFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, CA) databases. The signal sequence algorithm computes a secretion signal score based on the character ofthe DNA nucleotides surrounding the first and optionally the second methionine codon(s)
(ATG) at tlie 5'-end ofthe sequence or sequence fragment under consideration. The nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons. If tlie first ATG has the required amino acids, the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the EST sequence contains an authentic signal sequence, tlie DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals. Use of this algorithm resulted in the identification of numerous polypeptide-encoding nucleic acid sequences.
6.4. EXAMPLE 4: Isolation of cDNA clones Encoding Human PRO Polypeptides Using the techniques described in Examples 1 to 3 above, numerous full-length cDNA clones were identified as encoding PRO polypeptides as disclosed herein. These cDNAs were then deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, USA (ATCC) as shown in Table 7 below.
Table 7
Material ATCC Pep. No. Deposit Date
23330-1390 209775 4/14/1998
23339-1130 209282 9/18/1997
26846-1397 203406 10/27/1998
26847-1395 209772 4/14/1998
27865-1091 209296 9/23/1997
30868-1156 1437-PTA 3/2/2000
30871-1157 209380 10/16/1997
32286-1191 209385 10/16/1997
33089-1132 209262 9/16/1997
33092-1202 209420 10/28/1997 33100-1159 209377 10/16/1997
33223-1136 209264 9/16/1997
34392-1170 209526 12/10/1997
34431-1177 209399 10/17/1997
34433-1308 209719 3/31/1998
34434-1139 209252 9/16/1997
35600-1162 209370 10/16/1997
35673-1201 209418 10/28/1997
35880-1160 209379 10/16/1997
35918-1174 209402 10/17/1997
36350-1158 209378 10/16/1997
36638-1056 209456 11/12/1997
38268-1188 209421 10/28/1997
40370-1217 209485 11/21/1997
40628-1216 209432 11/7/1997
43316-1237 209487 11/21/1997
44196-1353 209847 5/6/1998
45409-2511 203579 1/12/1999
45419-1252 209616 2/5/1998
46777-1253 209619 2/5/1998
48336-1309 209669 3/11/1998
48606-1479 203040 7/1/1998
49435-1219 209480 11/21/1997
49631-1328 209806 4/28/1998
50919-1361 209848 5/6/1998
50920-1325 209700 3/26/1998
50921-1458 209859 5/12/1998
52758-1399 . 209773 4/14/1998
53517-1366-1 209802 4/23/1998
53915-1258 209593 1/21/1998
53974-1401 209774 4/14/1998
53987-1438 209858 5/12/1998
56047-1456 209948 6/9/1998
56050-1455 203011 6/23/1998
56110-1437 203113 8/11/1998
56405-1357 209849 5/6/1998
56433-1406 209857 5/12/1998 56439-1376 209864 5/14/1998 56529-1647 203293 9/29/1998 56865-1491 203022 6/23/1998 56965-1356 209842 5/6/1998 57033-1403-1 209905 5/27/1998 57037-1444 209903 5/27/1998 57039-1402 209777 4/14/1998 57689-1385 209869 5/14/1998 57690-1374 209950 6/9/1998 57694-1341 203017 6/23/1998 57695-1340 203006 6/23/1998 57699-1412 203020 6/23/1998 57700-1408 203583 1/12/1999 57708-1411 203021 6/23/1998 57838-1337 203014 6/23/1998 58847-1383 209879 5/20/1998 58852-1637 203271 9/22/1998 58853-1423 203016 6/23/1998 '59212-1627 203245 9/9/1998 59220-1514 209962 6/9/1998 59493-1420 203050 7/1/1998 59497-1496 209941 6/4/1998 59586-1520 203288 9/29/1998 59588-1571 203106 8/11/1998 59620-1463 209989 6/16/1998 59622-1334 209984 6/16/1998 59777-1480 203111 8/11/1998 59848-1512 203088 8/4/1998 59849-1504 209986 6/16/1998 60621-1516 203091 8/4/1998 60622-1525 203090 8/4/1998 60764-1533 203452 11/10/1998 60783-1611 203130 8/18/1998 61755-1554 203112 8/11/1998 62306-1570 203254 9/9/1998 62312-2558 203836 3/9/1999 62814-1521 203093 8/4/1998 62872-1509 203100 8/4/1998
64883-1526 203253 9/9/1998
64886-1601 203241 9/9/1998
64889-1541 203250 9/9/1998
64896-1539 203238 9/9/1998
64897-1628 203216 9/15/1998
64903-1553 203223 9/15/1998
64908-1163-1 203243 9/9/1998
64950-1590 203224 9/15/1998
65402-1540 203252 9/9/1998
65404-1551 203244 9/9/1998
65405-1547 203476 11/17/1998
65410-1569 203231 9/15/1998
65412-1523 203094 8/4/1998
66307-2661 431-PTA 7/27/1999
66526-1616 203246 9/9/1998
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73736-1657 203466 11/17/1998
73739-1645 203270 9/22/1998
73742-1662 203316 10/6/1998
76385-1692 203664 2/9/1999
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76399-1700 203472 11/17/1998
76400-2528 203573 1/12/1999
76510-2504 203477 11/17/1998
76529-1666 203315 10/6/1998
76532-1702 203473 11/17/1998
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77624-2515 203553 12/22/1998
79230-2525 203549 12/22/1998
79862-2522 203550 12/22/1998
80145-2594 204-PTA 6/8/1999
80899-2501 203539 12/15/1998
81754-2532 203542 12/15/1998
81757-2512 203543 12/15/1998
81761-2583 203862 3/23/1999
82358-2738 510-PTA 8/10/1999
82364-2538 203603 1/20/1999
82403-2959 2317-PTA 8/1/2000
83500-2506 203391 10/29/1998
83560-2569 203816 3/2/1999
84210-2576 203818 3/2/1999
84920-2614 203966 4/27/1999
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92218-2554 203834 3/9/1999
92233-2599 134-PTA 5/25/1999
92256-2596 203891 3/30/1999
92265-2669 256-PTA 6/22/1999
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92929-2534-1 203586 1/12/1999
93011-2637 20-PTA 5/4/1999
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96872-2674 550-PTA 8/17/1999
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96889-2641 119-PTA 5/25/1999
100312-2645 44-PTA 5/11/1999
105782-2693 387-PTA 7/20/1999
105849-2704 473-PTA 8/3/1999
108725-2766 863-PTA 10/19/1999
108769-2765 861-PTA 10/19/1999
119498-2965 2298-PTA 7/25/2000
119535-2756 613-PTA 8/31/1999 125185-2806 1031-PTA 12/7/1999 131639-2874 1784-PTA 4/25/2000 139623-2893 1670-PTA 4/11/2000 143076-2787 1028-PTA 12/7/1999 143276-2975 2387-PTA 8/8/2000 164625-2890 1535-PTA 3/21/2000 167678-2963 2302-PTA 7/25/2000 170021-2923 1906-PTA 5/23/2000 170212-3000 2583-PTA 10/10/2000 177313-2982 2251-PTA 7/19/2000
These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposits will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures permanent and unrestricted availability ofthe progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC § 122 and the Commissioner's rules pursuant thereto (including 37 CFR § 1.14 with particular reference to 886 OG 638).
The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, tlie materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
6 > EXAMPLE 5: Isolation of cDNA clones Encoding Human PRQ1873. PRQ7223. PRQ7248.
PRO730. PRQ532. PRQ7261. PRQ734. PRQ77-1. PRO2010. PRQ5723.
PRQ3444. PRO9940. PRQ3562. PRO10008. PRO5730. PRO6008.
PRQ4527. PRQ4538 and PRQ4553
DNA molecules encoding the PR01873, PR07223, PR07248, PRO730, PR0532, PR07261, PR0734,
PR0771, PRO2010, PR05723, PR03444, PRO9940, PR03562, PRO10008, PRO5730, PRO6008, PR04527,
PR04538 and PR04553 polypeptides shown in the accompanying figures were obtained through GenBank.
6.6. EXAMPLE 6: Use of PRO as a Hybridization Probe
The following method describes use of a nucleotide sequence encoding PRO as a hybridization probe. DNA comprising the coding sequence of full-length or mature PRO (as shown in accompanying figures) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally- occurring variants of PRO) in human tissue cDNA libraries or human tissue genomic libraries.
Hybridization and wasliing of filters containing either library DNAs is performed under the following high- stringency conditions. Hybridization of radiolabeled probe derived from the gene encoding PRO polypeptide to the filters is perfoπned in a solution of 50% foπnamide, 5x SSC, 0.1 % SDS, 0.1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20 hours. Washing ofthe filters is performed in an aqueous solution of 0. lx SSC and 0.1% SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art.
6.7. EXAMPLE 7: Expression of PRO in E. coli
This example illustrates preparation of an unglycosylated form of PRO by recombinant expression in E. coli.
The DNA sequence encoding PRO is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector.
A variety ofexpression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see, Bolivar et al, Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, apoly-His leader (including the first six STII codons, poly-His sequence, and enterokinase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU gene.
The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al, supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight cultare may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.
After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in tlie art, and the solubilized PRO protein can then be purified using a metal chelating column under conditions that allow tight binding ofthe protein.
PRO may be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding PRO is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable franslation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30°C with shaking until an OD600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)2S04, 0.71 g sodium citrate»2H20, 1.07 g KCI, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgS04) and grown for approximately 20-30 hours at 30°C with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk cultare is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02 M, respectively, and the solution is stirred overnight at 4°C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman Ulfracentifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiagen Ni 2+-NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4°C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence. The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml. The refolding solution is stirred gently at 4°C for 12-36 hours. The refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration. The refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
Fractions containing the desired folded PRO polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.
Many ofthe PRO polypeptides disclosed herein were successfully expressed as descibed above. 6.8. EXAMPLE 8: Expression of PRO in mammalian cells
This example illustrates preparation of a potentially glycosylated form of PRO by recombinant expression in mammalian cells.
The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector. Optionally, the PRO DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO
DNA using ligation methods such as described in Sambrook et al. , supra. The resulting vector is called pRK5-PRO.
In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 μg ρRK5-PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al. , Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl,
0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25°C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C. The cultare medium is asphated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and tlie cells are incubated for about 5 days.
Approximately 24 hours after the transfections, the cultare medium is removed and replaced with cultare medium (alone) or cultare medium containing 200 μCi/ml 35S-cysteine and 200 μCi/ml 35S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence ofthe PRO polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, PRO may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al, Proc. Natl. Acad. Sci..12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re- introduced into the spinner flask containing tissue cultare medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine fransferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PRO can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
In another embodiment, PRO can be expressed in CHO cells. The pRK5-PRO can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dexfran. As described above, the cell cultures can be incubated, and the medium replaced with cultare medium (alone) or medium containing a radiolabel such as 35S- methionine. After determining the presence of a PRO polypeptide, the cultare medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed PRO polypeptide can then be concentrated and purified by any selected method.
Epitope-tagged PRO may also be expressed in host CHO cells. The PRO may be subcloned out ofthe pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-His tag into a Baculovirus expression vector. The poly-His tagged PRO insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the S V40 driven vector. Labeling may be performed, as described above, to verify expression. The cultare medium containing the expressed poly-His tagged PRO can then be concentrated and purified by any selected method, such as by Ni2+-chelate affinity chromatography. PRO may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure.
Stable expression in CHO cells is performed using the following procedure. The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g., extracellular domains) ofthe respective proteins are fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or as a poly-His tagged form.
Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al. , Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' ofthe DNA of interest to allow the convenient shuttling of cDNA's. The vector used in expression in CHO cells is as described in Lucas et al, Nucl. Acids Res.. 24:9 (1774-1779 (1996), and uses tlie SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance ofthe plasmid following transfection.
Twelve micrograms ofthe desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Qiagen), Dosper® or Fugeηe® (Boehringer Mannheim). The cells are grown as described hi Lucas et al. , supra. Approximately 3 x 107 cells are frozen in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexrng. The contents are pipetted into a centrifuge tube containing 10 ml of media and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and tlie cells are resuspended in 10 ml of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 ml spinner containing 90 ml of selective media. After 1-2 days, the cells are transferred into a 250 ml spinner filled with 150 ml selective growth medium and incubated at 37°C. After another 2-3 days, 250 ml, 500 ml and 2000 ml spinners are seeded with 3 x 105 cells/ml. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Patent No. 5,122,469, issued June 16, 1992 may actually be used. A 3L production spinner is seeded at 1.2 x 106 cells/ml. On day 0, the cell number and pH is determined. On day 1 , the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33°C, and 30 ml of 500 g/L glucose and 0.6 ml of 10% antifoam (e.g., 35% polydimetliylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability drops below 70%, the cell cultare is harvested by centrifugation and filtering through a 0.22 μm filter. The filtrate is either stored at 4°C or immediately loaded onto columns for purification. For the poly-His tagged constructs, tlie proteins are purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine 0°harmacia) column and stored at -80°C.
Immunoadliesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μl of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
Many ofthe PRO polypeptides disclosed herein were successfully expressed as descibed above.
6.9. EXAMPLE 9: Expression of PRO in Yeast
The following method describes recombinant expression of PRO in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PRO from the ADH2/GAPDH promoter. DNA encoding PRO and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO. For secretion, DNA encoding PRO can be cloned into the selected plasmid, together with DNA encoding tlie ADH2/GAPDH promoter, a native PRO signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PRO.
Yeast cells, such as yeast strain ABl 10, can then be transformed with tlie expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining ofthe gels with
Coomassie Blue stain.
Recombinant PRO can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate contamhig PRO may further be purified using selected column chromatography resins. Many ofthe PRO polypeptides disclosed herein were successfully expressed as described above. 6.10. EXAMPLE 10: Expression of PRO in Baculovirus-Infected Insect Cells
The following method describes recombinant expression in Baculo virus-infected insect cells.
The sequence coding for PRO is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PRO or the desired portion ofthe coding sequence of PRO (such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular) is amplified by PCR with primers complementary to tlie 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into tlie expression vector.
Recombinant baculovirus is generated by co-transfecting tlie above plasmid and BaculoGold™ virus DNA
(Pharmingen) into Spodopterafrugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al, Baculovirus expression vectors: A Laboratory Manual. Oxford: Oxford University Press (1994).
Expressedpoly-His tagged PRO can then be purified, for example, by Ni2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al, Nature. 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCI), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A NP-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of loading buffer. The filtered cell extract is loaded onto the column at 0.5 ml per minute. The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, tlie column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH
6.0), which elutes nonspecifically bound protein. After reaching A280baseline again, the column is developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer. One ml fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His,0-tagged PRO are pooled and -dialyzed against loading buffer. Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
Following PCR ampfification, the respective coding sequences are subcloned into a baculovirus expression vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins), and tlie vector and Baculogold® baculovirus DNA (Pharmingen) are co-transfected into 105 Spodopterafrugiperda ("Sf9") cells (ATCC CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG andpb.PH.His are modifications ofthe commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences. The cells are grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells are incubated for 5 days at 28°C. The supernatant is harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells are incubated for 3 days at 28°C. The supernatant is harvested and the expression of the constructs in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni 2+-NTA beads (QIAGEN) for histidine tagged proteins or Protein- A Sepharose CL-4B beads (Pharmacia) for
IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
The first viral amplification supernatant is used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells are incubated for 3 days at 28°C. The supernatant is harvested and filtered. Batch binding and SDS-PAGE analysis is repeated, as necessary, until expression ofthe spinner cultare is confirmed.
The conditioned medium from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein construct is purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM
Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine QPharmacia) column and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows. The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity ofthe proteins is verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.
Alternatively, a modified baculovirus procedure may be used incorporating high-5 cells. In this procedure, the DNA encoding the desired sequence is amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector. Such epitope tags include poly-
His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such aspIEl-1 (Novagen). ThepIEl-1 andρIEl-2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus iel promoter in stably- transformed insect cells (1). The plasmids differ only hi the orientation ofthe multiple cloning sites and contain all promoter sequences known to be important for iel -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element. pIE 1- 1 and pIE 1 -2 include the translation initiation site and can be used to produce fusion proteins. Briefly, the desired sequence or the desired portion ofthe 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 those selected restriction enzymes and subcloned into the expression vector. For example, derivatives of pIEl- 1 can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine (pb.PH.His) tag downstream (3'-of) the desired sequence. Preferably, the vector construct is sequenced for confirmation.
High-5 cells are grown to a confluency of 50% under tlie conditions of, 27°C, no C02, NO pen/strep. For each 150 mm plate, 30 μg of pIE based vector containing the sequence is mixed with 1 ml Ex-Cell medium (Media: Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401-78P (note: this media is light sensitive)), and in a separate tube, 100 μl ofCellFectin(CellFECTF (GibcoBRL#10362-010) (vortexed to mix)) is mixed with 1 ml of Ex-Cell medium. The two solutions are combined and allowed to incubate at room temperatare for 15 minutes. 8 ml of
Ex-Cell media is added to the 2 ml of DNA CellFECTIN mix and this is layered on high-5 cells that have been washed once with Ex-Cell media. The plate is then incubated in darkness for 1 hour at room temperature. The DNA/CellFECTIN mix is then asphated, and the cells are washed once with Ex-Cell to remove excess CellFECTIN, 30 ml of fresh Ex-Cell media is added and the cells are incubated for 3 days at 28°C. The supernatant is harvested and the expression ofthe sequence in the baculovirus expression vector is determined by batch binding of 1 ml of supernatent to 25 ml of Ni 2+-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
The conditioned media from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the protein comprising the sequence is purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 48°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is then subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows. The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containmg 275 ml of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity ofthe sequence is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary.
Many ofthe PRO polypeptides disclosed herein were successfully expressed as described above. 6.11. EXAMPLE 11: Preparation of Antibodies that Bind PRO
This example illustrates preparation of monoclonal antibodies which can specifically bind the PRO polypeptide or an epitope on the PRO polypeptide without substantially binding to any other polypeptide or polypeptide epitope. Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified PRO, fusion proteins containing PRO, and cells expressing recombinant PRO on the cell surface. Selection ofthe immunogen can be made by the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from tlie mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO antibodies.
After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63 AgU.1 , available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue cultare plates containing HAT (hypoxanthine, aminopterin, and tliymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against PRO. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO is within the skill in the art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue cultare flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.
6.12. EXAMPLE 12: Purification of PRO Polypeptides Using Specific Antibodies Native or recombinant PRO polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PRO polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin. Polyclonal immunoglobulms are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
Such an immunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
A soluble PRO polypeptide-containing preparation is passed over the immvmoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PRO polypeptide binding (e.g. , a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is collected.
6.13. EXAMPLE 13: Drug Screening
This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drug screening techniques. The PRO polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PRO polypeptide and its target cell or target receptors caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the PRO polypeptide or fragment, or (ii) for the presence of a complex between the PRO polypeptide or fra ment and the cell, by methods well known in the art. In such competitive binding assays, the PRO polypeptide or fragment is typically labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell complex.
Another technique for drag screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984.
Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PRO polypeptide, the peptide test compounds are reacted with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well known in the art. Purified PRO polypeptide can also be coated directly onto plates for use in the aforementioned drag screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding PRO polypeptide specifically compete with a test compound for binding to PRO polypeptide or fragments thereof. In this manner, the antibodies can be used to detect tiie presence of any peptide which shares one or more antigenic determinants with PRO polypeptide.
6.14. EXAMPLE 14: Rational Drug Design
The goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest (/. e. , a PRO polypeptide) or of small molecules with which they interact, e.g., agonists , antagonists , or inhibitors. Any of these examples can be used to fashion drags which are more active or stable forms ofthe PRO polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo (cf., Hodgson, Bio/Technology. 9: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO polypeptide, or of an PRO polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the PRO polypeptide must be ascertained to elucidate the- structure and to determine active site(s) ofthe molecule. Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins.
In both cases, relevant structural information is used to design analogous PRO polypeptide-like molecules or to identify efficient inhibitors. Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al, J. Biochem.. 113:742-746 (1993). It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge ofthe PRO polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography. 6.15. EXAMPLE 15: Stimulation of Endothelial Cell Proliferation (Assay 8)
This assay is designed to determine whether PRO polypeptides ofthe present invention show the ability to stimulate adrenal cortical capillary endothelial cell (ACE) growth. PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of conditions or disorders where angiogenesis would be beneficial including, for example, wound healing, and tlie like (as would agonists of these PRO polypeptides). Antagonists ofthe PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic freatment of cancerous tumors.
Bovine adrenal cortical capillary endothelial (ACE) cells (from primary cultare, maximum of 12-14 passages) were plated in 96-well plates at 500 cells/well per 100 microliter. Assay media included low glucose DMEM, 10% calf serum, 2 mM glutamine, and IX penicillin/streptomycin/fungizone. Control wells included the following: (1) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus VEGF (5 ng/ml); and (4) ACE cells plus FGF (5ng/ml). The control or test sample, (in 100 microliter volumes), was then added to the wells (at dilutions of 1%, 0.1% and 0.01%, respectively). The cell cultures were incubated for 6-7 days at 37°C/5% C02. After the incubation, the media in the wells was aspirated, and the cells were washed IX with PBS. An acid phosphatase reaction mixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1% Triton X-100, 10 mM p- nifrophenyl phosphate) was then added to each well. After a 2 hour incubation at 37°C, the reaction was stopped by addition of 10 microliters IN NaOH. Optical density (OD) was measured on a microplate reader at 405 nm.
The activity of a PRO polypeptide was calculated as the fold increase in proliferation (as determined by the acid phosphatase activity, OD 405 nm) relative to (1) cell only background, and (2) relative to maximum stimulation by VEGF. VEGF (at 3-10 ng/ml) and FGF (at 1-5 ng/ml) were employed as an activity reference for maximum stimulation. Results of the assay were considered "positive" if tlie observed stimulation was ≥ 50% increase over background. VEGF (5 ng/ml) control at 1% dilution gave 1.24 fold stimulation; FGF (5 ng/ml) control at 1% dilution gave 1.46 fold stimulation.
PR021 tested positive in this assay.
6.16. EXAMPLE 16: Inhibition of Vascular Endothelial Growth Factor (VEGF) Stimulated
Proliferation of Endothelial Cell Growth (Assay 9) The ability of various PRO polypeptides to inhibit VEGF stimulated proliferation of endothelial cells was tested. Polypeptides testing positive in this assay are useful for inhibiting endothelial cell growth in mammals where such an effect would be beneficial, e.g., for inhibiting tumor growth. Specifically, bovine adrenal cortical capillary endothelial cells (ACE) (from primary culture, maximum of 12-14 passages) were plated in 96-well plates at 500 cells/well per 100 microliter. Assay media included low glucose DMEM, 10% calf serum, 2 mM glutamine, and IX penicillm/streptomycm/fungizone. Control wells included the following: (1) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGF plus 5 ng/ml LIF. The test samples, poly-his tagged PRO polypeptides (in 100 microliter volumes), were then added to the wells (at dilutions of 1 %, 0.1 % and 0.01 %, respectively). The cell cultures were incubated for 6-7 days at 37°C/5 % C02. After the incubation, the media in the wells was aspirated, and the cells were washed IX with PBS. An acid phosphatase reaction mixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1 % Triton X-100, 10 mM p-nitrophenyl phosphate) was then added to each well. After a 2 hour incubation at 37°C, the reaction was stopped by addition of 10 microliters IN NaOH. Optical density (OD) was measured on a microplate reader at 405 nm.
The activity of PRO polypeptides was calculated as the percent inhibition of VEGF (3 ng/ml) stimulated proliferation (as determined by measuring acid phosphatase activity at OD 405 nm) relative to the cells without stimulation. TGF-beta was employed as an activity reference at 1 ng/ml, since TGF-beta blocks 70-90% of VEGF-stimulated ACE cell proliferation. The results are indicative of the utility of the PRO polypeptides in cancer therapy and specifically in inhibiting tumor angiogenesis. Numerical values (relative inhibition) are determined by calculating the percent inhibition of VEGF stimulated proliferation by the PRO polypeptides relative to cells without stimulation and then dividing that percentage into the percent inhibition obtained by TGF-β at 1 ng/ml which is known to block 70-90% of VEGF stimulated cell proliferation. The results are considered positive if the PRO polypeptide exhibits 30% or greater inhibition of VEGF stimulation of endothelial cell growth (relative inhibition 30% or greater).
PR0247, PRO720 and PRO4302 tested positive in this assay.
6.17. EXAMPLE 17: Enhancement of Heart Neonatal Hypertrophy Induced by LIF + ET-1 (Assay
75)
This assay is designed to determine whether PRO polypeptides of the present invention show the ability to enhance neonatal heart hypertrophy induced by LIF and endothelin-1 (ET-1). A test compound that provides a positive response in the present assay would be useful for the therapeutic treatment of cardiac insufficiency diseases or disorders characterized or associated with an undesired level of hypertrophy of the cardiac muscle.
Cardiac myocytes from 1-day old Harlan Sprague Dawley rats (180 μl at 7.5 x lOVml, serum <0.1, freshly isolated) are introduced on day 1 to 96-well plates previously coated with DMEM/F12 + 4%FCS. Test PRO polypeptide samples or growth medium alone (negative control) are then added directly to the wells on day
2 in 20 μl volume. LIF + ET-1 are then added to the wells on day 3. The cells are stained after an additional
2 days in culture and are then scored visually the next day. A positive in the assay occurs when the PRO polypeptide treated myocytes obtain a score greater than zero. A score of zero represents non-responsive cells whereas scores of 1 or 2 represent enhancement (i.e. they are visually larger on the average or more numerous than the untreated myocytes) .
PR021 polypeptides tested positive in this assay.
6.18. EXAMPLE 18: Detection of Endothelial Cell Apoptosis (FACS) (Assay 96)
The ability of PRO polypeptides of the present invention to induce apoptosis in endothelial cells was tested in human venous umbilical vein endothelial cells (HUVEC, Cell Systems) in gelatinized T175 flasks using HUVEC cells below passage 10. PRO polypeptides testing positive in this assay are expected to be useful for therapeutically treating conditions where apoptosis of endothelial cells would be beneficial including, for example, the therapeutic treatment of tumors.
On day one, the cells were split [420,000 cells per gelatinized 6 cm dishes - (11 x IO3 cells/cm2 Falcon,
Primaria)] and grown in media containing serum (CS-C, Cell System) overnight or for 16 hours to 24 hours. On day 2, the cells were washed lx with 5 ml PBS ; 3 ml of 0 % serum medium was added with VEGF
(100 ng/ml); and 30 μl of the PRO test compound (final dilution 1%) or 0% serum medium (negative control) was added. The mixtures were incubated for 48 hours before harvesting.
The cells were then harvested for FACS analysis. The medium was aspirated and the cells washed once with PBS. 5 ml of 1 x trypsin was added to the cells in a T-175 flask, and the cells were allowed to stand until they were released from the plate (about 5-10 minutes). Trypsinization was stopped by adding 5 ml of growth media. The cells were spun at 1000 rpm for 5 minutes at 4°C. The media was aspirated and the cells were resuspended in 10 ml of 10% serum complemented medium (Cell Systems), 5 μl of Annexin-FITC (BioVison) added and chilled tubes were submitted for FACS. A positive result was determined to be enhanced apoptosis in the PRO polypeptide treated samples as compared to the negative control. PRO4302 polypeptide tested positive in this assay.
6.19. EXAMPLE 19: Induction of c-fos in HUVEC Cells (Assay 123)
This assay is designed to determine whether PRO polypeptides show the ability to induce c-fos in HUVEC cells. PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of conditions or disorders where angiogenesis would be beneficial including, for example, wound healing, and the like (as would agonists of these PRO polypeptides) . Antagonists of the PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of cancerous tumors.
Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) in growth media (50% Ham's F12 w/o GHT: low glucose, and 50% DMEM without glycine: with NaHC03, 1% glutamine, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF) were plated on 96-well microtiter plates at a cell density of 5xl03 cells/well. The day after plating (day 2), the cells were starved for 24 hours by removing the growth media and replacing with serum free media. On day 3, the cells are treated with 100 μl/well test samples and controls (positive control = growth media; negative control = Protein 32 buffer = 10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8). One plate of cells was incubated for 30 minutes at 37° C, in 5% C02. Another plate of cells was incubated for 60 minutes at 37°C, in 5% C02. The samples were removed, and RNA was harvested using the RNeasy 96 kit (Qiagen). Next, the RNA was assayed for c-fos, egr-1 and GAPDH induction using Taqman.
The measure of activity of the fold increase over the negative control (Protein 32/HEPES buffer described above) value was by obtained by calculating the fold increase of the ratio of c-fos to GAPDH in test samples as compared to the negative control. The results are considered positive if the PRO polypeptide exhibits at least a two-fold value over the negative buffer control. PR01376 polypeptide tested positive in this assay. 6.20. EXAMPLE 20: Normal Human Iliac Artery Endothelial Cell Proliferation (Assay 138) This assay is designed to determine whether PRO polypeptides of the present invention show the ability to modulate proliferation of human iliac artery endothelial cells in culture and, therefore, function as useful growth or inhibitory factors. On day 0, human iliac artery endothelial cells (from cell lines, maximum of 12-14 passages) were plated in 96-well plates at 1000 cells/well per 100 microliter and incubated overnight in complete media [epithelial cell growth media (EGM, Clonetics), plus supplements: human epithelial growth factor (hEGF), bovine brain extract (BBE), hydrocortisone, GA-1000, and fetal bovine serum (FBS, Clonetics)]. On day 1, complete media was replaced by basal media [EGM plus 1 % FBS] and addition of PRO polypeptides at 1 % , 0.1 % and 0.01 % . On day 7, an assessment of cell proliferation was performed by Alamar Blue assay followed by Crystal Violet.
Results are expressed as % of the cell growth observed with control buffer.
The following PRO polypeptides stimulated proliferation in this assay: PR0214, PR0256, PR0363, PR0365, PR0791, PR0836, PRO1025, PROl 186, PROl 192, PR01272, PRO1306, PR01325, PR01329, PR01376, PR01411, PRO1508, PR01787, PR01868, PR04324, PR04333, PRO4408, PR04499, PR09821, PR09873, PRO10008, PRO10096, PRO19670, PRO20040, PRO20044 and PR021384.
The following PRO polypeptides inhibited proliferation in this assay: PR0238, PRO1029, PR01274, PR01279, PR01419, PRO1890, PRO6006 and PR028631.
6.21. EXAMPLE 21: Pooled Human Umbilical Vein Endothelial Cell Proliferation (Assay 139) This assay is designed to determine whether PRO polypeptides of the present invention show the ability to modulate proliferation of pooled human umbilical vein endothelial cells in culture and, therefore, function as useful growth or inhibitory factors.
On day 0, pooled human umbilical vein endothelial cells (from cell lines, maximum of 12-14 passages) were plated in 96-well plates at 1000 cells/well per 100 microliter and incubated overnight in complete media [epitiielial cell growth media (EGM, Clonetics), plus supplements: human epithelial growth factor (hEGF), bovine brain extract (BBE), hydrocortisone, GA-1000, and fetal bovine serum (FBS, Clonetics)]. On day 1, complete media was replaced by basal media [EGM plus 1 % FBS] and addition of PRO polypeptides at 1 %, 0.1 % and 0.01 % . On day 7, an assessment of cell proliferation was performed by Alamar Blue assay followed by Crystal Violet. Results are expresses as % of the cell growth observed with control buffer. The following PRO polypeptides stimulated proliferation in this assay: PR0181, PRO205, PR0221,
PR0231, PR0238, PR0241, PR0247, PR0256, PR0258, PR0263, PR0265, PR0295, PR0321, PR0322, PR0337, PR0363, PR0533, PR0697, PR0725, PR0771, PR0788, PR0819, PR0828, PR0846, PR0865, PRO1005, PRO1006, PRO1025, PRO1054, PRO1071, PRO1079, PRO1080, PR01114, PR01131, PR01155, PROl 160, PROl 192, PR01244, PR01272, PR01273, PR01279, PR01283, PR01286, PRO1306, PRO1309, PR01325, PR01329, PR01347, PR01356, PR01376, PR01382, PR01412, PRO1550, PR01556, PRO1760,
PR01787, PRO1801, PR01868, PR01887, PR03438, PR03444, PR04324, PR04341, PR04342, PR04353, PRO4354,PRO4356,PRO4371,PRO4422,PRO4425,PRO5723,PRO5737,PRO6029,PRO6071,PRO10096and PRO21055.
The following PRO polypeptides inhibited proliferation in this assay: PR0229, PR0444, PR0827, PRO1007, PRO1075, PROl 184, PROl 190, PROl 195, PR01419, PR01474, PR01477, PR01488, PR01782, PRO4302, PRO4405, PR05725, PR05776, PR07436, PR09771, PRO10008 and PR021384.
6.22. EXAMPLE 22: Human Coronary Artery Smooth Muscle Cell Proliferation (Assay 140) This assay is designed to determine whether PRO polypeptides of the present invention show the ability to modulate proliferation of human coronary artery smooth muscle cells in culture and, therefore, function as useful growth or inhibitory factors. On day 0, human coronary artery smooth muscle cells (from cell lines, maximum of 12-14 passages) were plated in 96-well plates at 1000 cells/well per 100 microliter and incubated overnight in complete media [smooth muscle growth media (SmGM, Clonetics), plus supplements: insulin, human epithelial growth factor (hEGF), human fibroblast growth factor (hFGF), GA-1000, and fetal bovine serum (FBS, Clonetics)]. On day 1 , complete media was replaced by basal media [SmGM plus 1 % FBS] and addition of PRO polypeptides at 1 % , 0.1 % and 0.01 % . On day 7, an assessment of cell proliferation was performed by Alamar Blue assay followed by Crystal Violet. Results are expresses as % of the cell growth observed with control buffer.
The following PRO polypeptides stimulated proliferation in this assay: PRO 162, PRO 182, PRO204, PR0221, PRO230, PR0256, PR0258, PR0533, PR0697, PR0725, PR0738, PR0826, PR0836, PRO840, PR0846, PR0865, PR0982, PRO1025, PRO1029, PRO1071, PRO1083, PROl 134, PROl 160, PROl 182, PROl 184, PROl 186, PROl 192, PR01274, PR01279, PR01283, PRO1306, PRO1308, PR01325, PR01337,
PR01338, PR01343, PR01376, PR01387, PR01411, PR01412, PR01415, PR01434, PR01474, PRO1550, PR01556, PR01567, PRO1600, PR01754, PR01758, PRO1760, PR01787, PR01865, PR01868, PR01917, PR01928, PR03438, PR03562, PR04333, PR04345, PR04353, PR04354, PRO4408, PRO4430, PRO4503, PR06714, PR09771, PRO9820, PRO9940, PRO10096, PRO21055, PR021184 and PR021366. The following PRO polypeptides inhibited proliferation in this assay: PR0181, PR0195, PRO1080,
PR01265, PRO1309, PR01488, PRO4302, PRO4405 and PR05725.
6.23. EXAMPLE 23: Microarray Analysis to Detect Overexpression of PRO Polypeptides in
HUVEC Cells Treated with Growth Factors This assay is designed to deteπnine whether PRO polypeptides ofthe present invention show the ability to induce angiogenesis by stimulating endothelial cell tube formation in HUVEC cells.
Nucleic acid microarrays, often containing thousands of gene sequences, are useful for identifying differentially expressed genes in tissues exposed to various stimuli (e.g. , growth factors) as compared to their normal, unexposed counterparts. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The cDNA probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene . If the hybridization signal of a probe from a test (exposed tissue) sample is greater than hybridization signal of a probe from a control (normal, unexposed tissue) sample, the gene or genes overexpressed in the exposed tissue are identified. The implication of this result is that an overexpressed protein in an exposed tissue may be involved in the functional changes within the tissue following exposure to the stimuli (e.g. , tube formation).
The methodology of hybridization of nucleic acids and microarray technology is well known in the art. In the present example, the specific preparation of nucleic acids for hybridization and probes, slides, and hybridization conditions are all detailed in U.S. Provisional Patent Application Serial No. 60/193,767, filed on March 31, 2000 and which is herein incorporated by reference.
In the present example, HUVEC cells grown in either collagen gels or fibrin gels were induced to form tubes by the addition of various growth factors. Specifically, collagen gels were prepared as described previously in Yang et al. , American J. Pathology, 1999, 155(3):887-895 and Xin et al , American J. Pathology, 2001, 158(3): 1111-1120. Following gelation of the HUVEC cells, IX basal medium containing M199 supplemented with 1 %FBS, IX ITS, 2 mM L-glutamine, 50 μg/ml ascorbic acid, 26.5 mM NaHC03, lOOU/ml penicillin and
100 U/ml streptomycin was added. Tube formation was elicited by the inclusion in the cultare media of either a mixture of phorbol myrsitate acetate (50 nM), vascular endothelial cell growth factor (40 ng/ml) and basic fibroblast growth factor (40 ng/ml) ("PMA growth factor mix") or hepatocyte growth factor (40 ng/ml) and vascular endothelial cell growth factor (40 ng/ml) (HGF/ VEGF mix) for the indicated period of time . Fibrin Gels were prepared by suspending Huvec (4 x 10s cells/ml) in M199 containing 1 % fetal bovine serum (Hyclone) and human fibrinogen (2.5mg/ml). Thrombin (50U/ml) was then added to the fibrinogen suspension at a ratio of 1 part thrombin solution:30 parts fibrinogen suspension. The solution was then layered onto 10 cm tissue culture plates (total volume: 15 ml/plate) and allowed to solidify at 37°C for 20 min. Tissue culture media (10 ml of BM containing PMA (50 nM), bFGF (40ng/ml) and VEGF (40 ng/ml)) was then added and the cells incubated at 37°C in 5 %C02 in air for the indicated period of time.
Total RNA was extracted from the HUVEC cells incubated for 0, 4, 8, 24, 40 and 50 hours in the different matrix and media combinations using a TRIzol extraction followed by a second purification using RNAeasy Mini Kit (Qiagen). The total RNA was used to prepare cRNA which was then hybridized to the microarrays. In the present experiments, nucleic acid probes derived from the herein described PRO polypeptide- encoding nucleic acid sequences were used in the creation of the microarray and RNA from the HUVEC cells described above were used for the hybridization thereto. Pairwise comparisons were made using time 0 chips as a baseline. Three replicate samples were analyzed for each experimental condition and time. Hence there were 3 time 0 samples for each treatment and 3 replicates of each successive time point. Therefore, a 3 by 3 comparison was performed for each time point compared against each time 0 point. This resulted in 9 comparisons per time point. Only those genes that had increased expression in all three non-time-0 replicates in each of the different matrix and media combinations as compared to any of the three time zero replicates were considered positive. Although this stringent method of data analysis does allow for false negatives, it minimizes false positives.
PR0178, PR0195, PR0228, PRO301, PRO302, PR0532, PR0724, PRO730, PR0734, PR0793, PR0871, PR0938, PRO1012, PROl 120, PROl 139, PROl 198, PR01287, PR01361, PR01864, PR01873, PRO2010, PR03579, PR04313, PR04527, PR04538, PR04553, PR04995, PRO5730, PRO6008, PR07223,
PR07248 and PR07261 tested positive in this assay.
6.24. EXAMPLE 24: In situ Hybridization
In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze* the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis, and aid in chromosome mapping.
In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision. 1: 169-176 (1994), using PCR-generated 33P-labeled riboprobes. Briefly, formalin-fixed, paraffin- embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37°C, and further processed for in situ hybridization as described by Lu and Gillett, supra. A (33-P)UTP- labeled antisense riboprobe was generated from a PCR product and hybridized at 55 °C overnight. The slides were dipped in Kodak NTB2™ nuclear track emulsion and exposed for 4 weeks.
6.24.1. — P-Riboprobe synthesis 6.0 μl (125 mCi) of 33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed-vacuum dried. To each tube containing dried 33P-UTP, the. following ingredients were added:
2.0 μl 5x transcription buffer LO μl DTT (lOO mM)
2.0 μl NTP mix (2.5 mM: 10 μl each of 10 mM GTP, CTP & ATP + 10 μl H20) 1.0 μl UTP (50 μM) 0 μl RNAsin
0 μl DNA template (1 μg) 0 μl H20
0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually) The tubes were incubated at 37°C for one hour. A total of 1.0 μl RQ1 DNase was added, followed by incubation at 37°C for 15 minutes. A total of 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) was added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a MICROCON-50™ ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, a total of 100 μl TE was added, then 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR II™. The probe was ran on a TBE/urea gel. A total of 1-3 μl of the probe or 5 μl of RNA Mrk III was added to 3 μl of loading buffer. After heating on a 95°C heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, and the sample was loaded and run at 180-250 volts for 45 minutes. The gel was wrapped in plastic wrap (SARAN™ brand) and exposed to XAR film with an intensifying screen in a - 70°C freezer one hour to overnight.
6.24.2. ^P-Hvbridization
6.24.2.1. Pretreatment of frozen sections
The slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes. The trays were placed in a 55 °C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H20). After deproteination in 0.5 μg/ml proteinase K for 10 minutes at 37°C (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0.5 x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, and 100% ethanol, 2 minutes each.
6.24.2.2. Pretreatment of paraffin-embedded sections
The slides were deparaffinized, placed in SQ H20, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNase-free RNase buffer; 37°C, 15 minutes) for human embryo tissue, or 8 x proteinase K (100 μl in 250 ml Rnase buffer, 37°C, 30 minutes) for formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
6.24.2.3. Prehybridization
The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filterpaper. The tissue was covered with 50 μl of hybridization buffer (3.75 g dextran sulfate + 6 ml SQ H20), vortexed, and heated in the microwave for 2 minutes with the cap loosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20 x SSC, and 9 ml SQ H20 were added, and the tissue was vortexed well and incubated at 42°C for 1-4 hours.
6.24.2.4. Hybridization
1.0 x 106 cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heated at 95°C for 3 minutes. The slides were cooled on ice, and 48 μl hybridization buffer was added per slide. After vortexing, 50 μl 33P mix was added to 50 μl prehybridization on the slide. The slides were incubated overnight at 55°C. 6.24.2.5. Washes
Washing was done for 2x10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0.25 M EDTA, Vt=4L), followed by RNAseA treatment at 37°C for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer = 20 μg/ml), The slides were washed 2 xlO minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55 °C, 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml
EDTA, Vf=4L).
6.24.2.6. Oligonucleotides
In situ analysis was performed on three of the DNA sequences disclosed herein. The primers used to generate the probes and/or the probes employed for these analyses are as follows:
DNA33100-pl: 5'GGA TTC TAA TAC GAC TCA CTA TAGGGC CGG GTG GAG GTG GAA CAGAAA
3' (SEQ ID NO:375) DNA33100-p2: 5' CTA TGAAAT TAA CCC TCA CTAAAGGGA CACAGA CAGAGC CCC ATA CGC 3' (SEQ ID NO:376)
DNA34431-pl: 5'GGA TTC TAA TAC GAC TCA CTA TAG GGC CAG GGA AAT CCG GAT GTC TC 3' (SEQ ID NO-.377)
DNA34431-p2: 5' CTATGAAAT TAA CCC TCA CTAAAGGGA GTAAGG GGATGC CAC CGA GTA 3' (SEQ ID NO:378)
DNA38268-pl: 5'GGA TTC TAA TAC GAC TCA CTA TAG GGC CAG CTA CCC GCA GGA GGA GG 3' (SEQ ID NO:379)
DNA38268-p2: 5'CTA TGA AAT TAA CCC TCA CTA AAG GGATCC CAG GTGATGAGG TCC AGA
3' (SEQ ID NO:380)
DNA64908 probe: 5'CCATCTCGGAGACCTTTGTGCAGCGTGTATACCAGCCTTACCTCACCA
CTTGCGACGGACACAGAGCCTGCAGCACCTACCGAACCATCTACCGGAC
TGCCTATCGCCGTAGCCCTGGGGTGACTCCCGCAAGCCTCGCTATGCTTG CTGCCCTGGTTGGAAGAGGACCAGTGGGCTCCCTGGGGCTTGTGGAGCA
GCAATATGCCAGCCTCCATGTGGGAATGGAGGGAGTTGCATCCGCCCAG GACACTGCCGCTGCCCTGTGGGATGGCAGGGAGATACTTGCCAGACAGA TGTTGATGAATGCAGTACAGGAGAGGCCAGTTGTCCCCAGCGCTGTGTC AATACTGTGGGAAGTTACTGGTGCCAGGGATGGGAGGGACAAAGCCCAT CTGCAGATGGGACGCGCTGCCTGTCTAAGGAGGGGCCCTCCCGGTGGCC CCAACCCCACAGCAGGAGTGGACAGCA3' (SEQ ID NO:381)
6.24.2.7. Results
In situ analysis was performed and the results from these analyses are as follows:
6.24.2.7.1. DNA33100-1159 (PR0229) (Scavenger-R/CD6 homologTNF motif) A specific positive signal was observed in mononuclear phagocytes (macrophages) of fetal and adult spleen, liver, lymph node and thymus. All other tissues were negative.
6.24.2.7.2. DNA34431-1177 (PRQ263) (CD44) A specific positive signal was observed in human fetal tissues and placenta over mononuclear cells, with strong expression in epithelial cells of the adrenal cortex. All adult tissues were negative.
6.24.2.7.3. DNA38268-1188 (PRQ295) (Integrin)
A specific positive signal was observed in human fetal ganglion cells , fetal neurons , adult adrenal medulla and adult neurons. All other tissues were negative.
6.24.2.7.4. DNA64908-1163-1 (PRQ1449)
A specific positive signal was observed in the developing vasculature (from E7-E11) , in endothelial cells and in progenitors of endothelial cells in wholemount in situ hybridizations of mouse embryos (Figure 375). Specific expression was also observed in a subset of blood vessels and epidermis from E12 onward. A mouse orthologue of PR01449 which has about 78% amino acid identity with PR01449 was used as the probe. In normal adult tissues, expression was low to absent. When present, expression was confined to the vasculatare (Figure 376). Figure 376 further shows that highest expression in adult tissues was observed regionally in vessels running witiiin tlie white matter ofthe brain. Elevated expression was also observed in vasculature of many inflamed and diseased tissues, including, but not limited to, tumor vasculature.
Following electroporation of the mouse orthologue of PRO 1449 into the choroid layer in the eyes of chicken embryos, new vessel formation was observed in the electroporated eye (top right), but not in the control side from the same embryo (top left), or an embryo that was electroporated with a control cDNA (bottom right) (Figure 377). 6.25. EXAMPLE 25: Inhibition of basic Fibroblast Growth Factor (bFGF) Stimulated Proliferation of Endothelial Cell Growth The ability of various PRO polypeptides to inhibit bFGF stimulated proliferation of endothelial cells was tested. Polypeptides testing positive in this assay are useful for inhibiting endothelial cell growth in mammals where such an effect would be beneficial, e.g., for inhibiting tumor growth.
Specifically, human venous umbilical vein endothelial cells (HUVEC, Cell Systems) in epithelial cell growth media (EGM, Clonetics) were plated on 96-well microtiter plates at a cell density of 5xl03 cells/well in a volume of lOOμl/well. The day after plating (day 2), the cells were starved for 24 hours by removing the growth media and replacing with starving media (M199 supplemented with 1 %FBS, 2 mM L-glutamine, lOOU/ml penicillin and 100 U/ml streptomycin). On day 5, the cells are treated with either: (1) M199 with 10% FBS; (2)
M199 with 1 % FBS; (3) M199 with 1 % FBS and 20 ng/ml bFGF; (4) M199 with 1 % FBS and 20 ng/ml bFGF and PRO polypeptide (500 M); (5) M199 with 1% FBS and 20 ng/ml bFGF and PRO polypeptide (50 nM); or (6) M199 with 1 % FBS and 20 ng/ml bFGF and PRO polypeptide (5 nM). On day 8, an assessment of cell proliferation was performed by Alamar Blue assay. Optical density (OD) was measured on a microplate reader at excitation 530 and emission at 590 nm.
The activity of PRO polypeptides was calculated as the percent inhibition of bFGF stimulated proliferation relative to the cells without stimulation. The results are indicative ofthe utility ofthe PRO polypeptides in cancer therapy and specifically in inhibiting tumor angiogenesis. Numerical values (relative inhibition) are determined by calculating the percent inhibition of bFGF stimulated proliferation by the PRO polypeptides relative to cells without stimulation. The results are considered positive if the PRO polypeptide exhibits 30% or greater inhibition of bFEGF stimulation of endothelial cell growth. PR05725 tested positive in this assay.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice tlie invention. The present invention is not to be limited in scope by the construct(s) deposited, since the deposited embodiment(s) is/are intended as single illusttation(s) of certain aspects of tlie invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material(s) herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect ofthe ivention, including the best mode thereof, nor is it to be construed as limiting the scope ofthe claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope ofthe appended claims.

Claims

WHAT IS CLAIMED IS:
1. An isolated nucleic acid molecule having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ
ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ
ID NO:54), Figure 56 (SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ
ID NO:92), Figure 94 (SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100), Figure 102 (SEQ ID NO: 102), Figure 104 (SEQ ID NO: 104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ ID NO: 108), Figure 110 (SEQ ID NO: 110), Figure 112 (SEQ ID NO: 112), Figure 114 (SEQ ID NO: 114), Figure 116 (SEQ ID NO: 116), Figure 118 (SEQ ID NO: 118), Figure 120 (SEQ ID NO: 120), Figure 122 (SEQ ID NO: 122), Figure 124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO: 126), Figure 128 (SEQ ID
NO: 128), Figure 130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO: 134), Figure 136 (SEQ ID NO:136), Figure 138 (SEQ ID NO:138), Figure 140 (SEQ ID NO:140), Figure 142 (SEQ ID NO: 142), Figure 144 (SEQ ID NO: 144), Figure 146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Figure 150 (SEQ ID NO: 150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO-.156), Figure 158 (SEQ ID NO:158), Figure 160 (SEQ ID NO-.160), Figure 162 (SEQ ID NO:162), Figure
164 (SEQ ID NO: 164), Figure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO: 168), Figure 170 (SEQ ID NO: 170), Figure 172 (SEQ ID NO: 172), Figure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID NO: 178), Figure 180 (SEQ ID NO: 180), Figure 182 (SEQ ID NO: 182), Figure 184 (SEQ ID NO: 184), Figure 186 (SEQ ID NO: 186), Figure 188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID
NO: 198), Figure 200 (SEQ ID NO:200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID NO:206), Figure 208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO-.212), Figure 214 (SEQ ID NO:214), Figure 216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO:220), Figure 222 (SEQ ID NO.-222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO:228), Figure 230 (SEQ ID NO:230), Figure 232 (SEQ ID NO:232), Figure
234 (SEQ ID NO:234), Figure 236 (SEQ ID NO:236), Figure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO-.240), Figure 242 (SEQ ID NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID NO:248), Figure 250 (SEQ ID NO:250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure 258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO:262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Figure 270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO:276), Figure 278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID
NO:282), Figure 284 (SEQ ID NO:284), Figure 286 (SEQ ID NO:286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID NO:290), Figure 292 (SEQ ID NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure 300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Figure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID NO:312), Figure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure
318 (SEQ ID NO-.318), Figure 320 (SEQ ID NO-.320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure 326 (SEQ ID NO:326), Figure 328 (SEQ ID NO:328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID NO:332), Figure 334 (SEQ ID NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Figure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO:346), Figure 348 (SEQ ID NO:348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID
NO:352), Figure 354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID NO:360), Figure 362 (SEQ ID NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure 368 (SEQ ID NO:368), Figure 370 (SEQ ID NO:370), Figure 372 (SEQ ID NO:372) and Figure 374 (SEQ ID NO:374).
2. An isolated nucleic acid molecule having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1), Figure 3 (SEQ ID NO:3), Figure 5 (SEQ ID NO:5), Figure 7 (SEQ ID NO:7), Figure 9 (SEQ ID NO:9), Figure 11 (SEQ ID NO: 11), Figure 13 (SEQ ID NO: 13), Figure 15 (SEQ ID NO: 15), Figure 17 (SEQ ID NO: 17), Figure 19 (SEQ ID NO: 19), Figure 21 (SEQ ID NO:21), Figure 23 (SEQ ID NO:23), Figure 25 (SEQ
ID NO:25), Figure 27 (SEQ ID NO:27), Figure 29 (SEQ ID NO:29), Figure 31 (SEQ ID NO:31), Figure 33 (SEQ ID NO:33), Figure 35 (SEQ ID NO:35), Figure 37 (SEQ ID NO:37), Figure 39 (SEQ ID NO:39), Figure 41 (SEQ ID NO:41), Figure 43 (SEQ ID NO:43), Figure 45 (SEQ ID NO:45), Figure 47 (SEQ ID NO:47), Figure 49 (SEQ ID NO:49), Figure 51 (SEQ ID NO:51), Figure 53 (SEQ ID NO:53), Figure 55 (SEQ ID NO:55), Figure 57 (SEQ ID NO:57), Figure 59 (SEQ ID NO:59), Figure 61 (SEQ ID NO:61), Figure 63 (SEQ
ID NO:63), Figure 65 (SEQ ID NO:65), Figure 67 (SEQ ID NO:67), Figure 69 (SEQ ID NO:69), Figure 71 (SEQ ID NO.-71), Figure 73 (SEQ ID NO:73), Figures 75A-75B (SEQ ID NO:75), Figure 77 (SEQ ID NO:77), Figure 79 (SEQ ID NO:79), Figure 81 (SEQ ID NO:81), Figure 83 (SEQ ID NO:83), Figure 85 (SEQ ID NO:85), Figure 87 (SEQ ID NO:87), Figure 89 (SEQ ID NO:89), Figure 91 (SEQ ID NO:91), Figure 93 (SEQ ID NO:93), Figure 95 (SEQ ID NO:95), Figure 97 (SEQ ID NO:97), Figure 99 (SEQ ID NO:99), Figure 101
(SEQ ID NO: 101), Figure 103 (SEQ ID NO: 103), Figure 105 (SEQ ID NO: 105), Figure 107 (SEQ ID NO: 107), Fi ure 109 (SEQ ID NO: 109), Figure 111 (SEQ ID NO: lll), Figure 113 (SEQ ID NO:113), Figure 115 (SEQ ID NO: 115), Figure 117 (SEQ ID NO: 117), Figure 119 (SEQ ID NO: 119), Figure 121 (SEQ ID NO: 121), Figure 123 (SEQ ID NO: 123), Figure 125 (SEQ ID NO: 125), Figure 127 (SEQ ID NO: 127), Figure 129 (SEQ ID NO: 129), Figure 131 (SEQ ID NO:131), Figure 133 (SEQ ID NO: 133), Figure 135 (SEQ ID NO: 135), Figure 137 (SEQ ID NO: 137), Figure 139 (SEQ ID NO: 139), Figure 141 (SEQ ID NO: 141), Figure 143 (SEQ ID NO: 143), Figure 145 (SEQ ID NO: 145), Figure 147 (SEQ ID NO: 147), Figure 149 (SEQ ID NO: 149), Figure
151 (SEQ ID NO:151), Figure 153 (SEQ ID NO:153), Figure 155 (SEQ ID NO:155), Figure 157 (SEQ ID NO: 157), Figure 159 (SEQ ID NO: 159), Figure 161 (SEQ ID NO: 161), Figure 163 (SEQ ID NO: 163), Figure 165 (SEQ ID NO: 165), Figure 167 (SEQ ID NO: 167), Figure 169 (SEQ ID NO: 169), Figure 171 (SEQ ID NO: 171), Figure 173 (SEQ ID NO: 173), Figure 175 (SEQ ID NO: 175), Figure 177 (SEQ ID NO: 177), Figure 179 (SEQ ID NO: 179), Figure 181 (SEQ ID NO:181), Figure 183 (SEQ ID NO:183), Figure 185 (SEQ ID
NO: 185), Figure 187 (SEQ ID NO: 187), Figure 189 (SEQ ID NO: 189), Figure 191 (SEQ ID NO: 191), Figure 193 (SEQ ID NO: 193), Figure 195 (SEQ ID NO: 195), Figure 197 (SEQ ID NO: 197), Figure 199 (SEQ ID NO: 199), Figure 201 (SEQ ID NO:201), Figure 203 (SEQ ID NO:203), Figure 205 (SEQ ID NO:205), Figure 207 (SEQ ID NO:207), Figure 209 (SEQ ID NO:209), Figure 211 (SEQ ID NO:211), Figure 213 (SEQ ID NO:213), Figure 215 (SEQ ID NO:215), Figure 217 (SEQ ID NO:217), Figure 219 (SEQ ID NO:219), Figure
221 (SEQ ID NO:221), Figure 223 (SEQ ID NO:223), Figure 225 (SEQ ID NO:225), Figure 227 (SEQ ID NO:227), Figure 229 (SEQ ID NO:229), Figure 231 (SEQ ID NO:231), Figure 233 (SEQ ID NO:233), Figure 235 (SEQ ID NO:235), Figure 237 (SEQ ID NO:237), Figure 239 (SEQ ID NO:239), Figure 241 (SEQ ID NO:241), Figure 243 (SEQ ID NO:243), Figure 245 (SEQ ID NO:245), Figure 247 (SEQ ID NO:247), Figure 249 (SEQ ID NO:249), Figure 251 (SEQ ID NO:251), Figure 253 (SEQ ID NO:253), Figure 255 (SEQ ID
NO:255), Figure 257 (SEQ ID NO:257), Figure 259 (SEQ ID NO:259), Figure 261 (SEQ ID NO:261), Figure 263 (SEQ ID NO:263), Figure 265 (SEQ ID NO:265), Figure 267 (SEQ ID NO:267), Figure 269 (SEQ ID NO:269), Figure 271 (SEQ ID NO:271), Figure 273 (SEQ ID NO:273), Figure 275 (SEQ ID NO:275), Figure 277 (SEQ ID NO:277), Figure 279 (SEQ ID NO:279), Figure 281 (SEQ ID NO:281), Figure 283 (SEQ ID NO:283), Figure 285 (SEQ ID NO:285), Figure 287 (SEQ ID NO:287), Figures 289A-289B (SEQ ID NO:289),
Figure 291 (SEQ ID NO:291), Figure 293 (SEQ ID NO:293), Figure 295 (SEQ ID NO:295), Figure 297 (SEQ ID NO:297), Figure 299 (SEQ ID NO:299), Figure 301 (SEQ ID NO:301), Figure 303 (SEQ ID NO:303), Figure 305 (SEQ ID NO:305), Figure 307 (SEQ ID NO:307), Figure 309 (SEQ ID NO:309), Figures 311A-311B (SEQ ID NO:311), Figure 313 (SEQ ID NO:313), Figure 315 (SEQ ID NO:315), Figure 317 (SEQ ID NO:317), Figure 319 (SEQ ID NO:319), Figure 321 (SEQ ID NO:321), Figure 323 (SEQ ID NO:323), Figure 325 (SEQ ID
NO:325), Figure 327 (SEQ ID NO:327), Figure 329 (SEQ ID NO:329), Figure 331 (SEQ ID NO:331), Figure 333 (SEQ ID NO:333), Figure 335 (SEQ ID NO:335), Figure 337 (SEQ ID NO:337), Figure 339 (SEQ ID NO:339), Figure 341 (SEQ ID NO:341), Figure 343 (SEQ ID NO:343), Figure 345 (SEQ ID NO:345), Figure 347 (SEQ ID NO:347), Figure 349 (SEQ ID NO:349), Figures 351A-351B (SEQ ID NO:351), Figure 353 (SEQ ID NO:353), Figure 355 (SEQ ID NO:355), Figure 357 (SEQ ID NO:357), Figure 359 (SEQ ID NO:359), Figure
361 (SEQ ID NO:361), Figure 363 (SEQ ID NO:363), Figure 365 (SEQ ID NO:365), Figure 367 (SEQ ID NO:367), Figure 369 (SEQ ID NO:369), Figure 371 (SEQ ID NO:371) and Figure 373 (SEQ ID NO:373).
3. An isolated nucleic acid molecule having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the full-length coding sequence of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1), Figure 3 (SEQ ID NO:3), Figure 5 (SEQ ID NO:5), Figure 7 (SEQ ID NO:7), Figure 9 (SEQ ID NO:9), Figure 11 (SEQ ID NO: ll), Figure 13 (SEQ ID NO: 13), Figure 15 (SEQ ID NO: 15), Figure 17 (SEQ ID NO: 17), Figure 19 (SEQ ID NO: 19), Figure 21 (SEQ ID NO:21), Figure 23
(SEQ ID NO:23), Figure 25 (SEQ ID NO:25), Figure 27 (SEQ ID NO:27), Figure 29 (SEQ ID NO:29), Figure 31 (SEQ ID NO:31), Figure 33 (SEQ ID NO:33), Figure 35 (SEQ ID NO:35), Figure 37 (SEQ ID NO:37), Figure 39 (SEQ ID NO:39), Figure 41 (SEQ ID NO:41), Figure 43 (SEQ ID NO:43), Figure 45 (SEQ ID NO:45), Figure 47 (SEQ ID NO:47), Figure 49 (SEQ ID NO:49), Figure 51 (SEQ ID NO:51), Figure 53 (SEQ ID NO:53), Figure 55 (SEQ ID NO:55), Figure 57 (SEQ ID NO:57), Figure 59 (SEQ ID NO:59), Figure 61
(SEQ ID NO:61), Figure 63 (SEQ ID NO:63), Figure 65 (SEQ ID NO:65), Figure 67 (SEQ ID NO:67), Figure 69 (SEQ ID NO:69), Figure 71 (SEQ ID NO:71), Figure 73 (SEQ ID NO:73), Figures 75A-75B (SEQ ID NO:75), Figure 77 (SEQ ID NO:77), Figure 79 (SEQ ID NO:79), Figure 81 (SEQ ID NO:81), Figure 83 (SEQ ID NO:83), Figure 85 (SEQ ID NO:85), Figure 87 (SEQ ID NO:87), Figure 89 (SEQ ID NO:89), Figure 91 (SEQ ID NO:91), Figure 93 (SEQ ID NO:93), Figure 95 (SEQ ID NO:95), Figure 97 (SEQ ID NO:97), Figure
99 (SEQ ID NO:99), Figure 101 (SEQ ID NO: 101), Figure 103 (SEQ ID NO: 103), Figure 105 (SEQ ID NO:105), Figure 107 (SEQ ID NO:107), Figure 109 (SEQ ID NO:109), Figure 111 (SEQ ID NO:lll), Figure 113 (SEQ ID NO: 113), Figure 115 (SEQ ID NO: 115), Figure 117 (SEQ ID NO: 117), Figure 119 (SEQ ID NO: 119), Figure 121 (SEQ ID NO: 121), Figure 123 (SEQ ID NO: 123), Figure 125 (SEQ ID NO: 125), Figure 127 (SEQ ID NO:127), Figure 129 (SEQ ID NO:129), Figure 131 (SEQ ID NO:131), Figure 133 (SEQ ID
NO: 133), Figure 135 (SEQ ID NO: 135), Figure 137 (SEQ ID NO: 137), Fi ure 139 (SEQ ID NO: 139), Figure 141 (SEQ ID NO: 141), Figure 143 (SEQ ID NO: 143), Figure 145 (SEQ ID NO: 145), Figure 147 (SEQ ID NO:147), Figure 149 (SEQ ID NO:149), Figure 151 (SEQ ID NO:151), Figure 153 (SEQ ID NO:153), Figure 155 (SEQ ID NO: 155), Figure 157 (SEQ ID NO: 157), Figure 159 (SEQ ID NO: 159), Figure 161 (SEQ ID NO: 161), Figure 163 (SEQ ID NO: 163), Figure 165 (SEQ ID NO: 165), Figure 167 (SEQ ID NO: 167), Figure
169 (SEQ ID NO:169), Figure 171 (SEQ ID NO:171), Figure 173 (SEQ ID NO:173), Figure 175 (SEQ ID NO: 175), Figure 177 (SEQ ID NO: 177), Figure 179 (SEQ ID NO: 179), Figure 181 (SEQ ID NO: 181), Figure 183 (SEQ ID NO: 183), Figure 185 (SEQ ID NO: 185), Figure 187 (SEQ ID NO: 187), Figure 189 (SEQ ID NO: 189), Figure 191 (SEQ ID NO: 191), Figure 193 (SEQ ID NO: 193), Figure 195 (SEQ ID NO: 195), Figure 197 (SEQ ID NO: 197), Figure 199 (SEQ ID NO: 199), Figure 201 (SEQ ID NO:201), Figure 203 (SEQ ID
NO:203), Figure 205 (SEQ ID NO:205), Figure 207 (SEQ ID NO:207), Figure 209 (SEQ ID NO:209), Figure 211 (SEQ ID NO:211), Figure 213 (SEQ ID NO:213), Figure 215 (SEQ ID NO:215), Figure 217 (SEQ ID NO:217), Figure 219 (SEQ ID NO:219), Figure 221 (SEQ ID NO:221), Figure 223 (SEQ ID NO:223), Figure 225 (SEQ ID NO:225), Figure 227 (SEQ ID NO:227), Figure 229 (SEQ ID NO:229), Figure 231 (SEQ ID NO:231), Figure 233 (SEQ ID NO:233), Figure 235 (SEQ ID NO:235), Figure 237 (SEQ ID NO:237), Figure
239 (SEQ ID NO:239), Figure 241 (SEQ ID NO:241), Figure 243 (SEQ ID NO:243), Figure 245 (SEQ ID NO:245), Figure 247 (SEQ ID NO:247), Figure 249 (SEQ ID NO:249), Figure 251 (SEQ ID NO:251), Figure 253 (SEQ ID NO:253), Figure 255 (SEQ ID NO:255), Figure 257 (SEQ ID NO:257), Figure 259 (SEQ ID NO:259), Figure 261 (SEQ ID NO:261), Figure 263 (SEQ ID NO:263), Figure 265 (SEQ ID NO:265), Figure 267 (SEQ ID NO:267), Figure 269 (SEQ ID NO:269), Figure 271 (SEQ ID NO:271), Figure 273 (SEQ ID NO:273), Figure 275 (SEQ ID NO:275), Figure 277 (SEQ ID NO:277), Figure 279 (SEQ ID NO:279), Figure 281 (SEQ ID NO:281), Figure 283 (SEQ ID NO:283), Figure 285 (SEQ ID NO:285), Figure 287 (SEQ ID
NO:287), Figures 289A-289B (SEQ ID NO:289), Figure 291 (SEQ ID NO:291), Figure 293 (SEQ ID NO:293), Figure 295 (SEQ ID NO:295), Figure 297 (SEQ ID NO:297), Figure 299 (SEQ ID NO:299), Figure 301 (SEQ ID NO:301), Figure 303 (SEQ ID NO:303), Figure 305 (SEQ ID NO:305), Figure 307 (SEQ ID NO-.307), Figure 309 (SEQ ID NO:309), Figures 311A-311B (SEQ ID N0:311), Figure 313 (SEQ ID NO:313), Figure 315 (SEQ ID NO:315), Figure 317 (SEQ ID NO:317), Figure 319 (SEQ ID NO:319), Figure 321 (SEQ ID NO:321), Figure
323 (SEQ ID NO:323), Figure 325 (SEQ ID NO:325), Figure 327 (SEQ ID NO-.327), Figure 329 (SEQ ID NO:329), Figure 331 (SEQ ID NO:331), Figure 333 (SEQ ID NO:333), Figure 335 (SEQ ID NO:335), Figure 337 (SEQ ID NO:337), Figure 339 (SEQ ID NO:339), Figure 341 (SEQ ID NO:341), Figure 343 (SEQ ID NO:343), Figure 345 (SEQ ID NO:345), Figure 347 (SEQ ID NO:347), Figure 349 (SEQ ID NO:349), Figures 351A-351B (SEQ ID NO:351), Figure 353 (SEQ ID NO:353), Figure 355 (SEQ ID NO:355), Figure 357 (SEQ
ID NO:357), Figure 359 (SEQ ID NO:359), Figure 361 (SEQ ID NO:361), Figure 363 (SEQ ID NO:363), Figure 365 (SEQ ID NO:365), Figure 367 (SEQ ID NO:367), Figure 369 (SEQ ID NO:369), Figure 371 (SEQ ID NO:371) and Figure 373 (SEQ ID NO:373).
4. An isolated nucleic acid molecule having at least 80 % nucleic acid sequence identity to the full- length coding sequence of the DNA deposited under any ATCC accession number shown in Table 7.
5. A vector comprising the nucleic acid of Claim 1.
6. A host cell comprising the vector of Claim 5.
7. The host cell of Claim 6, wherein said cell is a CHO cell.
8. The host cell of Claim 6, wherein said cell is an E. coli.
9. The host cell of Claim 6, wherein said cell is a yeast cell.
10. A process for producing a PRO polypeptide comprising culturing the host cell of Claim 6 under conditions suitable for expression of said PRO polypeptide and recovering said PRO polypeptide from the cell culture.
11. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of tlie amino acid sequence shown in Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ
ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56 (SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64 (SEQ
ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94 (SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100), Figure 102
(SEQ ID NO: 102), Figure 104 (SEQ ID NO: 104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ ID NO: 108), Figure 110 (SEQ ID NO: 110), Figure 112 (SEQ ID NO:112), Figure 114 (SEQ ID NO: 114), Figure 116 (SEQ ID NO: 116), Figure 118 (SEQ ID NO: 118), Figure 120 (SEQ ID NO: 120), Figure 122 (SEQ ID NO: 122), Figure 124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO: 126), Figure 128 (SEQ ID NO: 128), Figure 130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO:134), Figure 136 (SEQ ID NO: 136), Figure
138 (SEQ ID NO: 138), Figure 140 (SEQ ID NO: 140), Figure 142 (SEQ ID NO: 142), Figure 144 (SEQ ID NO: 144), Figure 146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Figure 150 (SEQ ID NO: 150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO: 156), Figure 158 (SEQ ID NO: 158), Figure 160 (SEQ ID NO: 160), Figure 162 (SEQ ID NO: 162), Figure 164 (SEQ ID NO: 164), Figure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO: 168), Figure 170 (SEQ ID NO: 170), Figure 172 (SEQ ID
NO: 172), Figure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID NO: 178), Figure 180 (SEQ ID NO: 180), Figure 182 (SEQ ID NO: 182), Figure 184 (SEQ ID NO: 184), Figure 186 (SEQ ID NO: 186), Figure 188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID NO: 198), Figure 200 (SEQ ID NO:200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID NO:206), Figure
208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO:212), Figure 214 (SEQ ID NO:214), Figure 216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO.-220), Figure 222 (SEQ ID NO:222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO.-228), Figure 230 (SEQ ID NO:230), Figure 232 (SEQ ID NO:232), Figure 234 (SEQ ID NO:234), Figure 236 (SEQ ID NO:236), Figure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO:240), Figure 242 (SEQ ID
NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID NO:248), Figure 250 (SEQ ID NO:250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure 258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO:262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Figure 270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO:276), Figure 278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID NO:282), Figure 284 (SEQ ID NO:284), Figure 286 (SEQ ID NO:286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID NO:290), Figure
292 (SEQ ID NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure 300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Figure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID NO:312), Figure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure 318 (SEQ ID NO:318), Figure 320 (SEQ ID NO:320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure 326 (SEQ ID
NO:326), Figure 328 (SEQ ID NO:328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID NO:332), Figure 334 (SEQ ID NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Figure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO:346), Figure 348 (SEQ ID NO:348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID NO:352), Figure 354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID NO:360), Figure
362 (SEQ ID NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure 368 (SEQ ID NO:368), Figure 370 (SEQ ID NO:370), Figure 372 (SEQ ID NO:372) and Figure 374 (SEQ ID NO:374).
12. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence encoded by the full-length coding sequence of the DNA deposited under any ATCC accession number shown in Table 7.
13. A chimeric molecule comprising a polypeptide according to Claim 11 fused to a heterologous amino acid sequence.
14. The chimeric molecule of Claim 13, wherein said heterologous amino acid sequence is an epitope tag sequence.
15. The chimeric molecule of Claim 13, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.
16. An antibody which specifically binds to a polypeptide according to Claim 11.
17. The antibody of Claim 16, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.
18. An isolated nucleic acid molecule having at least 80% nucleic acid sequence identity to: (a) a nucleotide sequence encoding the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4
(SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure 34 (SEQ ID
NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56 (SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure 72 (SEQ ID
NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94 (SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100), Figure 102 (SEQ ID NO: 102), Figure 104 (SEQ ID NO: 104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ ID NO: 108), Figure 110 (SEQ
ID NO: 110), Figure 112 (SEQ ID NO: 112), Figure 114 (SEQ ID NO: 114), Figure 116 (SEQ ID NO: 116), Figure 118 (SEQ ID NO: 118), Figure 120 (SEQ ID NO: 120), Figure 122 (SEQ ID NO: 122), Figure 124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO: 126), Figure 128 (SEQ ID NO: 128), Figure 130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO: 134), Figure 136 (SEQ ID NO: 136), Figure 138 (SEQ ID NO: 138), Figure 140 (SEQ ID NO: 140), Figure 142 (SEQ ID NO: 142), Figure 144 (SEQ ID NO: 144), Figure
146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Figure 150 (SEQ ID NO: 150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO: 156), Figure 158 (SEQ ID NO: 158), Figure 160 (SEQ ID NO: 160), Figure 162 (SEQ ID NO: 162), Figure 164 (SEQ ID NO: 164), Figure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO: 168), Figure 170 (SEQ ID NO: 170), Figure 172 (SEQ ID NO: 172), Figure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID NO: 178), Figure 180 (SEQ ID
NO.-180), Figure 182 (SEQ ID NO:182), Figure 184 (SEQ ID NO:184), Figure 186 (SEQ ID NO:186), Figure 188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID NO: 198), Figure 200 (SEQ ID NO:200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID NO:206), Figure 208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO:212), Figure 214 (SEQ ID NO:214), Figure
216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO:220), Figure 222 (SEQ ID NO:222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO:228), Figure 230 (SEQ ID NO.-230), Figure 232 (SEQ ID NO:232), Figure 234 (SEQ ID NO:234), Figure 236 (SEQ ID NO:236), Figure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO:240), Figure 242 (SEQ ID NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID NO:248), Figure 250 (SEQ ID
NO:250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure 258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO:262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Figure 270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO:276), Figure 278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID NO:282), Figure 284 (SEQ ID NO:284), Figure 286 (SEQ ID NO.-286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID NO:290), Figure 292 (SEQ ID NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure
300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Figure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID NO:312), Figure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure 318 (SEQ ID NO:318), Figure 320 (SEQ ID NO:320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure 326 (SEQ ID NO:326), Figure 328 (SEQ ID NO:328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID NO:332), Figure 334 (SEQ ID
NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Figure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO:346), Figure 348 (SEQ ID NO.-348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID NO:352), Figure 354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID NO:360), Figure 362 (SEQ ID NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure 368 (SEQ ID NO:368), Figure
370 (SEQ ID NO:370), Figure 372 (SEQ ID NO:372) or Figure 374 (SEQ ID NO:374), lacking its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2
(SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO:10), Figure 12 (SEQ ID NO:12), Figure 14 (SEQ ID NO:14), Figure 16 (SEQ ID NO:16), Figure 18
(SEQ ID NO: 18), Fi ure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Fi ure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56
(SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94
(SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100), Figure 102 (SEQ ID NO: 102), Figure 104 (SEQ ID NO: 104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ ID NO:108), Figure 110 (SEQ ID NO: 110), Figure 112 (SEQ ID NO:112), Figure 114 (SEQ ID NO:114), Figure 116 (SEQ ID NO:116), Figure 118 (SEQ ID NO:118), Figure 120 (SEQ ID NO: 120), Figure 122 (SEQ ID NO: 122), Figure 124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO:126), Figure 128 (SEQ ID NO: 128), Figure
130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO: 134), Figure 136 (SEQ ID NO: 136), Figure 138 (SEQ ID NO: 138), Figure 140 (SEQ ID NO: 140), Figure 142 (SEQ ID NO: 142), Figure 144 (SEQ ID NO: 144), Figure 146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Figure 150 (SEQ ID NO: 150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO: 156), Figure 158 (SEQ ID NO: 158), Figure 160 (SEQ ID NO: 160), Figure 162 (SEQ ID NO: 162), Figure 164 (SEQ ID NO: 164), Figure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO:168), Figure 170 (SEQ ID NO: 170), Figure 172 (SEQ ID NO: 172), Figure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID
NO: 178), Figure 180 (SEQ ID NO: 180), Figure 182 (SEQ ID NO: 182), Figure 184 (SEQ ID NO: 184), Figure 186 (SEQ ID NO: 186), Figure 188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID NO: 198), Figure 200 (SEQ ID NO:200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID NO:206), Figure 208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO:212), Figure
214 (SEQ ID NO:214), Figure 216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO:220), Figure 222 (SEQ ID NO:222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO:228), Figure 230 (SEQ ID NO:230), Figure 232 (SEQ ID NO:232), Figure 234 (SEQ ID NO:234), Figure 236 (SEQ ID NO:236), Figure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO:240), Figure 242 (SEQ ID NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID
NO:248), Figure 250 (SEQ ID NO:250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure 258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO:262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Fi ure 270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO:276), Figure 278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID NO:282), Figure
284 (SEQ ID NO:284), Figure 286 (SEQ ID NO:286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID NO:290), Figure 292 (SEQ ID NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure 300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Figure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID NO:312), Figure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure 318 (SEQ ID
NO:318), Figure 320 (SEQ ID NO:320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure 326 (SEQ ID NO:326), Figure 328 (SEQ ID NO:328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID NO:332), Figure 334 (SEQ ID NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Figure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO.-346), Figure 348 (SEQ ID NO:348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID NO:352), Figure
354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID NO:360), Figure 362 (SEQ ID NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure 368 (SEQ ID NO:368), Figure 370 (SEQ ID NO:370), Figure 372 (SEQ ID NO:372) or Figure 374 (SEQ ID NO: 374), with its associated signal peptide; or (c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2
(SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56
(SEQ ID NO.-56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94
(SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100), Figure 102 (SEQ ID NO: 102), Figure 104 (SEQ ID NO: 104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ ID NO: 108), Figure 110 (SEQ ID NO: 110), Figure 112 (SEQ ID NO: 112), Figure 114 (SEQ ID NO: 114), Figure 116 (SEQ ID NO:116), Figure 118 (SEQ ID NO: 118), Figure 120 (SEQ ID NO:120), Figure 122 (SEQ ID NO: 122), Figure 124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO: 126), Figure 128 (SEQ ID NO: 128), Figure
130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO: 134), Figure 136 (SEQ ID NO.-136), Figure 138 (SEQ ID NO: 138), Figure 140 (SEQ ID NO:140), Figure 142 (SEQ ID NO: 142), Figure 144 (SEQ ID NO: 144), Figure 146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Figure 150 (SEQ ID NO:150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO:156), Figure 158 (SEQ ID NO: 158), Figure 160 (SEQ ID NO: 160), Figure 162 (SEQ ID NO: 162), Figure 164 (SEQ ID
NO: 164), Figure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO: 168), Figure 170 (SEQ ID NO: 170), Figure 172 (SEQ ID NO: 172), Fi ure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID NO:178), Figure 180 (SEQ ID NO: 180), Figure 182 (SEQ ID NO: 182), Figure 184 (SEQ ID NO: 184), Figure 186 (SEQ ID NO: 186), Figure 188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID NO: 198), Figure
200 (SEQ ID NO:200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID NO:206), Figure 208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO:212), Figure 214 (SEQ ID NO:214), Figure 216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO:220), Figure 222 (SEQ ID NO:222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO:228), Figure 230 (SEQ ID NO:230), Figure 232 (SEQ ID NO.-232), Figure 234 (SEQ ID
NO:234), Figure 236 (SEQ ID NO:236), Figure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO:240), Figure 242 (SEQ ID NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID NO:248), Figure 250 (SEQ ID NO:250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure 258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO:262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Figure
270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO.-276), Figure 278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID NO:282), Figure 284 (SEQ ID NO:284), Figure 286 (SEQ ID NO:286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID NO:290), Figure 292 (SEQ ID NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure 300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Fi ure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID NO:312), Figure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure 318 (SEQ ID
NO:318), Figure 320 (SEQ ID NO:320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure 326 (SEQ ID NO:326), Figure 328 (SEQ ID NO.-328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID NO:332), Figure 334 (SEQ ID NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Figure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO:346), Figure 348 (SEQ ID NO:348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID NO:352), Fi ure
354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID NO:360), Figure 362 (SEQ ID NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure 368 (SEQ ID NO:368), Figure 370 (SEQ ID NO:370), Figure 372 (SEQ ID NO:372) or Figure 374 (SEQ ID NO: 374), lacking its associated signal peptide.
19. An isolated polypeptide having at least 80% amino acid sequence identity to: (a) an amino acid sequence of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ
ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28
(SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56 (SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64 (SEQ ID NO:64), Figure 66
(SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94 (SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100), Figure 102 (SEQ ID NO: 102), Figure
104 (SEQ ID NO: 104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ ID NO: 108), Figure 110 (SEQ ID NO: 110), Figure 112 (SEQ ID NO: 112), Figure 114 (SEQ ID NO: 114), Figure 116 (SEQ ID NO: 116), Figure 118 (SEQ ID NO: 118), Figure 120 (SEQ ID NO: 120), Figure 122 (SEQ ID NO: 122), Figure 124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO: 126), Figure 128 (SEQ ID NO: 128), Figure 130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO: 134), Figure 136 (SEQ ID NO: 136), Figure 138 (SEQ ID
NO: 138), Figure 140 (SEQ ID NO: 140), Figure 142 (SEQ ID NO: 142), Figure 144 (SEQ ID NO: 144), Figure 146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Figure 150 (SEQ ID NO: 150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO: 156), Figure 158 (SEQ ID NO: 158), Figure 160 (SEQ ID NO: 160), Figure 162 (SEQ ID NO: 162), Figure 164 (SEQ ID NO: 164), Figure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO: 168), Figure 170 (SEQ ID NO: 170), Figure 172 (SEQ ID NO: 172), Figure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID NO: 178), Figure 180 (SEQ ID NO:180), Figure 182 (SEQ ID NO:182), Figure 184 (SEQ ID NO:184), Figure 186 (SEQ ID NO:186), Figure
188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID NO: 198), Figure 200 (SEQ ID NO:200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID NO:206), Figure 208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO:212), Figure 214 (SEQ ID NO:214), Figure 216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO:220), Figure 222 (SEQ ID
NO:222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO:228), Figure 230 (SEQ ID NO:230), Figure 232 (SEQ ID NO:232), Figure 234 (SEQ ID NO:234), Figure 236 (SEQ ID NO:236), Fi ure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO:240), Figure 242 (SEQ ID NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID NO:248), Figure 250 (SEQ ID NO.-250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure
258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO:262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Figure 270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO:276), Figure 278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID NO:282), Figure 284 (SEQ ID NO:284), Figure 286 (SEQ ID NO:286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID NO:290), Figure 292 (SEQ ID
NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure 300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Figure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID NO:312), Figure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure 318 (SEQ ID NO:318), Figure 320 (SEQ ID NO:320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure 326 (SEQ ID NO:326), Figure
328 (SEQ ID NO:328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID NO:332), Figure 334 (SEQ ID NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Figure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO:346), Figure 348 (SEQ ID NO:348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID NO:352), Figure 354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID NO:360), Figure 362 (SEQ ID
NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure 368 (SEQ ID NO:368), Figure 370 (SEQ ID NO.-370), Figure 372 (SEQ ID NO:372) or Figure 374 (SEQ ID NO:374), lacking its associated signal peptide;
(b) an amino acid sequence of an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID
NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56 (SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64
(SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94 (SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100), Figure 102
(SEQ ID NO: 102), Figure 104 (SEQ ID NO: 104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ ID NO: 108), Figure 110 (SEQ ID NO:110), Figure 112 (SEQ ID NO: 112), Figure 114 (SEQ ID NO.-114), Figure 116 (SEQ ID NO: 116), Figure 118 (SEQ ID NO: 118), Figure 120 (SEQ ID NO: 120), Figure 122 (SEQ ID NO: 122), Figure 124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO: 126), Figure 128 (SEQ ID NO: 128), Figure 130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO: 134), Figure 136 (SEQ ID NO: 136), Figure
138 (SEQ ID NO: 138), Figure 140 (SEQ ID NO: 140), Figure 142 (SEQ ID NO: 142), Figure 144 (SEQ ID NO: 144), Figure 146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Figure 150 (SEQ ID NO: 150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO: 156), Figure 158 (SEQ ID NO: 158), Figure 160 (SEQ ID NO: 160), Figure 162 (SEQ ID NO: 162), Figure 164 (SEQ ID NO: 164), Fi ure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO: 168), Figure 170 (SEQ ID NO: 170), Figure 172 (SEQ ID
NO: 172), Figure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID NO: 178), Fi ure 180 (SEQ ID NO: 180), Figure 182 (SEQ ID NO: 182), Figure 184 (SEQ ID NO: 184), Figure 186 (SEQ ID NO: 186), Figure 188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID NO: 198), Figure 200 (SEQ ID NO:200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID NO:206), Figure
208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO:212), Figure 214 (SEQ ID NO.-214), Figure 216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO:220), Figure 222 (SEQ ID NO:222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO:228), Figure 230 (SEQ ID NO:230), Figure 232 (SEQ ID NO:232), Figure 234 (SEQ ID NO:234), Figure 236 (SEQ ID NO:236), Figure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO:240), Figure 242 (SEQ ID
NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID NO:248), Figure 250 (SEQ ID NO:250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure 258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO:262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Figure 270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO:276), Figure
278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID NO:282), Figure 284 (SEQ ID NO:284), Figure 286 (SEQ ID NO:286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID NO:290), Figure 292 (SEQ ID NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure 300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Figure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID NO:312), Figure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure 318 (SEQ ID NO:318), Figure 320 (SEQ ID NO:320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure 326 (SEQ ID
NO:326), Figure 328 (SEQ ID NO:328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID NO:332), Figure 334 (SEQ ID NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Figure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO:346), Figure 348 (SEQ ID NO:348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID NO:352), Figure 354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID NO:360), Figure
362 (SEQ ID NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure 368 (SEQ ID NO:368), Figure 370 (SEQ ID NO:370), Figure 372 (SEQ ID NO:372) or Figure 374 (SEQ ID NO:374), with its associated signal peptide; or
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in Fi ure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID
NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Fi ure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID
NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56 (SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID
NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94 (SEQ ID NO.-94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100), Figure 102 (SEQ ID NO: 102), Figure 104 (SEQ ID NO: 104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ ID NO: 108), Figure 110 (SEQ ID NO:110), Figure 112 (SEQ ID NO:112), Figure 114 (SEQ ID NO: 114), Figure 116 (SEQ ID NO: 116), Figure 118 (SEQ ID NO: 118), Figure 120 (SEQ ID NO: 120), Figure 122 (SEQ ID NO: 122), Figure
124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO: 126), Figure 128 (SEQ ID NO: 128), Figure 130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO: 134), Figure 136 (SEQ ID NO: 136), Figure 138 (SEQ ID NO: 138), Figure 140 (SEQ ID NO: 140), Figure 142 (SEQ ID NO: 142), Figure 144 (SEQ ID NO: 144), Figure 146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Fi ure 150 (SEQ ID NO: 150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO: 156), Figure 158 (SEQ ID
NO: 158), Figure 160 (SEQ ID NO: 160), Figure 162 (SEQ ID NO: 162), Figure 164 (SEQ ID NO: 164), Figure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO: 168), Figure 170 (SEQ ID NO: 170), Figure 172 (SEQ ID NO: 172), Figure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID NO: 178), Figure 180 (SEQ ID NO: 180), Figure 182 (SEQ ID NO: 182), Figure 184 (SEQ ID NO: 184), Figure 186 (SEQ ID NO: 186), Figure 188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID NO: 198), Figure 200 (SEQ ID NO:200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID NO:206), Figure
208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO:212), Figure 214 (SEQ ID NO:214), Figure 216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO:220), Figure 222 (SEQ ID NO:222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO:228), Figure 230 (SEQ ID NO:230), Figure 232 (SEQ ID NO:232), Figure 234 (SEQ ID NO:234), Figure 236 (SEQ ID NO:236), Figure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO:240), Figure 242 (SEQ ID
NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID NO:248), Figure 250 (SEQ ID NO:250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure 258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO:262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Figure 270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO:276), Figure
278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID NO:282), Figure 284 (SEQ ID NO:284), Figure 286 (SEQ ID NO:286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID NO:290), Figure 292 (SEQ ID NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure 300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Figure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID
NO:312), Fi ure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure 318 (SEQ ID NO:318), Figure 320 (SEQ ID NO.-320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure 326 (SEQ ID NO:326), Figure 328 (SEQ ID NO:328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID NO:332), Figure 334 (SEQ ID NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Figure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO:346), Figure
348 (SEQ ID NO:348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID NO:352), Figure 354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID NO:360), Figure 362 (SEQ ID NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure 368 (SEQ ID NO:368), Figure 370 (SEQ ID NO:370), Figure 372 (SEQ ID NO:372) or Figure 374 (SEQ ID NO:374), lacking its associated signal peptide.
20. A method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO:10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18
(SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Fi ure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56 (SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70),
Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94 (SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100), Figure 102 (SEQ ID NO: 102), Figure 104 (SEQ ID NO:104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ
ID NO: 108), Figure 110 (SEQ ID NO: 110), Figure 112 (SEQ ID NO: 112), Figure 114 (SEQ ID NO: 114), Figure 116 (SEQ ID NO: 116), Figure 118 (SEQ ID NO: 118), Figure 120 (SEQ ID NO: 120), Figure 122 (SEQ ID NO:122), Figure 124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO:126), Figure 128 (SEQ ID NO: 128), Figure 130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO: 134), Figure 136 (SEQ ID NO: 136), Figure 138 (SEQ ID NO: 138), Figure 140 (SEQ ID NO: 140), Figure 142 (SEQ ID NO: 142), Figure
144 (SEQ ID NO: 144), Figure 146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Figure 150 (SEQ ID NO: 150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO: 156), Figure 158 (SEQ ID NO: 158), Figure 160 (SEQ ID NO: 160), Figure 162 (SEQ ID NO: 162), Figure 164 (SEQ ID NO: 164), Figure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO: 168), Figure 170 (SEQ ID NO: 170), Figure 172 (SEQ ID NO: 172), Figure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID
NO: 178), Figure 180 (SEQ ID NO: 180), Figure 182 (SEQ ID NO: 182), Figure 184 (SEQ ID NO: 184), Figure 186 (SEQ ID NO: 186), Figure 188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID NO: 198), Figure 200 (SEQ ID NO.-200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID NO:206), Figure 208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO:212), Figure
214 (SEQ ID NO:214), Figure 216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO-.220), Figure 222 (SEQ ID NO:222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO:228), Figure 230 (SEQ ID NO:230), Figure 232 (SEQ ID NO:232), Figure 234 (SEQ ID NO:234), Figure 236 (SEQ ID NO:236), Figure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO:240), Figure 242 (SEQ ID NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID
NO:248), Figure 250 (SEQ ID NO:250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure 258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO-.262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Figure 270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO:276), Figure 278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID NO:282), Figure
284 (SEQ ID NO:284), Figure 286 (SEQ ID NO:286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID NO:290), Figure 292 (SEQ ID NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure 300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Figure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID NO:312), Figure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure 318 (SEQ ID NO:318), Figure 320 (SEQ ID NO:320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure 326 (SEQ ID NO:326), Figure 328 (SEQ ID NO:328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID
NO:332), Figure 334 (SEQ ID NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Fi ure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO:346), Figure 348 (SEQ ID NO:348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID NO:352), Figure 354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID NO:360), Figure 362 (SEQ ID NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure
368 (SEQ ID NO:368), Figure 370 (SEQ ID NO:370), Figure 372 (SEQ ID NO:372) or Figure 374 (SEQ ID NO: 374), or agonist or antagonist thereof.
21. The method according to Claim 20, wherein the mammal is human.
22. The method of Claim 21 , wherein the human has suffered myocardial infarction.
23. The method of Claim 21 , wherein the human has cardiac hypertrophy, trauma, a cancer, or age- related macular degeneration.
24. The method of Claim 23 , wherein the cardiac hypertrophy is characterized by the presence of an elevated level of PGF2α.
25. The method of Claim 20, wherein the polypeptide is administered together with a cardiovascular, endothelial or angiogenic agent.
26. The method of Claim 23 , wherein the polypeptide is administered following primary angioplasty.
27. The method of Claim 20, wherein the cardiovascular, endothelial or angiogenic disorder is cancer.
28. The method of Claim 27, wherein the polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent or a cytotoxic agent.
29. The method of Claim 20, wherein said agonist is an antibody to said polypeptide.
30. The method of Claim 20, wherein said antagonist is an antibody to said polypeptide.
31. A method for freating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal a nucleic acid molecule that encodes a polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure
26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56 (SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure
64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94 (SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO: 100),
Figure 102 (SEQ ID NO: 102), Figure 104 (SEQ ID NO: 104), Figure 106 (SEQ ID NO: 106), Figure 108 (SEQ ID NO:108), Figure 110 (SEQ ID NO: 110), Figure 112 (SEQ ID NO: 112), Figure 114 (SEQ ID NO:114), Figure 116 (SEQ ID NO: 116), Figure 118 (SEQ ID NO: 118), Figure 120 (SEQ ID NO:120), Figure 122 (SEQ ID NO: 122), Figure 124 (SEQ ID NO: 124), Figure 126 (SEQ ID NO: 126), Figure 128 (SEQ ID NO: 128), Figure 130 (SEQ ID NO: 130), Figure 132 (SEQ ID NO: 132), Figure 134 (SEQ ID NO: 134), Figure 136 (SEQ ID
NO: 136), Figure 138 (SEQ ID NO: 138), Fi ure 140 (SEQ ID NO: 140), Figure 142 (SEQ ID NO: 142), Figure 144 (SEQ ID NO: 144), Figure 146 (SEQ ID NO: 146), Figure 148 (SEQ ID NO: 148), Figure 150 (SEQ ID NO: 150), Figure 152 (SEQ ID NO: 152), Figure 154 (SEQ ID NO: 154), Figure 156 (SEQ ID NO: 156), Figure 158 (SEQ ID NO: 158), Figure 160 (SEQ ID NO: 160), Figure 162 (SEQ ID NO: 162), Figure 164 (SEQ ID NO: 164), Figure 166 (SEQ ID NO: 166), Figure 168 (SEQ ID NO: 168), Figure 170 (SEQ ID NO: 170), Figure
172 (SEQ ID NO: 172), Figure 174 (SEQ ID NO: 174), Figure 176 (SEQ ID NO: 176), Figure 178 (SEQ ID NO: 178), Figure 180 (SEQ ID NO: 180), Figure 182 (SEQ ID NO: 182), Figure 184 (SEQ ID NO: 184), Figure 186 (SEQ ID NO: 186), Figure 188 (SEQ ID NO: 188), Figure 190 (SEQ ID NO: 190), Figure 192 (SEQ ID NO: 192), Figure 194 (SEQ ID NO: 194), Figure 196 (SEQ ID NO: 196), Figure 198 (SEQ ID NO: 198), Figure 200 (SEQ ID NO:200), Figure 202 (SEQ ID NO:202), Figure 204 (SEQ ID NO:204), Figure 206 (SEQ ID
NO:206), Figure 208 (SEQ ID NO:208), Figure 210 (SEQ ID NO:210), Figure 212 (SEQ ID NO:212), Figure 214 (SEQ ID NO-.214), Figure 216 (SEQ ID NO:216), Figure 218 (SEQ ID NO:218), Figure 220 (SEQ ID NO:220), Figure 222 (SEQ ID NO:222), Figure 224 (SEQ ID NO:224), Figure 226 (SEQ ID NO:226), Figure 228 (SEQ ID NO:228), Figure 230 (SEQ ID NO:230), Figure 232 (SEQ ID NO:232), Figure 234 (SEQ ID NO:234), Figure 236 (SEQ ID NO:236), Figure 238 (SEQ ID NO:238), Figure 240 (SEQ ID NO:240), Figure
242 (SEQ ID NO:242), Figure 244 (SEQ ID NO:244), Figure 246 (SEQ ID NO:246), Figure 248 (SEQ ID NO:248), Figure 250 (SEQ ID NO:250), Figure 252 (SEQ ID NO:252), Figure 254 (SEQ ID NO:254), Figure 256 (SEQ ID NO:256), Figure 258 (SEQ ID NO:258), Figure 260 (SEQ ID NO:260), Figure 262 (SEQ ID NO:262), Figure 264 (SEQ ID NO:264), Figure 266 (SEQ ID NO:266), Figure 268 (SEQ ID NO:268), Figure 270 (SEQ ID NO:270), Figure 272 (SEQ ID NO:272), Figure 274 (SEQ ID NO:274), Figure 276 (SEQ ID NO:276), Figure 278 (SEQ ID NO:278), Figure 280 (SEQ ID NO:280), Figure 282 (SEQ ID NO:282), Figure 284 (SEQ ID NO:284), Figure 286 (SEQ ID NO:286), Figure 288 (SEQ ID NO:288), Figure 290 (SEQ ID
NO:290), Figure 292 (SEQ ID NO:292), Figure 294 (SEQ ID NO:294), Figure 296 (SEQ ID NO:296), Figure 298 (SEQ ID NO:298), Figure 300 (SEQ ID NO:300), Figure 302 (SEQ ID NO:302), Figure 304 (SEQ ID NO:304), Figure 306 (SEQ ID NO:306), Figure 308 (SEQ ID NO:308), Figure 310 (SEQ ID NO:310), Figure 312 (SEQ ID NO:312), Figure 314 (SEQ ID NO:314), Figure 316 (SEQ ID NO:316), Figure 318 (SEQ ID NO:318), Figure 320 (SEQ ID NO:320), Figure 322 (SEQ ID NO:322), Figure 324 (SEQ ID NO:324), Figure
326 (SEQ ID NO:326), Figure 328 (SEQ ID NO:328), Figure 330 (SEQ ID NO:330), Figure 332 (SEQ ID NO:332), Figure 334 (SEQ ID NO:334), Figure 336 (SEQ ID NO:336), Figure 338 (SEQ ID NO:338), Figure 340 (SEQ ID NO:340), Figure 342 (SEQ ID NO:342), Figure 344 (SEQ ID NO:344), Figure 346 (SEQ ID NO.-346), Figure 348 (SEQ ID NO:348), Figure 350 (SEQ ID NO:350), Figure 352 (SEQ ID NO:352), Figure 354 (SEQ ID NO:354), Figure 356 (SEQ ID NO:356), Figure 358 (SEQ ID NO:358), Figure 360 (SEQ ID
NO:360), Figure 362 (SEQ ID NO:362), Figure 364 (SEQ ID NO:364), Figure 366 (SEQ ID NO:366), Figure 368 (SEQ ID NO:368), Figure 370 (SEQ ID NO:370), Figure 372 (SEQ ID NO:372) or Figure 374 (SEQ ID NO:374), or agonist or antagonist thereof.
32. The method of Claim 31 , wherein said agonist is an antibody to said polypeptide.
33. The method of Claim 31 , wherein said antagonist is an antibody to said polypeptide.
34. The method of Claim 31 , wherein the mammal is human.
35. The method of Claim 31, wherein the nucleic acid molecule is administered via ex vivo gene therapy.
36. A method for inhibiting endothelial cell growth in a mammal comprising administering to the mammal a PR0229, PRO 238, PR0247, PR0444, PRO720, PR0827, PRO1007, PRO1029, PRO1075, PROl 184,
PROl 190, PROl 195, PR01274, PR01279, PR01419, PR01474, PR01477, PR01488, PR01782, PRO1890,
PRO4302,PRO4405,PRO5725,PRO5776,PRO6006,PRO7436,PRO9771,PRO10008,PRO21384orPRO28631 polypeptide or agonist thereof, wherein endothelial cell growth in said mammal is inhibited.
37. A method for stimulating endothelial cell growth in a mammal comprising administering to the mammal a PR021, PROlδl, PRO205, PR0214, PR0221, PR0231, PR0238, PR0241, PR0247, PR0256, PR0258, PR0263, PR0265, PR0295, PR0321, PR0322, PR0337, PR0363, PR0365, PR0533, PR0697, PR0725, PR0771, PR0788, PR0791, PR0819, PR0828, PR0836, PR0846, PR0865, PRO1005, PRO1006, PRO1025, PRO1054, PRO1071, PRO1079, PRO1080, PROl 114, PR01131, PR01155, PRO1160, PR01186, PROl 192, PR01244, PR01272, PR01273, PR01279, PR01283, PR01286, PRO1306, PRO1309, PR01325, PR01329, PR01347, PR01356, PR01376, PR01382, PR01411, PR01412, PRO1508, PRO1550, PR01556, PRO1760, PR01787, PRO1801, PR01868, PR01887, PR03438, PR03444, PR04324, PR04333, PR04341, PR04342, PR04353, PR04354, PR04356, PR04371, PRO4408, PR04422, PR04425, PR04499, PR05723, PRQ5737, PRO6029, PRO6071, PR09821, PR09873, PRO10008, PRO10096, PRO19670, PRO20040, PRO20044, PRO21055 or PR021384 polypeptide, or agonist thereof, wherein endothelial cell growth in said mammal is stimulated.
38. A method for inducing cardiac hypertrophy in a mammal comprising administering to the mammal a PR021 polypeptide or agonist thereof, wherein cardiac hypertrophy in said mammal is induced.
39. A method for stimulating angiogenesis induced by a PR01376 or PR01449 polypeptide in a mammal comprising administering a therapeutically effective amount of said polypeptide to the mammal, wherein said angiogenesis is stimulated.
40. A method for inducing endothelial cell apoptosis comprising administering to the endothelial cell a PRO4302 polypeptide or agonist thereof, wherein apoptosis in said endothelial cell is induced.
41. A method for stimulating smooth muscle cell growth comprising administering to the smooth muscle cell a PR0162, PR0182, PRO204, PR0221, PRO230, PR0256, PR0258, PR0533, PR0697, PR0725, PR0738, PR0826, PR0836, PRO840, PR0846, PR0865, PR0982, PRO1025, PRO1029, PRO1071, PRO1083, PROH34, PRO1160, PR01182, PROH84, PR01186, PROH92, PR01274, PR01279, PR01283, PRO1306, PRO1308, PR01325, PR01337, PR01338, PR01343, PR01376, PR01387, PR01411, PR01412, PR01415,
PR01434, PR01474, PRO1550, PR01556, PR01567, PRO1600, PR01754, PR01758, PRO1760, PR01787, PR01865, PR01868, PR01917, PR01928, PR03438, PR03562, PR04333, PR04345, PR04353, PR04354, PRO4408, PRO4430, PRO4503, PR06714, PR09771, PRO9820, PRO9940, PRO10096, PRO21055, PR021184 or PR021366 polypeptide, or agonist thereof, wherein smooth muscle cell growth in said smooth muscle cell is stimulated.
42. A method for inhibiting smooth muscle cell growth comprising administering to the smooth muscle cell a PR0181, PR0195, PRO1080, PR01265, PRO1309, PR01488, PRO4302, PRO4405 or PR05725 polypeptide, or agonist thereof, wherein smooth muscle cell growth in said smooth muscle cell is stimulated.
43. A method for inducing endothelial cell tube formation comprising administering to the endothelial cell a PR0178, PR0195, PR0228, PRO301, PRO302, PR0532, PR0724, PRO730, PR0734, PR0793, PR0871, PR0938, PRO1012, PROl 120, PROl 139, PROl 198, PR01287, PR01361, PR01864, PR01873, PRO2010, PR03579, PR04313, PR04527, PR04538, PR04553, PR04995, PRO5730, PRO6008, PR07223, PR07248 or PR07261 polypeptide, or agonist thereof, wherein tabe formation in said endothelial cell is induced.
PCT/US2001/021735 1996-11-06 2001-07-09 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis WO2002008284A2 (en)

Priority Applications (698)

Application Number Priority Date Filing Date Title
JP2002514188A JP2004516013A (en) 2000-07-20 2001-07-09 Compositions and methods for diagnosing and treating disorders associated with angiogenesis
CA002416538A CA2416538A1 (en) 2000-07-20 2001-07-09 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
EP01951036A EP1309685A2 (en) 2000-07-20 2001-07-09 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
AU2001271973A AU2001271973A1 (en) 2000-07-20 2001-07-09 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US09/918,585 US20030060406A1 (en) 1997-10-17 2001-07-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/931,836 US7435793B2 (en) 1998-05-15 2001-08-16 Peptides that induce chondrocyte redifferentiation
MXPA03001643A MXPA03001643A (en) 2000-08-24 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor.
US09/938,418 US20020161199A1 (en) 1998-04-08 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor
CA002420193A CA2420193A1 (en) 2000-08-24 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor
KR1020037002672A KR100607611B1 (en) 2000-08-24 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor
EP01966255A EP1311668A2 (en) 2000-08-24 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor
AU8678501A AU8678501A (en) 2000-08-24 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor
AU2001286785A AU2001286785B2 (en) 2000-08-24 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor
JP2002522275A JP2004520810A (en) 2000-08-24 2001-08-23 Compositions and methods for tumor diagnosis and treatment
PCT/US2001/026626 WO2002016602A2 (en) 2000-08-24 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor
US09/941,992 US20030082546A1 (en) 1996-11-06 2001-08-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2001/027099 WO2002024888A2 (en) 2000-09-01 2001-08-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP07022176A EP1944317A3 (en) 2000-09-01 2001-08-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2002529483A JP4451059B2 (en) 2000-09-01 2001-08-29 Secreted and transmembrane polypeptides and nucleic acids encoding them
CA002632702A CA2632702A1 (en) 2000-09-01 2001-08-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002421056A CA2421056A1 (en) 2000-09-01 2001-08-29 A polypeptide for use as a diagnostic marker for the presence of colon tumours
AU2002216610A AU2002216610A1 (en) 2000-09-01 2001-08-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP01985272A EP1341814A2 (en) 2000-09-01 2001-08-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/946,374 US20030073129A1 (en) 1998-09-01 2001-09-04 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU1661002A AU1661002A (en) 2000-09-01 2001-09-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,295 US20020156006A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,192 US20020177553A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,189 US6972325B2 (en) 1997-10-17 2001-10-15 PRO273 polypeptides
US09/978,188 US20030139328A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,193 US20030073624A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,191 US20030050239A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,298 US20030134785A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,299 US20030199435A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,194 US20030195333A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,403 US20030050240A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,375 US7196165B2 (en) 1997-10-17 2001-10-16 PRO363 polypeptides
US09/978,544 US20030199436A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,665 US7294700B2 (en) 1997-10-17 2001-10-16 Anti-PRO846 antibodies
US09/978,824 US20050124789A9 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/981,915 US7285623B2 (en) 1997-10-17 2001-10-16 PRO337 polypeptides
US09/978,757 US20030083248A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,802 US20030199674A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,681 US20030195148A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/981,915 US20030054986A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,564 US7195760B2 (en) 1997-10-17 2001-10-16 Anti-pro363 antibodies
US09/978,643 US20030104998A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,585 US20030049633A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,697 US20020169284A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,423 US20030069178A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,824 US20030055216A1 (en) 1997-10-17 2001-10-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,388 US20040223964A1 (en) 1998-03-17 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/210,028 US20030203446A1 (en) 1998-10-07 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,749 US20040029218A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,124 US20030190701A1 (en) 1998-04-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/166,709 US20030104536A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/162,521 US7067628B2 (en) 1998-03-17 2001-10-19 PRO788 polypeptides
US10/160,502 US7220835B2 (en) 1998-07-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,067 US7279553B2 (en) 1998-05-13 2001-10-19 PRO1083 polypeptides
US10/145,017 US20030186365A1 (en) 1998-03-26 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,036 US20050227342A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,829 US20030194780A1 (en) 1998-04-29 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,247 US7112657B2 (en) 1998-10-07 2001-10-19 PRO697 polypeptides
US10/164,929 US20030194781A1 (en) 1998-03-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,089 US7208575B2 (en) 1998-10-07 2001-10-19 PRO531 polypeptides
US10/143,029 US7105640B2 (en) 1997-10-17 2001-10-19 Anti-pro792 antibodies
US10/164,728 US20030186368A1 (en) 1998-05-13 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,922 US20030195345A1 (en) 1997-10-17 2001-10-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US7132283B2 (en) 1997-10-17 2001-10-24 PRO273 polypeptides
US10/017,084 US20030203402A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/020,445 US20030198994A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US20020192706A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,081 US20030049684A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,833 US6916648B2 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,834 US20030064407A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,086 US7122375B2 (en) 1997-10-17 2001-10-24 PRO274 nucleic acids
US09/999,830 US20030077700A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,829 US20030195344A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,083 US20030148376A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/002,967 US20030148373A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,085 US6974696B2 (en) 1997-10-17 2001-10-24 PRO853 nucleic acids
US10/013,928 US20030215905A1 (en) 1998-10-07 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/016,177 US20030073131A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,925 US7037710B2 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,927 US7189529B2 (en) 1997-10-17 2001-10-25 PRO792 nucleic acids
US10/013,920 US20040006219A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,923 US7169912B2 (en) 1997-10-17 2001-10-25 PRO1017 nucleic acids
US10/013,917 US7029874B2 (en) 1998-03-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,918 US20030211091A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,926 US7074593B2 (en) 1998-04-01 2001-10-25 PRO 703 nucleic acids
US10/013,921 US20030068648A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,929 US7019124B2 (en) 1997-10-17 2001-10-25 PRO788 nucleic acids
US10/000,157 US20020182673A1 (en) 1998-05-15 2001-10-30 IL-17 homologous polypedies and therapeutic uses thereof
US09/991,163 US20020132253A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,687 US20020198149A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,436 US20020198148A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,711 US20030032023A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,442 US20020132252A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,521 US20030083461A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,438 US20030027754A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,726 US20030054359A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,456 US20020137890A1 (en) 1997-03-31 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,427 US20030073809A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,562 US20030027985A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,443 US20030054987A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,073 US20020127576A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,748 US20030069403A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,598 US6956108B2 (en) 1997-06-16 2001-11-14 PRO1184 antibodies
US09/993,667 US20030022187A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,444 US6930170B2 (en) 1997-06-16 2001-11-14 PRO1184 polypeptides
US09/991,854 US20030059780A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,469 US20030068623A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,583 US7074897B2 (en) 1997-06-16 2001-11-14 Pro943 polypeptides
US09/990,440 US20030060407A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,384 US20030087305A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,333 US6953836B2 (en) 1997-06-16 2001-11-15 PRO844 polypeptides
US09/997,683 US20030059783A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,641 US7112656B2 (en) 1997-06-16 2001-11-15 PRO1312 polypeptides
US09/997,384 US7119177B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,601 US20030054404A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,614 US20030124531A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,601 US7189814B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,156 US20030044806A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,585 US20030119055A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,666 US20030027163A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,514 US7019116B2 (en) 1997-06-16 2001-11-15 PRO 1387 polypeptides
US09/998,041 US7309775B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,628 US20030059782A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,428 US20030027162A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/002,796 US20030032057A1 (en) 1997-08-26 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,529 US7309761B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,653 US7034122B2 (en) 1997-06-16 2001-11-15 Antibodies to PRO1159 polypeptides
US09/997,666 US7244816B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,440 US20030059833A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,349 US7034106B2 (en) 1997-06-16 2001-11-15 Pro1159 polypeptides
US09/997,573 US20030049682A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,559 US20030054403A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,542 US20030068647A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,641 US20030224358A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,529 US20030134284A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,857 US20030064375A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,585 US7166282B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,041 US20030119001A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,172 US20030050457A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,150 US20030194760A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,181 US6913919B2 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,157 US7101687B2 (en) 1997-06-16 2001-11-16 Nucleic acids encoding PRO943
US09/990,441 US7041804B2 (en) 1997-06-16 2001-11-16 Antibodies to PRO1387 polypeptides
US09/990,437 US20030045463A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,862 US20030130182A1 (en) 1997-11-05 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,726 US7018811B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO189 polypeptides
US09/989,732 US7037679B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO1184 polypeptides
US09/989,721 US20020142961A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,735 US6972185B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO844 polypeptides
US09/989,727 US20020072497A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,722 US20020072067A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,729 US20030059831A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,723 US20020072092A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,734 US7491529B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,279 US7083978B2 (en) 1997-06-16 2001-11-19 Nucleic acid encoding PRO1111 polypeptides
US09/989,328 US7056736B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,293 US7034136B2 (en) 1997-06-16 2001-11-20 Nucleic acids encoding PRO1159 polypeptides
US09/989,724 US7060812B2 (en) 1997-06-16 2001-11-20 PRO1312 nucleic acids
US09/989,725 US20030139329A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,731 US20020103125A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,730 US7157247B2 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,728 US7029873B2 (en) 1997-06-16 2001-11-20 Nucleic acids to PRO1387 polypeptides
US10/001,054 US20020192209A1 (en) 1997-09-17 2001-11-30 Methods and compositions for inhibiting neoplastic cell growth
US10/007,236 US7034123B2 (en) 1998-09-01 2001-12-06 Anti-PRO1347 antibodies
US10/006,063 US20030114652A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,485 US7026448B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,856 US7538086B2 (en) 1998-09-01 2001-12-06 PRO1303 polypeptides
US10/006,818 US20030054406A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,768 US6936697B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,867 US7160985B2 (en) 1997-10-29 2001-12-06 Pro180 polypeptide
US10/006,117 US7071304B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,130 US7098312B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/007,194 US7041805B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,116 US20030082626A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,746 US7026449B2 (en) 1999-01-05 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,041 US6951921B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,172 US7081514B2 (en) 1998-09-01 2001-12-06 PRO1347 polypeptides
US10/012,753 US7488796B2 (en) 1998-09-01 2001-12-07 PRO1269 polypeptides
US10/012,121 US7022817B2 (en) 1998-09-01 2001-12-07 PRO1325 polypeptides
US10/011,692 US20030109672A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,101 US20030187239A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/011,795 US7012131B2 (en) 1998-09-01 2001-12-07 Pro1410 polypeptides
US10/012,149 US7038019B2 (en) 1998-09-01 2001-12-07 Antibodies to PRO1410
US10/012,752 US7026455B2 (en) 1998-09-01 2001-12-07 Anti-pro 1343 antibodies
US10/012,137 US20030187189A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/011,671 US20030096954A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,755 US20030096955A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,231 US6924355B2 (en) 1998-09-01 2001-12-07 PRO1343 polypeptides
US10/012,754 US20030187191A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,754 US7375184B2 (en) 1998-09-01 2001-12-07 PRO1382 polypeptides
US10/012,237 US20030191281A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,064 US6953841B2 (en) 1999-01-05 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/011,833 US6951920B2 (en) 1998-09-01 2001-12-07 PRO1340 polypeptides
US10/013,915 US20030204053A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,909 US20030186318A1 (en) 1999-01-05 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,913 US20030083462A1 (en) 1999-01-05 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,822 US20030130491A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,911 US20030187193A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,910 US7057018B2 (en) 1999-01-05 2001-12-10 Pro 1474 polypeptides
US10/013,907 US20030064925A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,906 US20030191282A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,430 US20030092883A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,393 US6951737B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,519 US7033785B2 (en) 1998-09-01 2001-12-11 PRO1347 nucleic acids
US10/015,480 US7074912B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,869 US7189530B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,671 US6946263B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,389 US6936436B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,869 US20030073130A1 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,653 US20030187195A1 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,499 US20030065142A1 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,388 US20030191299A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,386 US7022498B2 (en) 1998-09-01 2001-12-12 Pro 1325 nucleic acids
US10/015,385 US20030195347A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,391 US20030120053A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,390 US20030216562A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,715 US7033786B2 (en) 1998-09-01 2001-12-12 Pro1340 nucleic acids
US10/015,387 US20030135034A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,527 US20030082628A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2001/048938 WO2003000729A2 (en) 2001-06-20 2001-12-13 Secreted polypeptide and their use in the treatment of bone disorders
EP01990229A EP1397383A2 (en) 2001-06-20 2001-12-13 Secreted polypeptide and their use in the treatment of bone disorders
US10/017,867 US20030180792A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU2002229091A AU2002229091A1 (en) 2001-06-20 2001-12-13 Secreted polypeptide and their use in the treatment of bone disorders
US10/017,610 US20030113795A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/020,063 US20030119097A1 (en) 1999-01-05 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/020,419 US20030044897A1 (en) 1999-05-04 2001-12-13 Novel secreted polypeptide and methods of treatment of bone disorders
US10/017,407 US20030125535A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,306 US20030170718A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/036,150 US7256039B2 (en) 1998-05-15 2001-12-26 PRO4405 nucleic acids
US10/036,342 US7193045B2 (en) 1998-05-15 2001-12-26 Polypeptides that induce cell proliferation
US10/036,063 US20030092063A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/035,958 US7241862B2 (en) 1998-05-15 2001-12-26 Polypeptides that induce cell proliferation or induce fetal hemoglobin
US10/035,719 US20030036114A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/036,041 US20020192751A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/036,160 US7125959B2 (en) 1998-05-15 2001-12-26 PRO4405 polypeptides
US10/035,855 US7105639B2 (en) 1998-05-15 2001-12-26 Anti-PRO 4405 antibodies
US10/036,214 US20030032061A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/035,977 US20030134327A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/052,586 US20020127584A1 (en) 1997-09-18 2002-01-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/053,107 US20020192752A1 (en) 1998-09-09 2002-01-17 Compositions and methods for the treatment of immune related diseases
US09/978,187 US20030096744A1 (en) 1997-10-17 2002-01-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,273 US7317092B2 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,203 US20030180796A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,494 US20030032063A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,193 US20030044902A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,211 US20030044844A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,269 US20030040014A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,500 US20020177165A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding
US10/066,198 US20030170721A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,051 US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,059 US20030190721A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 US20030077776A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,045 US20030073210A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,040 US20030082759A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,042 US20030096386A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,046 US20030194791A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,047 US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,048 US20030199051A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,060 US20030190722A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,058 US20030190720A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,050 US20030054516A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,054 US20030199054A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,056 US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,061 US20030082761A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,055 US20030190718A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,053 US20030199053A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,057 US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,062 US20030077779A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,049 US20030022239A1 (en) 1997-06-18 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,063 US20030199055A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,052 US20030199052A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,261 US20030068796A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,262 US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,236 US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,291 US20030199058A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,322 US20030199059A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,108 US7635478B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,235 US20030082762A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,154 US20030190724A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,771 US20030199060A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 US20030199057A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,292 US20030073211A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,213 US7193048B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,109 US20030190723A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,155 US20030068794A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,157 US20030190725A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,156 US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/123,906 US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,905 US20030087344A1 (en) 1997-06-18 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/123,912 US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,903 US20030073212A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/124,813 US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,821 US20030199023A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,823 US20030199062A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,817 US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,805 US20030194794A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,816 US20030190728A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,814 US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,818 US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,820 US20030190729A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,819 US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/124,822 US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/125,927 US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/127,831 US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,689 US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/137,865 US20030032155A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,868 US20030082764A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,474 US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 US20030022331A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/140,024 US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,928 US20030068798A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,808 US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/140,865 US20030207420A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/140,809 US20030207418A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,860 US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,651 US7193057B2 (en) 1997-10-29 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in rectal tumor
US10/140,864 US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,925 US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,760 US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/146,792 US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,730 US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,536 US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 US20030082765A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/153,934 US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,843 US20030207805A1 (en) 1997-06-18 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 US20030207429A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,782 US20030082766A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,693 US20030073169A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,695 US20030032101A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,692 US20030166188A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,706 US20030022293A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,696 US20030082767A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,705 US20030032103A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,707 US20030166110A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,691 US20030166106A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,690 US20030166105A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,697 US20030032102A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,698 US20030166108A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,701 US20030104538A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,703 US20030170794A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,704 US20030170795A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,702 US20030170793A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,689 US20030166104A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,699 US20030166109A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,694 US20030166107A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,700 US20030027262A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,585 US20030032105A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,587 US20030166113A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,589 US20030166114A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,576 US7125962B2 (en) 1997-09-18 2002-06-18 Anti-Pro268 antibodies
US10/174,582 US20030027265A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,579 US20030027264A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,583 US7211645B2 (en) 1997-09-18 2002-06-18 PRO268 polypeptides
US10/174,588 US20030027266A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,578 US20030073170A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,572 US20030027263A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,569 US20030166111A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,591 US20030166115A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,574 US20030170796A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,586 US20030032106A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,581 US7153939B2 (en) 1997-09-18 2002-06-18 PRO354 antibodies
US10/174,590 US20030008352A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,570 US20030211572A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,751 US20030166122A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,739 US20030027267A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,741 US20030073171A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,745 US20030166120A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,736 US20030166117A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,753 US20030077732A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,742 US20030166118A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,738 US20030022294A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,754 US20030166123A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,743 US20030027269A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,735 US20030082715A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,748 US20030166121A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,747 US20030032107A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,752 US20030022295A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,744 US20030166119A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,749 US20050196832A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,746 US20030027270A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,750 US20030073172A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,749 US20030017542A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,993 US20030027280A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,988 US20030170802A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,485 US20030032109A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,917 US20030044918A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,491 US20030087373A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,759 US20030166128A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,989 US20030170803A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,493 US20030032111A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,921 US20030027276A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,757 US7317082B2 (en) 1997-09-18 2002-06-20 PRO1018 polypeptides
US10/176,913 US20030022298A1 (en) 1997-09-15 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,483 US20030017541A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,911 US20030032113A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,918 US7495083B2 (en) 1997-09-18 2002-06-20 PRO940 antibodies
US10/176,747 US20030027273A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,914 US20030017543A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,919 US20030032114A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,482 US20030022296A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,920 US20030166129A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,484 US20030059876A9 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,479 US20030040054A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,981 US20030170800A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,753 US20030044917A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,487 US20030032110A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,746 US20030068680A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,754 US7709602B2 (en) 1997-09-18 2002-06-20 PRO1078 polypeptides
US10/176,490 US20030170798A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,481 US20030032108A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,985 US20030027277A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,983 US20030170801A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,480 US20030166124A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,982 US20030044919A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,750 US20030027274A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,752 US20030170799A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,978 US20030032116A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,924 US20030166131A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,986 US20030073173A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,991 US20030027324A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,916 US20030040056A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,492 US20030027272A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,922 US20030166130A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,923 US20030068681A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,748 US20030040055A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,987 US20030027278A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,915 US20030017544A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,760 US7339033B2 (en) 1998-06-26 2002-06-21 Pro1481
US10/176,489 US20030166125A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,979 US20030087374A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,751 US20030036117A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,925 US20030032115A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,755 US20030166127A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,756 US20030032112A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,488 US20030027271A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,758 US20030008353A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,992 US20030027279A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,486 US7354999B2 (en) 1997-09-18 2002-06-21 PRO1481 polypeptides
US10/179,510 US20030032117A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,522 US20030044923A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,516 US20030040058A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,523 US20030215909A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,511 US20030104539A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,519 US7339024B2 (en) 1997-09-18 2002-06-24 PRO1772 polypeptides
US10/179,525 US20030040060A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,506 US20030044920A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,514 US20030044922A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,507 US20030040057A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,513 US20030044921A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,515 US20030166135A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,509 US20030207392A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,508 US20030166133A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,526 US20030100061A1 (en) 1998-06-26 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,517 US20030170805A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,512 US20030166134A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,518 US20030104540A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,520 US20030096353A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,521 US20030170806A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,557 US20030022301A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,560 US20030044925A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,551 US20030036123A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,554 US20050202526A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,550 US20030064440A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,549 US20030032122A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,556 US7355000B2 (en) 1997-09-18 2002-06-25 PRO1380 polypeptides
US10/180,544 US20030032119A1 (en) 1998-06-26 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,545 US20030040062A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,547 US20030032121A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,553 US7365156B2 (en) 1997-09-18 2002-06-25 PRO1316 polypeptides
US10/180,555 US20030032123A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,541 US20030036120A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,543 US20030032118A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,559 US20030032124A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,546 US20030032120A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,540 US20030040061A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,548 US7696319B2 (en) 1997-09-18 2002-06-25 PRO1772 antibodies
US10/180,552 US7348415B2 (en) 1997-09-18 2002-06-25 PRO1316 antibodies
US10/180,542 US20030036121A1 (en) 1998-06-26 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,019 US7425605B2 (en) 1997-09-18 2002-06-26 PRO1486 polypeptides
US10/183,017 US20030040065A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,018 US20030104541A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,002 US20030054454A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,012 US7718770B2 (en) 1997-09-18 2002-06-26 PRO1305-polypeptides
US10/183,011 US20030068682A1 (en) 1998-06-26 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/181,000 US7319137B2 (en) 1997-09-18 2002-06-26 PRO1339 polypeptides
US10/183,016 US20030082717A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,999 US7297767B2 (en) 1997-09-18 2002-06-26 PRO1374 polypeptides
US10/183,013 US7309769B2 (en) 1997-09-18 2002-06-26 PRO1487 polypeptides
US10/183,014 US20030064441A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,001 US7084255B2 (en) 1997-09-18 2002-06-26 PRO1278 polypeptides
US10/183,009 US7339034B2 (en) 1997-09-18 2002-06-26 PRO1305 antibodies
US10/183,010 US20030032126A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,015 US20030044926A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,006 US7297776B2 (en) 1997-09-18 2002-06-26 PRO1374 antibodies
US10/183,008 US20030040064A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,005 US7317093B2 (en) 1997-09-18 2002-06-26 PRO1339 antibodies
US10/180,998 US7087421B2 (en) 1997-09-18 2002-06-26 Pro1278 polypeptides
US10/183,003 US20030082716A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,652 US20030032134A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,616 US20030036128A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,654 US7378486B2 (en) 1997-09-18 2002-06-27 PRO1482 antibodies
US10/184,640 US7271250B2 (en) 1998-06-26 2002-06-27 PRO1757 antibodies
US10/184,641 US20030073174A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,612 US20030036127A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,627 US20030040070A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,614 US20030032128A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,631 US20030036134A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,613 US20030119105A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,642 US7332573B2 (en) 1997-09-18 2002-06-27 PRO1571 polypeptides
US10/184,630 US20030036133A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,627 US7282569B2 (en) 1997-09-18 2002-06-27 PRO1508 antibodies
US10/184,619 US20030049738A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,633 US20030068683A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,615 US20030044927A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,638 US20030054456A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,651 US7291704B2 (en) 1997-09-18 2002-06-27 PRO1758 polypeptides
US10/184,618 US7393917B2 (en) 1997-09-18 2002-06-27 PRO1482 polypeptides
US10/184,630 US7304143B2 (en) 1997-09-18 2002-06-27 PRO1571 antibodies
US10/184,628 US7309770B2 (en) 1997-09-18 2002-06-27 PRO1757 polypeptides
US10/184,650 US20030036138A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,617 US20030036129A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,624 US20030104542A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,643 US20030044929A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,636 US20030036136A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,656 US20030044931A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,647 US20030032133A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,626 US20030040069A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,632 US20030036135A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,623 US20030032129A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,621 US20030054455A1 (en) 1998-06-26 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,634 US20030068684A1 (en) 1998-06-26 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,629 US20030036132A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,645 US7291718B2 (en) 1998-06-26 2002-06-28 PRO1758 antibodies
US10/184,620 US20030044928A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,646 US20030032132A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,655 US20030040073A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,625 US20030040068A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,644 US20030044930A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,658 US20030027281A1 (en) 1998-06-26 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,637 US20030032131A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,657 US20030104543A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,635 US20030032130A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,622 US20030036130A1 (en) 1997-09-18 2002-06-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,747 US7291707B2 (en) 1997-09-18 2002-07-01 PRO1337 polypeptides
US10/187,601 US7291705B2 (en) 1997-09-18 2002-07-01 PRO19645 polypeptides
US10/187,594 US7294335B2 (en) 1998-06-26 2002-07-01 PRO19645 antibodies
US10/187,739 US7291706B2 (en) 1998-06-26 2002-07-01 PRO4352 polypeptides
US10/187,884 US20030036155A1 (en) 1997-09-18 2002-07-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,597 US20030036141A1 (en) 1997-09-18 2002-07-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,598 US20030036142A1 (en) 1997-09-18 2002-07-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,588 US7351795B2 (en) 1998-06-26 2002-07-01 PRO19563 polypeptides
US10/187,887 US7285645B2 (en) 1997-09-18 2002-07-01 PRO4356 antibodies
US10/187,886 US7291708B2 (en) 1997-09-18 2002-07-01 PRO1785 polypeptides
US10/187,746 US20030036149A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,774 US20030040074A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,767 US7312310B2 (en) 1997-09-18 2002-07-02 PRO6015 polypeptides
US10/188,770 US7358340B2 (en) 1997-09-18 2002-07-02 PRO19563 antibodies
US10/188,780 US7268217B2 (en) 1998-06-26 2002-07-02 PRO4421 polypeptides
US10/187,754 US20030036153A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,596 US20030032136A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,743 US20030036148A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,747 US20030036150A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,741 US20030036147A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,885 US20030032138A1 (en) 1998-06-24 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,775 US20030040075A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,773 US20030036159A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,603 US20030036146A1 (en) 1998-06-26 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,745 US7250490B2 (en) 1997-09-18 2002-07-02 PRO1480 polypeptides
US10/187,753 US20030036152A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,757 US7276578B2 (en) 1997-09-18 2002-07-02 PRO4334 polypeptides
US10/187,751 US20030036151A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,766 US7351804B2 (en) 1998-06-26 2002-07-02 Antibodies against PRO4421
US10/188,781 US20030036160A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,769 US20030036157A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,600 US20030036143A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,602 US20030036145A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/192,010 US20030044932A1 (en) 1997-09-18 2002-07-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/194,361 US20030036161A1 (en) 1998-06-26 2002-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/194,461 US20030054459A1 (en) 1998-06-26 2002-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/194,423 US7339025B2 (en) 1998-06-26 2002-07-12 PRO6246 polypeptides
US10/194,462 US7388073B2 (en) 1998-06-26 2002-07-12 PRO9835 polypeptides
US10/194,365 US7381791B2 (en) 1998-06-26 2002-07-12 PRO9739 polypeptides
US10/195,894 US20030043176A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,889 US7534856B2 (en) 1998-06-26 2002-07-15 PRO19624 antibodies
US10/195,901 US20030036165A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,888 US20060073545A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,883 US20060073544A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,902 US20030038826A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,897 US20030036164A1 (en) 1997-09-18 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,892 US7385033B2 (en) 1998-06-26 2002-07-15 PRO12970 polypeptides
US10/195,893 US20030206188A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/196,762 US20030040078A1 (en) 1998-06-26 2002-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/196,743 US20030038827A1 (en) 1998-06-26 2002-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/196,756 US7304145B2 (en) 1998-06-26 2002-07-16 PRO19646 antibodies
US10/196,745 US7423120B2 (en) 1997-09-18 2002-07-16 PRO19814 polypeptides
US10/196,760 US7408034B2 (en) 1998-06-26 2002-07-16 PRO20025 polypeptides
US10/196,759 US20030071835A1 (en) 1998-06-26 2002-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/197,942 US20030175882A1 (en) 1998-09-10 2002-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/198,768 US20030049756A1 (en) 1998-06-26 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/199,464 US20030032140A1 (en) 1997-09-18 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/199,316 US20030068726A1 (en) 1998-06-26 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/199,462 US20030054468A1 (en) 1998-06-26 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/205,904 US20030073813A1 (en) 1998-06-26 2002-07-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/213,199 US7381809B2 (en) 1998-09-09 2002-08-05 Compositions and methods for the treatment of immune related diseases
US10/223,082 US7164007B2 (en) 2001-06-20 2002-08-16 Anti-PR020044 antibodies
US10/223,090 US7250495B2 (en) 2001-06-20 2002-08-16 PRO20044 polypeptides
US10/223,089 US20030125521A1 (en) 2001-06-20 2002-08-16 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/223,085 US20030100497A1 (en) 2001-06-20 2002-08-16 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/223,088 US7265210B2 (en) 2000-09-15 2002-08-16 Anti-PRO9821 antibodies
US10/223,084 US7164009B2 (en) 2000-09-15 2002-08-16 Pro9821 polypeptides
US10/223,087 US7135334B2 (en) 2001-06-20 2002-08-16 PRO20044 nucleic acids
US10/223,081 US7115415B2 (en) 2000-09-15 2002-08-16 PRO9821 nucleic acids
US10/223,083 US20030119112A1 (en) 2001-06-20 2002-08-16 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/226,739 US7390879B2 (en) 1999-06-15 2002-08-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/242,505 US20030138898A1 (en) 1998-12-01 2002-09-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/244,990 US20030113852A1 (en) 2000-09-01 2002-09-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/272,051 US20030108544A1 (en) 1999-09-01 2002-10-16 Compositions and methods for the diagnosis and treatment of tumor
US10/617,573 US7771719B1 (en) 2000-01-11 2003-07-11 Pharmaceutical compositions, kits, and therapeutic uses of antagonist antibodies to IL-17E
US10/884,091 US20040265966A1 (en) 2000-03-01 2004-07-01 Secreted and transmebrane polypeptides and nucleic acids encoding the same
US10/805,667 US20050037458A1 (en) 1999-03-10 2004-07-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/934,892 US20050037465A1 (en) 1999-05-04 2004-09-03 Novel secreted polypeptide and methods of treatment of bone disorders
US10/972,317 US7208321B2 (en) 1998-06-02 2004-10-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/025,607 US20050181478A1 (en) 1998-09-01 2004-12-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/100,159 US7425613B2 (en) 1997-11-05 2005-04-05 PRO1375 polypeptides
US11/189,442 US20060246465A1 (en) 1998-06-04 2005-07-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/240,891 US20060246540A1 (en) 1997-08-26 2005-09-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/263,278 US20080286821A1 (en) 1999-05-14 2005-10-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2005363921A JP2006104212A (en) 2000-08-24 2005-12-16 Composition and method for diagnosis and treatment of tumor
US11/323,117 US20070092941A1 (en) 1998-09-16 2005-12-29 PRO1298 polypeptides
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
JP2006024604A JP2006223304A (en) 2000-09-01 2006-02-01 Secreted and transmembrane polypeptide and nucleic acid encoding same
US11/457,750 US20070031901A1 (en) 1999-03-08 2006-07-14 Compositions and methods for the diagnosis and treatment of tumor
JP2006252159A JP2007077155A (en) 2000-08-24 2006-09-19 Composition and method for diagnosing and treating tumor
US11/529,324 US8273703B2 (en) 1998-05-15 2006-09-29 IL-17 receptor-like polypeptides and therapeutic uses thereof
US11/538,754 US20070098634A1 (en) 1999-09-01 2006-10-04 Compositions and methods for the diagnosis and treatment of tumor
US11/786,466 US20080182275A1 (en) 1998-12-22 2007-04-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/796,725 US20090197301A1 (en) 1998-09-01 2007-04-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/949,633 US20080160021A1 (en) 2000-01-11 2007-12-03 Il-17 homologous polypeptides and therapeutic uses thereof
US12/549,497 US8034342B2 (en) 2000-01-11 2009-08-28 Pharmaceutical compositions, kits, and therapeutic uses of antagonist antibodies to IL-17E
US13/270,006 US20120064073A1 (en) 2000-01-11 2011-10-10 Il-17 homologous polypeptides and therapeutic uses thereof
US13/269,984 US20120058909A1 (en) 2000-01-11 2011-10-10 Il-17 homologous polypeptides and therapeutic uses thereof
US13/593,362 US20130064827A1 (en) 1998-05-15 2012-08-23 Il-17 homologous polypeptides and thereapeutic uses thereof
US14/489,167 US20150141283A1 (en) 2000-01-11 2014-09-17 Il-17 homologous polypeptides and therapeutic uses thereof
US14/581,971 US20150266953A1 (en) 1998-05-15 2014-12-23 Il-17 homologous polypeptides and therapeutic uses thereof

Applications Claiming Priority (72)

Application Number Priority Date Filing Date Title
US21955600P 2000-07-20 2000-07-20
US60/219,556 2000-07-20
US22062400P 2000-07-25 2000-07-25
US22066400P 2000-07-25 2000-07-25
US60/220,624 2000-07-25
US60/220,664 2000-07-25
PCT/US2000/020710 WO2001009327A2 (en) 1999-07-28 2000-07-28 Method of preventing the injury or death of retinal cells and treating ocular diseases
USPCT/US00/20710 2000-07-28
US22269500P 2000-08-02 2000-08-02
US60/222,695 2000-08-02
US09/643,657 2000-08-17
US09/643,657 US6642024B1 (en) 1998-01-29 2000-08-17 Guanylate-binding protein
PCT/US2000/023522 WO2001016319A2 (en) 1999-08-31 2000-08-23 Compositions and methods for the treatment of immune related diseases
USPCT/US00/23522 2000-08-23
PCT/US2000/023328 WO2001016318A2 (en) 1999-09-01 2000-08-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US00/23328 2000-08-24
US23097800P 2000-09-07 2000-09-07
US60/230,978 2000-09-07
US60/000,000 2000-09-15
US66461000A 2000-09-18 2000-09-18
US66535000A 2000-09-18 2000-09-18
US09/664,610 2000-09-18
US09/665,350 2000-09-18
US24292200P 2000-10-24 2000-10-24
US60/242,922 2000-10-24
US70923800A 2000-11-08 2000-11-08
PCT/US2000/030952 WO2001049715A2 (en) 2000-01-06 2000-11-08 Methods and compositions for inhibiting neoplastic cell growth
US09/709,238 2000-11-08
USPCT/US00/30952 2000-11-08
USPCT/US00/30873 2000-11-10
PCT/US2000/030873 WO2001040465A2 (en) 1999-11-30 2000-11-10 Compositions and methods for the treatment of immune related diseases
USPCT/US00/32678 2000-12-01
PCT/US2000/032678 WO2001040466A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/034956 WO2001046420A2 (en) 1999-12-23 2000-12-20 Il-17 and il-17r homologous polypeptides and therapeutic uses thereof
US09/747,259 US6569645B2 (en) 1999-05-14 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
USPCT/US00/34956 2000-12-20
US09/747,259 2000-12-20
US09/767,609 US20020042367A1 (en) 1997-11-25 2001-01-22 Fibroblast growth factor-19 (FGF-19) nucleic acids and polypeptides and methods of use for the treatment of obesity and related disorders
US09/767,609 2001-01-22
US79649801A 2001-02-28 2001-02-28
USPCT/US01/06520 2001-02-28
PCT/US2001/006520 WO2001068848A2 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/796,498 2001-02-28
PCT/US2001/006666 WO2001066740A2 (en) 2000-03-03 2001-03-01 Compositions and methods for the treatment of immune related diseases
USPCT/US01/06666 2001-03-01
US80270601A 2001-03-09 2001-03-09
US09/802,706 2001-03-09
US80868901A 2001-03-14 2001-03-14
US09/808,689 2001-03-14
US09/816,744 US6579520B2 (en) 1998-05-15 2001-03-22 IL-17 related mammalian cytokine polypeptides (IL-17E)
US09/816,744 2001-03-22
US09/828,366 2001-04-05
US09/828,366 US20020010137A1 (en) 1997-09-18 2001-04-05 Methods and compositions for inhibiting neoplastic cell growth
US09/854,280 2001-05-10
US09/854,208 US7217412B2 (en) 1998-05-15 2001-05-10 IL-17C related mammalian cytokine polypeptides
US09/854,208 2001-05-10
US09/854,280 US7115398B2 (en) 1998-05-15 2001-05-10 IL-17 homologous polypeptides and therapeutic uses thereof
US09/866,034 US20030170864A1 (en) 2000-05-30 2001-05-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/866,028 US6642360B2 (en) 1997-12-03 2001-05-25 Secreted polypeptides that stimulate release of proteoglycans from cartilage
US09/866,034 2001-05-25
PCT/US2001/017092 WO2001092331A2 (en) 2000-05-30 2001-05-25 Compositions and methods for the treatment of immune related diseases
US09/866,028 2001-05-25
USPCT/US01/17092 2001-05-25
USPCT/US01/17443 2001-05-30
US09/870,574 2001-05-30
US09/870,574 US6551799B2 (en) 1999-12-07 2001-05-30 Interleukin-22 polypeptides, nucleic acids encoding the same and methods for the treatment of pancreatic disorders
PCT/US2001/017443 WO2002016611A2 (en) 2000-08-24 2001-05-30 Interleukin-22 polypeptides, nucleic acids encoding the same and methods for the treatment of pancreatic disorders
PCT/US2001/017800 WO2001093983A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US01/17800 2001-06-01
USPCT/US01/19692 2001-06-20
PCT/US2001/019692 WO2002000690A2 (en) 2000-06-23 2001-06-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
USPCT/US01/00000 2001-06-28

Related Parent Applications (5)

Application Number Title Priority Date Filing Date
PCT/US2001/019692 Continuation WO2002000690A2 (en) 1996-11-06 2001-06-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
PCT/US2001/019692 Continuation-In-Part WO2002000690A2 (en) 1996-11-06 2001-06-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
PCT/US2001/021006 Continuation-In-Part WO2002002418A1 (en) 2000-06-30 2001-06-29 Base portion of a plastic container
PCT/US2001/021066 Continuation-In-Part WO2002008288A2 (en) 1996-11-06 2001-06-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2001/021066 Continuation WO2002008288A2 (en) 1996-11-06 2001-06-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Related Child Applications (14)

Application Number Title Priority Date Filing Date
PCT/US2000/004341 Continuation-In-Part WO2000053756A2 (en) 1996-11-06 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/004342 Continuation-In-Part WO2000078961A1 (en) 1997-03-31 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/034956 Continuation-In-Part WO2001046420A2 (en) 1997-03-31 2000-12-20 Il-17 and il-17r homologous polypeptides and therapeutic uses thereof
US09/747,259 Continuation-In-Part US6569645B2 (en) 1997-03-31 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
US09/908,827 Continuation-In-Part US20030054442A1 (en) 1997-03-31 2001-07-18 IL-17 homologous polypeptides and therapeutic uses thereof
US09/908,827 Continuation US20030054442A1 (en) 1997-03-31 2001-07-18 IL-17 homologous polypeptides and therapeutic uses thereof
US09/918,585 Continuation-In-Part US20030060406A1 (en) 1997-09-15 2001-07-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/938,418 Continuation US20020161199A1 (en) 1998-04-08 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor
US09/938,418 Continuation-In-Part US20020161199A1 (en) 1998-04-08 2001-08-23 Compositions and methods for the diagnosis and treatment of tumor
US09/946,374 Continuation-In-Part US20030073129A1 (en) 1997-09-15 2001-09-04 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/002,796 Continuation-In-Part US20030032057A1 (en) 1997-08-26 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/081,056 Continuation US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/197,942 Continuation US20030175882A1 (en) 1998-03-27 2002-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/272,051 Continuation-In-Part US20030108544A1 (en) 1999-09-01 2002-10-16 Compositions and methods for the diagnosis and treatment of tumor

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