WO2001019987A1 - Promotion ou inhibition de l'angiogenese et de la vascularisation cardiaque - Google Patents

Promotion ou inhibition de l'angiogenese et de la vascularisation cardiaque

Info

Publication number
WO2001019987A1
WO2001019987A1 PCT/US1999/028214 US9928214W WO0119987A1 WO 2001019987 A1 WO2001019987 A1 WO 2001019987A1 US 9928214 W US9928214 W US 9928214W WO 0119987 A1 WO0119987 A1 WO 0119987A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
pro230
pro302
acid sequence
seq
Prior art date
Application number
PCT/US1999/028214
Other languages
English (en)
Inventor
Sherman Fong
Mary E. Gerritsen
Audrey Goddard
Austin L. Gurney
Kenneth J. Hillan
P. Mickey Williams
William I. Wood
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/US1999/020944 external-priority patent/WO2000015792A2/fr
Priority claimed from PCT/US1999/021090 external-priority patent/WO2000015796A2/fr
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU17471/00A priority Critical patent/AU1747100A/en
Priority to PCT/US1999/030999 priority patent/WO2001005836A1/fr
Priority to AU23907/00A priority patent/AU2390700A/en
Priority to PCT/US2000/004414 priority patent/WO2001004311A1/fr
Priority to AU28839/00A priority patent/AU2883900A/en
Priority to PCT/US2000/005841 priority patent/WO2000053758A2/fr
Priority to JP2000603379A priority patent/JP2004516227A/ja
Priority to EP00913764A priority patent/EP1220905A2/fr
Priority to CA002362427A priority patent/CA2362427A1/fr
Priority to AU35144/00A priority patent/AU3514400A/en
Priority to KR1020017011406A priority patent/KR20010103046A/ko
Priority to AU63910/00A priority patent/AU6391000A/en
Priority to PCT/US2000/020710 priority patent/WO2001009327A2/fr
Publication of WO2001019987A1 publication Critical patent/WO2001019987A1/fr
Priority to US09/902,572 priority patent/US20030108983A1/en
Priority to US09/902,634 priority patent/US20030082540A1/en
Priority to US09/902,759 priority patent/US20030077654A1/en
Priority to US09/902,979 priority patent/US20030113718A1/en
Priority to US09/902,903 priority patent/US20030044839A1/en
Priority to US09/902,713 priority patent/US20030082541A1/en
Priority to US09/902,736 priority patent/US20030049676A1/en
Priority to US09/902,853 priority patent/US20020192659A1/en
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Priority to US09/904,011 priority patent/US20030003530A1/en
Priority to US09/903,943 priority patent/US20030054349A1/en
Priority to US09/903,562 priority patent/US6965015B2/en
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Priority to US09/903,823 priority patent/US20030104381A1/en
Priority to US09/903,640 priority patent/US7208308B2/en
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Priority to US09/905,291 priority patent/US20020160374A1/en
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Priority to US09/907,728 priority patent/US20030190611A1/en
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Priority to US09/907,925 priority patent/US20030054352A1/en
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Priority to US09/907,824 priority patent/US20020197671A1/en
Priority to US09/907,652 priority patent/US20030104469A1/en
Priority to US09/907,794 priority patent/US6635468B2/en
Priority to US09/909,320 priority patent/US7074592B2/en
Priority to US09/908,576 priority patent/US20040005553A1/en
Priority to US09/909,064 priority patent/US6818449B2/en
Priority to US09/909,204 priority patent/US20030036061A1/en
Priority to US09/909,088 priority patent/US20020146709A1/en
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Priority to US10/028,072 priority patent/US20030004311A1/en
Priority to US10/081,056 priority patent/US20040043927A1/en
Priority to US10/121,044 priority patent/US20030190717A1/en
Priority to US10/121,042 priority patent/US20030096386A1/en
Priority to US10/121,045 priority patent/US20030073210A1/en
Priority to US10/121,047 priority patent/US20030077778A1/en
Priority to US10/121,046 priority patent/US20030194791A1/en
Priority to US10/121,051 priority patent/US20030092147A1/en
Priority to US10/121,041 priority patent/US20030077776A1/en
Priority to US10/121,040 priority patent/US20030082759A1/en
Priority to US10/121,059 priority patent/US20030190721A1/en
Priority to US10/121,055 priority patent/US20030190718A1/en
Priority to US10/121,048 priority patent/US20030199051A1/en
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Priority to US10/121,063 priority patent/US20030199055A1/en
Priority to US10/121,043 priority patent/US7220831B2/en
Priority to US10/121,062 priority patent/US20030077779A1/en
Priority to US10/121,057 priority patent/US20030190719A1/en
Priority to US10/121,054 priority patent/US20030199054A1/en
Priority to US10/121,050 priority patent/US20030054516A1/en
Priority to US10/121,060 priority patent/US20030190722A1/en
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Priority to US10/121,049 priority patent/US20030022239A1/en
Priority to US10/121,058 priority patent/US20030190720A1/en
Priority to US10/121,056 priority patent/US20030082760A1/en
Priority to US10/123,213 priority patent/US7193048B2/en
Priority to US10/123,214 priority patent/US7343721B2/en
Priority to US10/123,322 priority patent/US20030199059A1/en
Priority to US10/123,109 priority patent/US20030190723A1/en
Priority to US10/123,235 priority patent/US20030082762A1/en
Priority to US10/123,213 priority patent/US20030199057A1/en
Priority to US10/123,155 priority patent/US20030068794A1/en
Priority to US10/123,771 priority patent/US20030199060A1/en
Priority to US10/123,154 priority patent/US20030190724A1/en
Priority to US10/123,156 priority patent/US20030194792A1/en
Priority to US10/123,292 priority patent/US20030073211A1/en
Priority to US10/123,291 priority patent/US20030199058A1/en
Priority to US10/123,236 priority patent/US20030068795A1/en
Priority to US10/123,108 priority patent/US7635478B2/en
Priority to US10/123,212 priority patent/US7276577B2/en
Priority to US10/123,215 priority patent/US7291329B2/en
Priority to US10/123,261 priority patent/US20030068796A1/en
Priority to US10/123,157 priority patent/US20030190725A1/en
Priority to US10/123,262 priority patent/US20030049816A1/en
Priority to US10/123,910 priority patent/US7329404B2/en
Priority to US10/123,908 priority patent/US7335728B2/en
Priority to US10/123,907 priority patent/US7084258B2/en
Priority to US10/123,909 priority patent/US7193049B2/en
Priority to US10/123,906 priority patent/US20030190726A1/en
Priority to US10/123,905 priority patent/US20030087344A1/en
Priority to US10/123,912 priority patent/US20030100087A1/en
Priority to US10/123,905 priority patent/US7285625B2/en
Priority to US10/123,904 priority patent/US20030022328A1/en
Priority to US10/123,911 priority patent/US7408032B2/en
Priority to US10/123,902 priority patent/US20030077781A1/en
Priority to US10/123,903 priority patent/US20030073212A1/en
Priority to US10/123,913 priority patent/US20030203462A1/en
Priority to US10/124,821 priority patent/US20030199023A1/en
Priority to US10/124,816 priority patent/US20030190728A1/en
Priority to US10/124,818 priority patent/US20030082763A1/en
Priority to US10/124,819 priority patent/US7285626B2/en
Priority to US10/125,795 priority patent/US7304131B2/en
Priority to US10/124,824 priority patent/US20030077659A1/en
Priority to US10/124,820 priority patent/US20030190729A1/en
Priority to US10/124,823 priority patent/US20030199062A1/en
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Priority to US10/125,704 priority patent/US7357926B2/en
Priority to US10/124,813 priority patent/US7312307B2/en
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Priority to US10/125,927 priority patent/US20030190731A1/en
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Priority to US10/125,931 priority patent/US20030199063A1/en
Priority to US10/127,831 priority patent/US20030082689A1/en
Priority to US10/128,689 priority patent/US20030087365A1/en
Priority to US10/131,817 priority patent/US7291701B2/en
Priority to US10/131,823 priority patent/US7304132B2/en
Priority to US10/131,825 priority patent/US7282566B2/en
Priority to US10/137,865 priority patent/US20030032155A1/en
Priority to US10/137,867 priority patent/US20030207349A1/en
Priority to US10/137,868 priority patent/US20030082764A1/en
Priority to US10/140,020 priority patent/US20030207415A1/en
Priority to US10/139,980 priority patent/US7247710B2/en
Priority to US10/140,023 priority patent/US20030207416A1/en
Priority to US10/140,474 priority patent/US20030032156A1/en
Priority to US10/140,470 priority patent/US20030022331A1/en
Priority to US10/140,024 priority patent/US20040058424A1/en
Priority to US10/139,963 priority patent/US7288625B2/en
Priority to US10/140,928 priority patent/US20030068798A1/en
Priority to US10/140,921 priority patent/US7317080B2/en
Priority to US10/140,805 priority patent/US20030207417A1/en
Priority to US10/140,864 priority patent/US20030207419A1/en
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Priority to US10/140,925 priority patent/US20030073215A1/en
Priority to US10/140,860 priority patent/US7307151B2/en
Priority to US10/141,755 priority patent/US7297764B2/en
Priority to US10/141,756 priority patent/US7488586B2/en
Priority to US10/141,701 priority patent/US20030207421A1/en
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Priority to US10/142,430 priority patent/US7309766B2/en
Priority to US10/142,417 priority patent/US7304133B2/en
Priority to US10/143,114 priority patent/US20030036180A1/en
Priority to US10/143,113 priority patent/US7329730B2/en
Priority to US10/142,425 priority patent/US20030207424A1/en
Priority to US10/142,423 priority patent/US20030049817A1/en
Priority to US10/142,419 priority patent/US7153941B2/en
Priority to US10/143,032 priority patent/US7408033B2/en
Priority to US10/142,431 priority patent/US7285629B2/en
Priority to US10/146,730 priority patent/US20030207427A1/en
Priority to US10/146,792 priority patent/US20030207428A1/en
Priority to US10/147,528 priority patent/US20030219885A1/en
Priority to US10/147,492 priority patent/US20030082765A1/en
Priority to US10/147,519 priority patent/US20030077791A1/en
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Priority to US10/152,395 priority patent/US7189534B2/en
Priority to US10/153,934 priority patent/US20030129695A1/en
Priority to US10/156,843 priority patent/US20030207805A1/en
Priority to US10/157,782 priority patent/US20030077792A1/en
Priority to US10/157,786 priority patent/US20030208055A1/en
Priority to US10/160,498 priority patent/US20030073216A1/en
Priority to US10/158,782 priority patent/US20030082766A1/en
Priority to US10/158,791 priority patent/US20030207429A1/en
Priority to US10/176,913 priority patent/US20030022298A1/en
Priority to US10/771,187 priority patent/US7355002B2/en
Priority to US10/970,823 priority patent/US7307152B2/en
Priority to US11/341,175 priority patent/US7468427B2/en
Priority to US11/518,609 priority patent/US20070077623A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to compositions and methods useful for promoting or inhibiting angiogenesis and/or cardiovascuiarization in mammals in need of such biological effect. This includes the diagnosis and treatment of cardiovascular disorders as well as oncological disorders.
  • 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 1 16 percent.
  • CHF congestive heart failure
  • 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 of the 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.
  • 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).
  • cardiac hypertrophy an enlargement of the heart that is activated by both mechanical and hormonal stimuli and enables the heart to adapt to demands for increased cardiac output.
  • 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 of the individual cells.
  • non-myocytes On a cellular level, the heart is composed of myocytes and surrounding support cells, generically called non-myocytes. While non-myocytes are primarily fibroblast/mesenchymal cells, they also include endothelial and smooth muscle cells. Indeed, although myocytes make up most of the adult myocardial mass, they represent only about 30% of the total cell numbers present in heart. In response to hormonal, physiological, hemodynamic, and pathological stimuli, adult ventricular muscle cells can adapt to increased workloads through the activation of a hypertrophic process. This response is characterized by an increase in myocyte cell size and contractile protein content of individual cardiac muscle cells, without concomitant cell division and activation of embryonic genes, including the gene for atrial natriuretic peptide (ANP). Chien et l., FASEB J., 5: 3037-3046 (1991); Chien et al.,
  • 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.
  • LIF leukocyte inhibitory factor
  • CTL1 cardiotrophin-1
  • catecholamines catecholamines
  • adrenocorticosteroids angiotensin
  • prostaglandins prostaglandins
  • 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
  • beta-blockers e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, carvedilol, etc
  • verapamil have been used extensively in the treatment of hypertrophic cardiomyopathy
  • 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)
  • 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)
  • 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 are calcium antagonists, e g , nitrendipine, adrenergic receptor blocking agents, e g , those listed above
  • Endothelin is a vasoconstricting peptide comprising 21 amino acids, isolated from swine arterial endothelial culture supernatant and structurally determined. Yanagisawa et al., Nature. 332: 41 1 -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.
  • ACE angiotensin-converting enzyme
  • 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.
  • animal model data suggests that the ACE/Ang II pathway, while clearly involved in cardiac hypertrophy, is not the only, or even the primary pathway active in this role.
  • Mouse genetic "knockout" models have been made to test individual components of the pathway. In one such model, the primary cardiac receptor for Ang II, AT sub IA, has been genetically deleted; these mice do not develop hypertrophy when Ang II is given experimentally
  • 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
  • t-PA tissue plasminogen activator
  • 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)
  • FGF basic and acidic fibroblast growth factors
  • PD-ECGF platelet-derived endothelial cell growth factor
  • VEGF vascular endothelial growth factor
  • hVEGF human VEGF
  • hVEGF-related proteins The 121-am ⁇ no acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 1 16 and 159 in hVEGF
  • the 189-am ⁇ no acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 1 16 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF)
  • hVPF human vascular permeability factor
  • the 206-ammo acid protein differs from h VEGF by virtue of an insertion of 41 amino acids at residue 1 16 in hVEGF Houck et al
  • angiogenesis which involves the formation of new blood vessels from preexisting endothelium, is implicated in the pathogenesis of a variety of disorders These include solid tumors and metastasis, atherosclerosis, retrolental 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 Folkman etal .
  • proliferative retinopathies e g
  • diabetic retinopathy diabetic retinopathy
  • AMD age-related macular degeneration
  • neovascular glaucoma immune rejection of transplanted corneal tissue and other tissues
  • rheumatoid arthritis rheumatoid arthritis
  • Garner A "Vascular diseases” In Pathobiology of Ocular Disease A Dynamic Approach. Garner A , K ntworth GK, eds , 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710
  • angiogenesis appears to be crucial for the transition from hyperplasia to neoplasm, and for providing nourishment to the growing solid tumor Folkman et al . Nature, 339 58 (1989)
  • the neovascula ⁇ zation allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors Weidner et al , N Engl J Med, 324 1-6 (1991 ), Horak et al , Lancet, 340 1 120-1 124 (1992), Macchia ⁇ m et al , Lancet, 340 145-146 (1992)
  • the search for positive regulators of angiogenesis has yielded many candidates, including aFGF, bFGF, TGF- ⁇ , TGF- ⁇ , HGF, TNF- ⁇ , angiogenm, IL-8, etc Folkman et al ,
  • VEGF vascular endothelial cell proliferation
  • Ferrara et ⁇ l vascular permeability and angiogenesis
  • Ferrara et ⁇ l vascular permeability and angiogenesis
  • VEGF has been shown to be a key mediator of neovascula ⁇ zation associated with tumors and intraocular disorders Ferrara et ⁇ l , Endocr Rev .
  • 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 et al . Cancer Res , 53 4727-4735 (1993). Mattern e/ ⁇ / , Brit J Cancer, 73 931-934 (1996), Dvorak et al .
  • 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.
  • IGFBPs insulin-like growth factor binding proteins
  • IGF insulin-like growth factor
  • vascular endothelial cell growth and angiogenesis In view of the 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.
  • the present invention concerns compositions and methods for promoting or inhibiting angiogenesis and/or cardiovascuiarization in mammals.
  • the present invention is based on the identification of proteins that test positive in various cardiovascular assays that test promotion or inhibition of certain biological activities. Accordingly, the proteins are believed to be useful drugs for the diagnosis and/or treatment (including prevention) of disorders where such effects are desired, such as the promotion or inhibition of angiogenesis, inhibition or stimulation of vascular endothelial cell growth, stimulation of growth or proliferation of vascular endothelial cells, inhibition of tumor growth, inhibition of angiogenesis-dependent tissue growth, stimulation of angiogenesis-dependent tissue growth, inhibition of cardiac hypertrophy and stimulation of cardiac hypertrophy, e.g., for the treatment of congestive heart failure.
  • the present invention provides a composition comprising a PRO polypeptide in admixture with a pharmaceutically acceptable carrier.
  • the composition comprises a therapeutically effective amount of the polypeptide.
  • the composition comprises a further active ingredient, namely, a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent.
  • the composition is sterile.
  • the PRO polypeptide may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability. Preserved liquid pharmaceutical formulations might contain multiple doses of PRO polypeptide, and might, therefore, be suitable for repeated use.
  • 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 with a pharmaceutically acceptable carrier.
  • the present invention provides a composition comprising an agonist or antagonist of a PRO polypeptide in admixture with a pharmaceutically acceptable carrier.
  • the composition comprises a therapeutically effective amount of the agonist or antagonist.
  • the composition comprises a further active ingredient, namely, a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent.
  • the composition is sterile.
  • the PRO polypeptide agonist or antagonist may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability. Preserved liquid pharmaceutical formulations might contain multiple doses of a PRO polypeptide agonist or antagonist, and might, therefore, be suitable for repeated use.
  • 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 or antagonist with a pharmaceutically acceptable carrier.
  • the present invention concerns a composition
  • a composition comprising an anti-PRO antibody in admixture with a pharmaceutically acceptable carrier.
  • the composition comprises a therapeutically effective amount of the antibody.
  • the composition comprises a further active ingredient, namely, a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent.
  • the composition is sterile.
  • the composition may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability. Preserved liquid pharmaceutical formulations might contain multiple doses of the anti-PRO antibody, and might, therefore, be suitable for repeated use.
  • the antibody is a monoclonal antibody, an antibody fragment, a humanized antibody, or a single-chain antibody.
  • the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of an anti-PRO antibody with a pharmaceutically acceptable carrier.
  • the present invention provides an article of manufacture comprising:
  • composition of matter comprising a PRO polypeptide or agonist or antagonist thereof;
  • the present invention provides a method for identifying an agonist of a PRO polypeptide comprising:
  • the present invention provides a method for identifying an agonist of a PRO polypeptide comprising:
  • 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 of the
  • PRO polypeptide is inhibited.
  • either the test compound or the PRO polypeptide is immobilized on a solid support.
  • the non-immobilized component carries a detectable label. In a preferred aspect, this method comprises the steps of:
  • test compound (b) determining the induction of said cellular response to determine if the test compound is an effective antagonist.
  • this process comprises the steps of:
  • 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:
  • 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 of the present invention is directed to an agonist or an antagonist of a PRO polypeptide which may optionally be identified by the methods described above.
  • the invention provides an isolated antibody that binds a PRO polypeptide.
  • the antibody is a monoclonal antibody, which preferably has non-human complementarity-determining-region (CDR) residues and human framework-region (FR) residues.
  • CDR non-human complementarity-determining-region
  • FR human framework-region
  • the antibody may be labeled and may be immobilized on a solid support.
  • the antibody is an antibody fragment, a single-chain antibody, or a humanized antibody.
  • the antibody specifically binds to the polypeptide.
  • 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 of the presence of said disease or susceptibility to said disease.
  • the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises analyzing the level of expression of a gene encoding a 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 of the same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in said mammal.
  • 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.
  • 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.
  • 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 of the 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.
  • 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.
  • 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.
  • the invention provides a cardiovascular, endothelial or angiogenic disorder diagnostic kit comprising an anti-PRO antibody and a carrier in suitable packaging.
  • kit further comprises instructions for using said antibody to detect the presence of the PRO polypeptide.
  • the carrier is a buffer, for example.
  • the cardiovascular, endothelial or angiogenic disorder is cancer.
  • 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.
  • the disorder is cardiac hypertrophy, trauma such as wounds or burns, or a type of cancer.
  • 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.
  • the mammal is human, preferably one who is at risk of developing cardiac hypertrophy and more preferably has suffered myocardial infarction.
  • the cardiac hypertrophy is characterized by the presence of an elevated level of PGF 2 ⁇ .
  • the cardiac hypertrophy may be induced by myocardial infarction, wherein preferably the administration of the PRO polypeptide is initiated within 48 hours, more preferably within 24 hours, following myocardial infarction.
  • the cardiovascular, endothelial or angiogenicdisorder 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 pu ⁇ ose 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 thrombolytic agent is recombinant human tissue plasminogen activator.
  • 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.
  • 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.
  • the invention concerns a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of an agonist of a PRO polypeptide.
  • the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration.
  • the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the agonist.
  • the invention concerns a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of an antagonist of a PRO polypeptide.
  • the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration.
  • the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the antagonist.
  • the invention concerns a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of an anti-PRO antibody.
  • the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration.
  • the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the antibody.
  • 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.
  • the mammal is human.
  • the gene is administered via ex vivo gene therapy.
  • the gene is comprised within a vector, more preferably an adenoviral, adeno-associated viral, lentiviral, or retroviral vector.
  • the invention provides 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
  • the signal sequence is from a mammal, such as from a native PRO polypeptide
  • 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 of the polypeptide, wherein said producer cell packages the retroviral vector in association with the structural proteins to produce recombinant retroviral particles
  • 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-
  • 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
  • the mammal is human and the cardiac hypertrophy has been induced by myocardial infarction
  • 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
  • the mammal is human who suffers from congestive heart failure
  • the invention provides a method for inhibiting angiogenesis induced by a PRO polypeptide in a mammal comprising administering a therapeutically effective amount of an anti-PRO antibody to the mammal
  • the mammal is a human, and more preferably the mammal has a tumor or a retinal disorder
  • 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
  • the mammal is a human, and more preferably angiogeneisis would promote tissue regeneration or wound healing B.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide.
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence i ldueen ⁇ tuitiyy,, y yeeti mmo ⁇ rree prreefiecriaabDliyy a ati l iecaassti a abuouuuti 8 o7 /% vo s seeqquueennccee i ldueeniitiintyy,, y yeetu miiourree prreefieerraabDliyy a ati l leeaassti ab ⁇ oouuti 8 o8a%/o s seeq
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99%
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at
  • Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is eithertransmembrane 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 of the 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.
  • nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 80 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 1 10 nucleotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length, yet more preferably at least about 150 nucleotides in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucleo
  • 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 fragment(s) are novel. All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PRO polypeptide fragments encoded by these nucleotidemolecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody.
  • the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
  • the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about preferably at least about
  • the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81 % sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91 % sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity and yet more preferably at least about 99%
  • the invention concerns an isolated PRO polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81% positives, more preferably at least about 82% positives, yet more preferably at least about 83% positives, yet more preferably at least about 84% positives, yet more preferably at least about 85% positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89% positives, yet more preferably at least about 90% positives, yet more preferably at least about 91% positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98% positives and yet more preferably at least about 99% positives when
  • the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described Processes for producing 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 of the 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 for producing 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 of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
  • the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein
  • the agonist or antagonist is an anti-PRO antibody or a small molecule
  • the invention concerns 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
  • the PRO polypeptide is a native PRO polypeptide
  • the invention concerns 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
  • the carrier is a pharmaceutically acceptable carrier
  • Another embodiment of the 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
  • the invention provides vectors comprising DNA encoding any of the herein described polypeptides
  • Host cell comprising any such vector are also provided
  • the host cells may be CHO cells, E colt, yeast, or Baculovirus-infected insect cells
  • a process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
  • the invention provides chimeric molecules comprising any of the 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.
  • the invention provides an antibody which specifically binds to any of the above orbelow described polypeptides.
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
  • 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.
  • Figure 1 shows a nucleotide sequence (SEQ ID NO: l ) of a native sequence PRO230 cDNA, wherein SEQ ID NO: l is a clone designated herein as "DNA33223-1 136".
  • Figure 2 shows the amino acid sequence (SEQ ID NO:2) derived from the coding sequence of SEQ ID NO: 1 shown in Figure 1.
  • Figure 3 shows a nucleotide sequence (SEQ ID NO:6) of a native sequence PR0216 cDNA, wherein SEQ
  • ID NO:6 is a clone designated herein as "DNA33087”.
  • Figure 4 shows the amino acid sequence (SEQ ID NO:7) derived from the coding sequence of SEQ ID NO:6 shown in Figure 3.
  • Figure 5 shows a nucleotide sequence (SEQ ID NO: l 1) of a native sequence PRO302 cDNA, wherein SEQ ID NO: l l is a clone designated herein as "DNA40370-1217".
  • Figure 6 shows the amino acid sequence (SEQ ID NO: 12) derived from the coding sequence of SEQ ID NO: l 1 shown in Figure 5.
  • cardiovascular, endothelial and angiogenic disorder cardiac, endothelial and angiogenic disorder
  • cardiac, endothelial and angiogenic dysfunction cardiac, endothelial or angiogenic disorder
  • cardiovascular, endothelial or angiogenic disfunction are used interchangeably and refer in part to systemic disorders that affect vessels, such as diabetes mellitus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins, and/or lymphatics. This would include indications that stimulate angiogenesis and/or cardiovascuiarization, and those that inhibit angiogenesis and/or cardiovascuiarization.
  • 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
  • lymphangiosarcoma tumor angiogenesis
  • trauma such as wounds, burns, and other injured tissue
  • implant fixation scarring
  • ischemia reperfusion injury rheumatoid arthritis
  • cerebrovascular disease renal diseases such as acute renal failure, and osteoporosis.
  • renal diseases such as acute renal failure, and osteoporosis.
  • “Hypertrophy”, as used herein, is defined as an increase in mass of an organ or structure independent of natural growth that does not involve tumor formation. Hypertrophy of an organ or tissue is due either to an increase in the mass of the individual cells (true hypertrophy), or to an increase in the number of cells making up the tissue (hype ⁇ lasia), or both. Certain organs, such as the heart, lose the ability to divide shortly after birth. Accordingly, "cardiac hypertrophy” is defined as an increase in mass of the heart, which, in adults, is characterized by an increase in myocyte cell size and contractile protein content without concomitant cell division.
  • the character of the stress responsible for inciting the hypertrophy (e.g., increased preload, increased afterload, loss of myocytes, as in myocardial infarction, or primary depression of contractility), appears to play a critical role in determining the nature of the response.
  • the early stage of cardiac hypertrophy is usually characterized mo ⁇ hologically 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.
  • cardiac hypertrophy is used to include all stages of the progression of this condition, characterized by various degrees of structural damage of the heart muscle, regardless of the underlying cardiac disorder. Hence, the term also includes physiological conditions instrumental in the development 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.
  • CHF Congestive heart failure
  • Myocardial infarction generally results from atherosclerosis of the coronary arteries, often with superimposed coronary thrombosis. It may be divided into two major types: transmural infarcts, in which myocardial necrosis involves the full thickness of the ventricular wall, and subendocardial (nontransmural) infarcts, in which the necrosis involves the subendocardium, the intramural myocardium, or both, without extending all the way through the ventricular wall to the epicardium 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
  • hypotrophic cardiomyopathy Another complex cardiac disease associated with cardiac hypertrophy is "hypertrophic cardiomyopathy” This condition is characterized by a great diversity of mo ⁇ hologic, functional, and clinical features (Maron et al ,
  • 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 hype ⁇ lasia of medial smooth muscle are prominent features of this disorder It has been reported that molecular variants of the 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 of the cardiac valves Various diseases, like rheumatic fever, can cause the shrinking or pulling apart of the valve orifice, while other diseases may result in endocarditis, an inflammation of the endocardium or lining membrane of the at ⁇ o ventricular
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth
  • 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 and non-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, endomet ⁇ al carcinoma, salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms'
  • 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, py ⁇ midine analogs, 5-fluorourac ⁇ l, cisplatin, pu ⁇ ne nucleosides, amines, amino acids, triazol nucleosides, or corticosteroids
  • Specific examples include Ad ⁇ amycin, Doxorubicin, 5-Fluorourac ⁇ l, Cytos e arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Tenipos
  • Esperamicins see U S Pat No 4,675,187
  • Melphalan and other related nitrogen mustards
  • hormonal agents that act to regulate or inhibit hormone action on tumors, such as tamoxifen and onap ⁇ stone
  • a “growth-inhibitory agent” when used herein refers to a compound or composition that inhibits growth of a cell, such as an Wnt-overexpressing cancer cell, either in vitro or in vivo
  • the growth-inhibitory agent is one which significantly reduces the percentage of malignant cells in S phase
  • growth-inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G 1 arrest and M-phase arrest
  • Classical M-phase blockers include the vincas (vinc ⁇ stine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, daunorubicin, etoposide, and bleomycin
  • Those agents that arrest Gl also spill over mto S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorourac ⁇ l, and
  • tumor necrosis factor an antibody capable of inhibiting or neutralizing the angiogenic activity of acidic or basic FGF or hepatocyte growth factor (HGF), an antibody capable of inhibiting or neutralizing the coagulant activities of tissue factor, protein C, or protein S (see, WO 91/01753, published 21 February 1991), or an antibody capable of binding to HER2 receptor (WO 89/06692), such as the 4D5 antibody (and functional equivalents thereof) (e g , WO 92/22653)
  • TNF tumor necrosis factor
  • HGF hepatocyte growth factor
  • 4D5 antibody and functional equivalents thereof
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a cardiovascular, endothelial, and angiogenic disorder
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a cardiovascular, endothelial, and angiogenic disorder such as hypertrophy
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented
  • the disorder may result from any cause, including ldiopathic, cardiotrophic, or myotrophic causes, or ischemia or lschemic insults, such as myocardial infarction
  • Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial effect, such as an anti-hypertrophic effect, for an extended period of time
  • “Mammal” for pu ⁇ oses 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
  • the mammal is human
  • Administration in combination with one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order
  • cardiovascular agents refers gene ⁇ cally to any drug that acts in treating cardiovascular, endothelial, and angiogenic disorders
  • 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
  • ang ⁇ otens ⁇ n-II receptor antagonists include ang ⁇ otens ⁇ n-II receptor antagonists, endothelin receptor antagonists such as, for example, BOSENTANTM and MOXONODINTM, interferon-gamma (IFN- ⁇ ), des-aspartate-angiotens 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 T103N, Nl 17Q, KHRR(296-299)AAAA t-
  • inotropic or hypertensive agents such as digoxigenin and ⁇ -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., chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlo ⁇ henamide, acetazolamide,
  • ACE angiotensin converting enzyme
  • Angiogenic agents and “endothelial agents” are active agents that promote angiogenesis and/or endothelial cell growth, or, if applicable, vasculogenesis. This would include factors that accelerate wound healing, such as growth hormone, insulin-like growth factor-I (IGF-I), VEGF, VIGF, PDGF, epidermal growth factor (EGF), CTGF and members of its family, FGF, and TGF- ⁇ and TGF- ⁇ .
  • IGF-I insulin-like growth factor-I
  • VEGF VEGF
  • VIGF vascular endothelial growth
  • PDGF epidermal growth factor
  • CTGF epidermal growth factor
  • 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-CD20, and other bioactive and organic chemical agents.
  • 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-CD20, and other bioactive and organic chemical agents.
  • a "therapeutically effective amount" of an active agent such as a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto or an anti- PRO230, anti-PR0216 or anti-PRO302 antibody, refers to an amount effective in the treatment of a cardiovascular, endothelial or angiogenic disorder in a mammal and can be determined empirically.
  • an "effective amount" of an active agent such as a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto or an anti-PRO230, anti-PR0216 or anti-PRO302 antibody, refers to an amount effective for carrying out a stated pu ⁇ ose, wherein such amounts may be determined empirically for the desired effect.
  • 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.
  • 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 of the 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.
  • 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 “ECD” refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have less than 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein.
  • 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 contemplated by the present invention.
  • 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 of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
  • PRO230 variant polypeptide means an active PRO230 polypeptide (other than a native sequence PRO230 polypeptide) as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 17 to
  • PR0216 variant polypeptide means an active PR0216 polypeptide (other than a native sequence PR0216 polypeptide) as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO:7) or (b) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7).
  • PRO302 variant polypeptide means an active PRO302 polypeptide (other than a native sequence PRO302 polypeptide) as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12), (b) X to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12), wherein X is any amino acid residue from 21 to 30 of Figure 6 (SEQ ID NO: 12) or (c) another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO: 12).
  • PRO230, PR0216 or PRO302 variant polypeptides include, for instance, PRO230, PR0216 or PRO302 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C-terminus, as well as within one or more internal domains, of the sequence of Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7) or Figure 6 (SEQ ID NO: 12), respectively.
  • a PRO230 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about
  • amino acid sequence identity with (a) residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 17 to 26 of Figure 2 (SEQ ID NO:2), or (c) another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO:2).
  • a PR0216 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid
  • a PRO302 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid
  • PRO230, PR0216 and PRO302 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 300 amino acids in length, or more.
  • Table 1 provides the complete source code forthe 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.
  • Tables 2A-2D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) 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.
  • filel and file2 are two dna or two protein sequences.
  • 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
  • the program may create a tmp file in /tmp to hold info about traceback.
  • static nm matches in core — for checking */ static Imax; /* lengths of stripped file names */ static ⁇ j[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static n ⁇ [2]; /* current elem number — for gapping */ static s ⁇ z[2], static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char out[2][P_LINE], /* output line */ static char star[P_LINE], /* set by stars() *//
  • */ *po[ ⁇ ] *ps[ ⁇ ]; if ( ⁇ slower(*ps[ ⁇ ]))
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]); po[ ⁇ ]+ + ; ps[ ⁇ ] + + ,
  • *py+ + *px; else if ( ⁇ slower(*px))
  • Percent (%) amino acid sequence identity with respect to the PRO230, PR0216 and PRO302 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO230, PR0216 or PRO302 sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for 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.
  • ALIGN-2 sequence comparison computer program
  • Table 1 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.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • % 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.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • a % ammo acid sequence identity value is determined by dividing (a) the number of matching identical ammo acids residues between the ammo acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (; e , the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptid
  • PRO230 variant polynucleotide or "PRO230 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PRO230 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes amino acids X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any amino acid residue from 17 to 26 of Figure 2 (SEQ ID NO 2), or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2)
  • a PRO230 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity
  • PR0216 variant polynucleotide or "PR0216 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PR0216 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO:7) or (b) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7).
  • a PR0216 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91 % nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence
  • PR0216 polynucleotide variants do not encompass the native PR0216 nucleotide sequence.
  • PRO302 variant polynucleotide or "PRO302 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PRO302 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12), (b) a nucleic acid sequence which encodes amino acids X to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12), wherein X is any amino acid residue from 21 to 30 of Figure 6 (SEQ ID NO: 12), or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO: 12).
  • a PRO302 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91 % nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence
  • PRO302 polynucleotide variants do not encompass the native PRO302 nucleotide sequence.
  • PRO230, PR0216 and PRO302 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more.
  • Percent (%) nucleic acid sequence identity with respect to the PRO230, PR0216 and PRO302 polypeptide- encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO230, PR0216 or PRO302 polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for 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 (DN ASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For 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, Ine , 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, Ine , South San Francisco, California or may be compiled from
  • % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows
  • % 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))
  • NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov
  • % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
  • a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide- encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide- encoding nucleic acid and the comparison nucleic acid molecule of interest (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 of the PRO polypeptide- encoding nucleic acid molecule of interest.
  • nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.
  • PRO230, PR0216 and PRO302 variant polynucleotides are nucleic acid molecules that encode an active PRO230, PR0216 or PRO302 polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO230, PR0216 or PRO302 polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO: 12), respectively.
  • PRO230, PR0216 and PRO302 variant polypeptides may be those that are encoded by a PRO230, PR0216 or PRO302 variant polynucleotide.
  • amino acid residues in the sequences compared that are not only identical, but also those that have similar properties are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) of the amino acid residue of interest.
  • the % value of positives of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y
  • isolated when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified (1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO230, PR0216 or PRO302 natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • An "isolated" nucleic acid molecule encoding a PRO230, PR0216 or PRO302 polypeptide or an "isolated” nucleic acid molecule encoding an anti-PRO230, anti-PR0216 or anti-PRO302 antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PRO230-, PR0216- or PRO302-encoding nucleic acid or the natural source of the anti-PRO230-, anti-PR0216- or anti-PRO302-encoding nucleic acid.
  • the isolated nucleic acid is free of association with all components with which it is naturally associated.
  • An isolated PRO230-, PR0216- or PRO302-encoding nucleic acid molecule or an isolated anti-PRO230-, anti-PR0216- or anti-PRO302-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 PRO230-, PR0216- or PRO302-encoding nucleic acid molecule or from the anti-PRO230-, anti-PR0216- or anti-PRO302-encoding nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule encoding a PRO230, PR0216 or PRO302 polypeptide or an isolated nucleic acid molecule encoding an anti-PRO230, anti-PR0216 or anti-PRO302 antibody includes PRO230-, PR0216- or PRO302-nucleic acid molecules or anti-PRO230-, anti-PR0216- or anti-PRO302-nucleic acid molecules contained in cells that ordinarily express PRO230, PR0216 or PRO302 polypeptides or anti- PRO230, anti-PR0216 or anti-PRO302 antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • 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 include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a PRO230, PR0216 or PRO302 polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • “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 environment 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 ⁇ g/ml), 0.1% SDS, and
  • Moderately-stringent conditions may be identified as described by Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Press, 1989), and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength, and % SDS) less stringent than those described above.
  • washing solution and hybridization conditions e.g., temperature, ionic strength, and % SDS
  • moderately stringent conditions is overnight incubation at 37 °C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 M 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 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.
  • epitope-tagged when used herein refers to a chimeric polypeptide comprising a PRO230, PR0216 or PRO302 polypeptide fused to a "tag polypeptide".
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused.
  • the tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
  • Active or “activity” in the context of PRO230, PR0216 or PRO302 variants refers to form(s) of PRO230, PR0216 or PRO302 proteins that retain the biologic and/or immunologic activities of a native or naturally- occurring PRO230, PR0216 or PRO302 polypeptide.
  • Bioactivity 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 polypeptides 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 of the arteries, capillaries, veins, and/or lymphatics, and cancer.
  • Antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes one or more of the biological activities of a native 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 other retinopathies, retrolental fibroplasia, age- related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation.
  • tumors and especially solid malignant tumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age- related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue
  • the antagonists also are useful for the treatment of diseases or disorders characterized by undesirable excessive vascular permeability, such as edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardial effusion (such as that associated with pericarditis), and pleural effusion.
  • vascular permeability such as edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardial effusion (such as that associated with pericarditis), and pleural effusion.
  • 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.
  • PRO polypeptide receptor 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 are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • 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 immunoglobulins” 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 light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody to and for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or 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).
  • CDRs complementarity-determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -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, abat et al, NIH Publ. No.91-3242, Vol. 1, pages 647-669 (1991 ).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies (Zapata etal. , 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 V H -V L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH I ) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the 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 (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM; and several of these may be further divided into subclasses (isotypes), e.g., IgG 1 , IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , e, ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, / ' . e. , the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the 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.
  • 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. Patent No.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 (immunoglobulins) 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 of the cham(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
  • chimeric antibodies immunoglobulins 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 of the cham(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
  • Humanized forms of non-human (e g , murine) antibodies are chimenc immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-bindmg subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity
  • Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues
  • humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences
  • Single-chain Fv or “sFv” antibody fragments comprise the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain
  • the F v polypeptide further comprises a polypeptide linker between the V H and V L domains that enables the sFv to form the desired structure for antigen binding
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H - V L )
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites
  • Diabodies are described more fully in, for example, EP 404,097, WO 93/1 1 161 , and Holhnger ef ⁇ /, 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
  • 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
  • label when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled” antibody
  • the label may be detectable by itself (e g , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable
  • Radionuclides that can serve as detectable labels include, for example, 1-131, 1-123, 1-125, Y-90, Re- 188, At-21 1 , Cu-67, B ⁇ -212, and Pd- 109
  • the label may also be a non- detectable entity such as a toxin
  • solid phase is meant a non-aqueous matrix to which an antibody of the present invention can adhere
  • solid phases encompassed herein include those formed partially or entirely of glass (e g controlled pore glass), polysaccha ⁇ des (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and si cones
  • the solid phase can comprise the well of an assay plate, m 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 "hposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant that is useful for delivery of a drug (such as the PRO230, PR0216 or PRO302 polypeptide or antibodies thereto disclosed herein) to a mammal
  • a drug such as the PRO230, PR0216 or PRO302 polypeptide or antibodies thereto disclosed herein.
  • the components of the hposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes
  • the term "lmmunoadhesin” designates antibody-like molecules that combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity that is other than the antigen recognition and binding site of an antibody (/ e , is “heterologous"), and an immunoglobulin constant domain sequence
  • the adhesin part of an lmmunoadhesin molecule typically is a contiguous am o acid sequence comprising at least the binding site of a receptor or a ligand
  • the immunoglobulin constant domain sequence in the lmmunoadhesin may be obtained from any immunoglobulin, such as IgG-1 , IgG- 2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA -2), IgE, IgD
  • PRO230, PRQ216 and PRO302 Variants In addition to the full-length native sequence PRO230, PR0216 and PRO302 polypeptides described herein, it is contemplated that PRO230, PR0216 and PRO302 variants can be prepared PRO230, PR0216 and PRO302 variants can be prepared by introducing appropriate nucleotide changes into the PRO230, PR0216 or PRO302 DNA, and/or by synthesis of the desired PRO230, PR0216 or PRO302 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO230, PR0216 or PRO302, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
  • Variations in the native full-length sequence PRO230, PR0216 or PRO302 or in various domains of the PRO230, PR0216 or PRO302 described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U S Patent No 5,364,934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO230, PR0216 or PRO302 that results in a change in the amino acid sequence of the PRO230, PR0216 or PRO302 as compared with the native sequence PRO230, PR0216 or PRO302
  • the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO230, PR0216 or PRO302
  • Guidance in determining which ammo acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO230, PR0216 or PRO302 with that of homologous known protein molecules and minimizing
  • conservative substitutions of interest are shown in Table 3 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 3, or as further described below in reference to amino acid classes, are introduced and the products screened
  • Leu (L) norleucine lie, val, met, ala, phe lie
  • Substantial modifications in function or immunological identity of the PRO230, PR0216 or PRO302 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • 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 et al, Philos. Trans. R. Soc. London SerA, 317:415 ( 1986)] or other known techniques can be performed on the cloned DNA to produce the PRO230, PR0216 or PRO302 variant DNA.
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • amino acids include alanine, glycine, serine, and cysteine.
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)].
  • Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed 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.
  • Covalent modifications of PRO230, PR0216 and PRO302 are included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of a PRO230, PR0216 or PRO302 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO230, PR0216 or PRO302.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking PRO230, PR0216 or PRO302 to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO230, anti-PR0216 or anti-PRO302 antibodies, and vice-versa.
  • crosslinkingagents include, e.g., l ,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicy lie acid, homobifunctional imidoesters, includingdisuccinimidy 1 esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l ,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
  • esters with 4-azidosalicy lie acid
  • homobifunctional imidoesters includingdisuccinimidy 1 esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l ,8-oc
  • Another type of covalent modification of the PRO230, PR0216 or PRO302 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
  • "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO230, PR0216 or PRO302 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO230, PR0216 or PRO302.
  • the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
  • Addition of glycosylation sites to the PRO230, PR0216 or PRO302 polypeptide may be accomplished by altering the amino acid sequence.
  • the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO230, PR0216 or PRO302 (for O-linked glycosylation sites).
  • the PRO230, PR0216 or PRO302 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO230, PR0216 or PRO302 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the PRO230, PR0216 or PRO302 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 1 1 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
  • Removal of carbohydrate moieties present on the PRO230, PR0216 or PRO302 polypeptide may be accomplished chemically or enzymatically or by mutational substitutionof 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. Biophys., 259:52 ( 1987) and by Edge et al., Anal. Biochem., 1 18: 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, Meth. Enzymol.. 138:350 (1987).
  • Another type of covalent modification of PRO230, PR0216 or PRO302 comprises linking the PRO230, PR0216 or PRO302 polypeptide to one of a variety of 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.
  • PEG polyethylene glycol
  • PRO302 polypropylene glycol
  • polyoxyalkylenes polyoxyalkylenes
  • PRO230, PR0216 or PRO302 of the present invention may also be modified in a way to form a chimeric molecule comprising PRO230, PR0216 or PRO302 fused to another, heterologous polypeptide or amino acid sequence.
  • such a chimeric molecule comprises a fusion of the PRO230, PR0216 or PRO302 with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO230, PR0216 or PRO302. The presence of such epitope-tagged forms of the PRO230, PR0216 or PRO302 can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO230, PR0216 or PRO302 to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • 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 9E 10 antibodies thereto [Evan et al, Molecular and Cellular Biology.
  • tag polypeptides include the Flag-peptide [Hopp et al, BioTechnology. 6: 1204- 1210 ( 1988)]; the K.T3 epitope peptide [Martin et al, Science. 255: 192-194 (1992)]; an ⁇ -tubulin epitope peptide [Skinner et al, J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al. Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
  • the chimeric molecule may comprise a fusion of the PRO230, PR0216 or PRO302 with an immunoglobulin or a particular region of an immunoglobulin.
  • an immunoglobulin or a particular region of an immunoglobulin.
  • a bivalent form of the chimeric molecule also referred to as an "immunoadhesin”
  • Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO230, PR0216 or PRO302 polypeptide in place of at least one variable region within an Ig molecule.
  • the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI , CH2 and CH3 regions of an IgG l molecule.
  • the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI , CH2 and CH3 regions of an IgG l molecule.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO230, PR0216 or PRO302.
  • cDNAs encoding PRO230, PR0216 or PRO302 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below.
  • PRO230, PR0216 or PRO302 proteins produced in separate expression rounds may be given different PRO numbers but the U Q number is unique for any given DNA and the encoded protein, and will not be changed However, for sake of simplicity, in the present specification the protein encoded by DNA33223-1 136, DNA33087-1 158 or DNA40370-1217, as well as all further native homologues and variants included in the foregoing definition of PRO230, PR0216 or PRO302, will be referred to as "PRO230, PR0216 or PRO302", respectively, regardless of their origin or mode of preparation
  • PRO230, PR0216 or PRO302 polypeptides by culturing cells transformed or transfected with a vector containing nucleic acid encoding PRO230, PR0216 or PRO302 polypeptides It is, of course, contemplated that alternative methods that are well known in the art may be employed to prepare PRO230, PR0216 or PRO302
  • the PRO230, PR0216 or PRO302 polypeptide sequence, or portions thereof may be produced by direct peptide synthesis using solid-phase techniques See, e g , Stewart et al , Solid-Phase Peptide Synthesis (W H Freeman Co San Francisco, CA, 1969), Merrifield, J Am Chem Soc , 85 2149-2154 (1963)
  • In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, with an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions
  • DNA encoding PRO230, PR0216 or PRO302 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA encoding PRO230, PR0216 or PRO302 and to express it at a detectable level Accordingly, DNAs encoding human PRO230, PR0216 or PRO302 can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in the Examples The gene encoding PRO230, PR0216 or PRO302 polypeptide may also be obtained from a genomic library or by oligonucleotide synthesis
  • Probes such as antibodies to the PRO230, PR0216 or PRO302 polypeptide or oligonucleotides of at least about 20-80 bases
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , supra
  • An alternative means to isolate the gene encoding PRO230, PR0216 or PRO302 is to use PCR methodology Sambrook et al , supra, Dieffenbach et al , PCR Primer A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1995)
  • the 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 radiolabels like 32 P-labeled ATP, biotinylation, or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supra
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases
  • Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as 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 mRN A that may not have been reverse-transcribed into cDN A
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO230, PR0216 or PRO302 production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences
  • the culture conditions such as media, temperature, pH, and the like, can be selected by the skilled artisan without undue experimentation
  • 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
  • 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, eubacte ⁇ a, such as Gram-negative or Gram- positive organisms, for example, Enterobacte ⁇ aceae such as E coli
  • E coli strains are publicly available, such as E coli K12 strain MM294 (ATCC 31 ,446), E coli XI 776 (ATCC 31 ,537), E coli strain W31 10 (ATCC 27,325), and K5 772 (ATCC 53,635)
  • Other suitable prokaryotic host cells include Enterobacte ⁇ aceae such as Eschenchia, e g , E coli, Enterobacter, Erwima, Klebstella, Proteus, Salmonella, e g , Salmonella typhtmurium, Serratta, e g
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding PRO230, PR0216 or PRO302 Saccharomyces cerevistae is a commonly used lower eukaryotichostmicroorganism Others include Schizosaccharomvces pombe (Beach and Nurse, Nature, 290 140 [1981 ], EP 139,383 published 2 May 1985), Kluyveromyces hosts (U S Patent No 4,943,529, Fleer et al , Bio/Technology, 9 968-975 (1991 )) such as, e g , K lactis (MW98-8C, CBS683, CBS4574, Louvencourt et al , J Bacte ⁇ ol .
  • Schizosaccharomvces pombe Beach and Nurse, Nature, 290 140 [1981 ], EP 139,383 published 2 May 1985
  • Kluyveromyces hosts U S Patent No 4,943,529, Fleer
  • K frasilts (ATCC 12,424) K bulgartcus (ATCC 16,045), K wickeramu (ATCC 24, 178), K waltu (ATCC 56,500), K drosophilarum (ATCC 36,906, Van den Berg et al , Bio/Technology, 8 135 (1990)), K thermotolerans, and K marxianus, yarrowia (EP 402,226), P ichia pastoris (EP 183,070, Sreek ⁇ shna et al , J Basic Microbiol .
  • Candida Trtchoderma reesia EP 244,234
  • Neurospora crassa Case et al , Proc Natl Acad Sci USA, 76 5259-5263 [1979]
  • Schwanniomyces such as Schwanniomyces occidentals (EP 394,538 published 31 October 1990)
  • filamentousfungi such as, e g , Neurospora Penicitlium, Tolypocladium (WO 91/00357 published 10 January 1991 ), and Aspergillus hosts such as A mdulans (Ballance et al , Biochem Biophvs Res Commun , 1 12 284-289 [1983], Tilburn et al , Gene, 26 205-221 [ 1983] Yelton et al , Proc Natl Acad Sci USA.
  • 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, Ptchia Saccharomyces Torulopsts, and Rhodotorula A list of specific species that are exemplary of this class of yeasts may be found in C Anthony, The Biochemistry of Methylotrophs. 269 ( 1982)
  • Suitable host cells for the expression of nucleic acid encoding glycosylated PRO230, PR0216 or PRO302 are derived from multicellular organisms
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al , J Gen Virol , 36 59 (1977)), Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasm, Proc Natl Acad Sci USA, 77 4216 ( 1980)), mouse sertoli cells (TM4, Mather, Biol Reprod .
  • the nucleic acid (e.g. , cDNA or genomic DNA) encoding PRO230, PR0216 or PRO302 may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression.
  • a replicable vector for cloning (amplification of the DNA) or for expression.
  • Various vectors are publicly available.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction 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 PRO230, PR0216 or PRO302 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal sequence may be a component of the vector, or it may be a part of the DNA encoding PRO230, PR0216 or PRO302 that is inserted into the vector.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • the signal sequence may be, e.g. , the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces ⁇ -factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362, 179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990.
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells.
  • Selection genes will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or 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.
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the nucleic acid encoding PRO230, PR0216 or PRO302, such as DHFR or thymidine kinase.
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al, Proc. Natl. Acad. Sci. USA. 77: 4216 (1980).
  • a suitable selection gene for use in yeast is the trp] gene present in the yeast plasmid YRp7. Stinchcomb et al.
  • Ihe trp ⁇ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4- 1. Jones, Genetics, 85: 12 (1977).
  • Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding PRO230, PR0216 or PRO302 to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems (Chang et al, Nature, 275: 615 ( 1978); Goeddel et al, Nature, 281 : 544 (1979)), alkaline phosphatase, a tryptophan (t ⁇ ) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP 36,776), and hybrid promoters such as the tac promoter.
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO230, PR0216 or PRO302.
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase (Hitzeman et al, J. Biol. Chem.. 255: 2073 ( 1980)) or other glycolytic enzymes (Hess et al, J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry.
  • J7: 4900 ( 1978) such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • enolase such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • yeast promoters that are inducible promoters having the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • PRO230, PR0216 or PRO302 nucleic acid transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1 ,504 published 5 July 1 89), 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.
  • viruses such as polyoma virus, fowlpox virus (UK 2,21 1 ,504 published 5 July 1 89), adenovirus (such as Adenovirus 2), bovine papilloma virus, avi
  • 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, -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the sequence coding for PRO230, PR0216 or PRO302, but is preferably located at a site 5' from the promoter
  • Expression vectors used in eukaryotic host cells 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 PRO230, PR0216 or PRO302
  • 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 the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected
  • Gene expression may be measured by immunological methods, such as lmmunohistochemical 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 lmmunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native-sequence PRO230, PR0216 or PRO302 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA encoding PRO230, PR0216 or PRO302 and encoding a specific antibody epitope
  • PRO230, PR0216 or PRO302 polypeptides may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e g TRITON-XTM 100) or by enzymatic cleavage Cells employed in expression of nucleic acid encoding the PRO230, PR0216 or PRO302 polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell-lysing agents
  • PRO230, PR0216 or PRO302 polypeptide may be desired to purify the PRO230, PR0216 or PRO302 polypeptide from recombinant cell proteins or polypeptides
  • the following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation- exchange resin such as DEAE, chromatofocus ⁇ ng,SDS-PAGE,ammon ⁇ um sulfate precipitation, gel filtration using, for example, Sephadex G-75, protein A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope-tagged forms of the PRO230, PR0216 or PRO302 polypeptide
  • Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990), Scopes, Protein Purification Principles and Practice (Springer- Verlag New York, 1982)
  • PRQ216 or PRO302 polypeptides l Assays for Cardiovascular, Endothelial, and Angiogenic Activity
  • Assays for testing for endothelin antagonist activity include a rat heart ventricle binding assay where the polypeptide is tested for its ability to inhibit lodinized 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 intra-cellular levels of second messengers, an arachidomc acid release assay that measuresthe ability of added compounds to reduce endothelin-stimulated arachidomc 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. Ma ⁇ bach, HI and Rovee, DT, eds (Year Book Medical Publishers, Ine , Chicago), pp 71-1 12, as modified by the article of Eaglstein and Mertz, J Invest Dermatol , 7 I_ 382-384 ( 1978)
  • An assay to screen for a test molecule relating to a PRO230, PR0216 or PRO302 polypeptide that binds an endothelin B , (ETB , ) receptor polypeptide and modulates signal transduction activity involves providing a host cell transformed with a DNA encoding endothelin B, 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
  • n vitro assays include induction of spreading of adult rat cardiac myocytes
  • ventricular myocytes are isolated from a single (male Sprague-Dawley) rat, essentially following a modification of the procedure described in detail by Piper et al , "Adult ventricular rat heart muscle cells" in Cell Culture Techniques in Heart and Vessel Research, H M Piper, ed (Berlin Springer- Verlag, 1990), pp 36-60
  • This procedure permits the isolation of adult ventricular myocytes and the long-term culture of these cells in the rod-shaped phenotype Phenyleph ⁇ ne and Prostaglandin F 2 ⁇ (PGF 2 ⁇ ) have been shown to induce a spreading response in these adult cells
  • PGF 2 ⁇ or PGF 2 ⁇ analogs e g fluprostenol
  • phenyleph ⁇ ne by various potential inhibitors of cardiac hypertrophy
  • an in vivo assay is a test for inhibiting cardiac hypertrophy induced by fluprostenol in vivo
  • This pharmacological model tests the ability of the 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 PGF 2 bother)
  • rats with pathologic cardiac hypertrophy induced by myocardial infarction have chronically elevated levels of extractable PGF 2 ⁇ in their myocardium Lai et al , Am J Physiol (Heart Circ Physiol ), 271 H2197- H2208 (1996)
  • factors that can inhibit the effects of fluprostenol on myocardial growth in vivo are potentially useful for treating cardiac hypertrophy
  • the effects of the PRO polypeptide on cardiac hypertrophy are determined by measuring the weight of heart, ventricles, and left ventricle (normalized by body weight) relative to fluprostenol-treated rats
  • an in vivo assay is the pressure-overload cardiac hypertrophy assay
  • rats e g , male Wistar or Sprague-Dawley
  • the abdominal aorta of each rat is narrowed down just below the diaphragm Beznak M , Can J Biochem Physiol .
  • Animal models of tumors and cancers 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 , subcutaneous injection, tail vein injection, spleen implantation, intrapentoneai 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 ,
  • the cells introduced into such animals can be derived from known tumor/cancer cell lines, such as any of the above-listed tumor cell lines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene), r s-transfected NIH-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 trochar, or as cell suspensions
  • tumor tissue fragments of suitable size are introduced into the s c space
  • Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously Tumor cells can also be injected as subdermal implants In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s c tissue
  • Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogene was initially isolated), or rtew-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al Proc Nat Acad Sci USA, 83 9129-9133 (1986)
  • animal models of colon cancer can be generated by passaging colon cancer cells in animals, e g , nude mice, leading to the appearance of tumors in these animals
  • An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang e/ ⁇ / , 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”TM sold by AntiCancer, Ine , (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
  • 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 the anti-tumor activities of various agents Palladmo 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 l OxlO 6 to 10xl0 7 cells/ml The animals are then infected subcutaneously with 10 to 100 l 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 vestigational tumor model Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients
  • 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 of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula However, the measurement of tumor size is very inaccurate The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay Another important variable in the description of tumor growth is the tumor volume doubling time Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc 6th Int Workshop on Immune-Deficient Animals.
  • recombinant (transgenic) animal models can be engineered by introducing the coding portion of the PRO230, PR0216 or PRO302 genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals
  • Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g baboons, chimpanzees and monkeys
  • Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (U S Patent No 4,873, 191 ), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al , Proc Natl Acad Sci USA.
  • 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, the technique of Lasko et al , Proc Natl Acad Sci USA, 89 6232-636 ( 1992)
  • transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or lmmunocytochemistry The animals are further examined for signs of tumor or cancer development Alternatively, "knock-out" animals can be constructed that have a defective or altered gene encoding a PRO230, PR0216 or PRO302 polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the PRO230, PR0216 or PRO302 polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal.
  • cDNA encoding a particular PRO230, PR0216 or PRO302 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques.
  • a portion of the genomic DNA encoding a particular PRO230, PR0216 or PRO302 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration.
  • another gene such as a gene encoding a selectable marker that can be used to monitor integration.
  • several kilobases of unaltered flanking DNA are included in the vector. See, e.g., Thomas and Capecchi, Cell, 5 L 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. 1 13-152.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock-out" animal.
  • Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • Knockout animals can be characterized, for instance, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to absence of the PRO230, PR0216 or PRO302 polypeptide.
  • SCC feline oral squamous cell carcinoma
  • Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor.
  • These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor.
  • each cat 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.
  • CT computed tomography
  • Each cat is treated repeatedly, over a longer period of time. Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck.
  • 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.
  • fibrosarcoma adenocarcinoma
  • lymphoma chondroma
  • leiomyosarcoma of dogs, cats, and baboons
  • mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans.
  • the use of this model is limited by the rare occurrence of this type of tumor in animals.
  • cardiovascular, endothelial, and angiogenic assays herein can be verified by further studies, such as by determining mRNA expression in various human tissues.
  • 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 (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • 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 may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
  • Antibodies useful for immunohistochemical staining and/orassay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native-sequence PRO230, PR0216 or PRO302 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO230, PR0216 or PRO302 DNA and encoding a specific antibody epitope.
  • General techniques for generating antibodies, and special protocols for in situ hybridization are provided hereinbelow.
  • the results of the cardiovascular, endothelial, and angiogenic study can be further verified by antibodybinding studies, in which the ability of anti-PRO230, anti-PR0216 or anti-PRO302 antibodies to inhibit the effect of the PRO230, PR0216 or PRO302 polypeptides on endothelial cells or other cells used in the cardiovascular, endothelial, and angiogenic assays is tested.
  • Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow.
  • Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques (CRC Press, Inc., 1987), pp.147-158.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected
  • 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
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobu n antibody that is labeled with a detectable moiety (indirect sandwich assay)
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme
  • the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example
  • 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 PRO230, PR0216 or PRO302 polypeptide herein
  • Such cells include, for example, those set forth in the Examples below
  • suitable tumor cells include, for example, stable tumor cells lines such as the B104- 1- 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and r ⁇ s-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumo ⁇ genic growth
  • stable tumor cells lines such as the B104- 1- 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and r ⁇ s-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumo ⁇ genic growth
  • Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumo ⁇ genic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC) Cells transfected with the coding sequences of the genes identified here
  • ADCC antibody-
  • PRO230, PR0216 or PRO302 polypeptide 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
  • nucleic acid (optionally contained in a vector) into the patient's cells in vivo and ex vivo
  • the nucleic acid is injected directly into the patient, usually at the sites where the PRO230, PR0216 or PRO302 polypeptide is required, / e , the site of synthesis of the PRO230, PR0216 or PRO302 polypeptide, if known, and the site (e g , wound) where biological activity of PRO230, PR0216 or PRO302 polypeptide is needed
  • 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 withm porous membranes that are implanted into the patient (see, e g , U S Pat Nos 4,892,538 and 5,283,187)
  • techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred
  • the currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral vectors (such as adenovirus, lentivirus, He ⁇ es simplex I virus, or adeno-associated virus (AAV)) and lipid-based systems (useful lipids for hpid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi, see, e g , Tonkmson et al , Cancer Investigation, 14(1) 54-65 (1996))
  • the most preferred vectors for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses
  • a viral vector such as a retroviral vector includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger
  • a viral vector such as a retroviral vector includes a
  • the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc
  • 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)
  • Anderson et al Science, 256 808-813 (1992) See also WO
  • This invention is also related to the use of the gene encoding the PRO230, PR0216 or PRO302 polypeptide as a diagnostic Detection of a mutated form of the PRO230, PR0216 or PRO302 polypeptide will allow a diagnosis of a cardiovascular, endothelial, and angiogenic disease or a susceptibility to a card ⁇ ovascular,endothel ⁇ al, and angiogenic disease, such as a tumor, since mutations in the PRO230, PR0216 or PRO302 polypeptide may cause tumors
  • Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy, and autopsy material
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al , Nature, 324 163-166 (1986)) prior to analysis RNA or cDNA may also be used for the same pu ⁇ ose
  • PCR primers complementary to the nucleic acid encoding the PRO230, PR0216 or PRO302 polypeptide can be used to identify and analyze PRO230, PR0216 or PRO302 polypeptide mutations
  • deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA encoding the PRO230, PR0216
  • 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 el al , Proc Natl Acad Sci USA, 85 4397-4401 (1985)
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes, e g , restriction fragment length polymo ⁇ hisms (RFLP), and Southern blotting of genomic DNA
  • mutations can also be detected by in situ analysis
  • nucleic acid encoding the PRO polypeptide may be linked to vascular disease or neovascula ⁇ zation 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 neovascula ⁇ zation associated with tumor formation, since an altered level of this PRO polypeptide may be indicative of such disorders
  • sequences of the present invention are also valuable for chromosome identification
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome Moreover, there is a current need for identifying particular sites on the chromosome Few chromosome marking reagents based on actual sequence data (repeat polymo ⁇ hisms) 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
  • 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 the primer will yield an amplified fragment
  • mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner
  • Other mapping strategies that can similarly be used to map to its chromosome include 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 of the clones from which the gene encoding the PRO230, PR0216 or PRO302 polypeptide was derived, and the longer the better For example, 2,000 bp is good, 4,000 bp is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time
  • Verma et al Human Chromosomes a Manual of Basic Techniques (Pergamon Press, New York, 1988)
  • 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)
  • This invention encompasses methods of screening compounds to identify those that mimic the PRO230, PR0216 or PRO302 polypeptide (agonists) or prevent the effect of the PRO230, PR0216 or PRO302 polypeptide (antagonists)
  • Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO230, PR0216 or PRO302 polypeptide encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins
  • Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art
  • the interaction is binding and the complex formed can be isolated or detected in the reaction mixture
  • the PRO230, PR0216 or PRO302 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
  • a solid phase e g
  • Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the PRO230, PR0216 or PRO302 polypeptide and drying
  • an immobilized antibody e g a monoclonal antibody, specific for the PRO230, PR0216 or PRO302 polypeptide to be immobilized can be used to anchor it to a solid surface.
  • the assay is performed 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.
  • the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected.
  • the detection of label immobilized on the surface indicates that complexing occurred.
  • complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
  • the candidate compound interacts with but does not bind to a particular PRO230, PR0216 or PRO302 polypeptide encoded by a gene identified herein
  • its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions.
  • assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns.
  • 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.
  • 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.
  • GALl-/ ⁇ cZ 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 ⁇ -galactosidase.
  • a complete kit (MATCHMAKERTM) for identifying protein-protein interactions between two specific proteins using the two-hybrid techniqueiscommerciallyavailable 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.
  • a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products.
  • a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound.
  • a placebo may be added to a third reaction mixture, to serve as positive control.
  • the binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.
  • the PRO polypeptide has the ability to stimulate the proliferation of endothelial cells in the presence of the co-mitogen ConA
  • a screening method takes advantage of this ability.
  • human umbilical vein endothelial cells are obtained and cultured in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) and supplemented with a reaction mixture appropriate for facilitating proliferation of the cells, the mixture containing Con-A (Calbiochem, La Jolla, CA).
  • Con-A and the compound to be screened are added and after incubation at 37°C, cultures are pulsed with 3 ⁇ -thymidine and harvested onto glass fiber filters (phD; Cambridge Technology, Watertown, MA).
  • 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 of the compound to inhibit 3 ⁇ (H)thymidine inco ⁇ oration in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide.
  • 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 the potential antagonist.
  • the gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Coligan et al. Current Protocols in Immun., 1(2): Chapter 5 ( 1991).
  • 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, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub- pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
  • 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 amino 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.
  • mammalian cells or a membrane preparation expressing the receptor would be incubated with the labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
  • 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.
  • 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.
  • 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 inhibiting the action of the PRO polypeptide.
  • Another potential PRO polypeptide antagonist or agonist 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 translation.
  • 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.
  • the 5' coding portion of the 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 of the gene involved in transcription (triple helix - see, Lee et al, Nucl. Acids Res., 6:3073 (1979); Cooney et al. Science. 241 : 456 (1988); Dervan et al, Science, 251 : 1360 ( 1991 )), thereby preventing transcription and the production of the PRO polypeptide.
  • 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 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.
  • antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
  • Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases 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 in length, or more.
  • Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biology, 4: 469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997).
  • Nucleic acid molecules in 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 purines or pyrimidines on one strand of a duplex.
  • Hoogsteen base-pairing rules which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • the PRO230, PR0216 or PRO302 polypeptides, or agonists or antagonists thereto, that have activity in the cardiovascular, angiogenic, and endothelial assays described herein, and/or whose gene product has been found to be localized to the cardiovascular system, are likely to have therapeutic uses in a variety of cardiovascular, endothelial, and angiogenic disorders, including systemic disorders that affect vessels, such as diabetes mellitus. Their therapeutic utility could include diseases of the arteries, capillaries, veins, and/or lymphatics.
  • Examples of treatments hereunder include treating muscle wasting disease, treating osteoporosis, aiding in implant fixation to stimulate the growth 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.
  • tissue such as connective tissue, skin, bone, cartilage, muscle, lung, or kidney
  • 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 antagonist 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.
  • the present invention concerns the treatment of cardiac hypertrophy, regardless of the underlying cause, by administering a therapeutically effective dose of the PRO polypeptide, or agonist or antagonist thereto.
  • 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 of the progression of cardiac hypertrophy, with or without structural damage of the heart muscle, regardless of the underlying cardiac disorder.
  • vascular tumors such as haemangioma, tumor angiogenesis, neovascula ⁇ zation in the retina, choroid, or cornea, associated with diabetic retinopathy or premature infant retinopathy or macular degeneration and proliferative vitreoretmopathy, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation, psoriasis, endomet ⁇ osis associated with neovascula ⁇ zation, restenosis subsequent to balloon angioplasty, scar tissue ove ⁇ r
  • 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
  • an antagonist thereof would be used for treatment of those conditions where angiogenesis is desired
  • 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 arte ⁇ tis, Takayasu's arte ⁇ tis, polyarte ⁇ tis nodosa (including the microangiopathic form), Kawasaki's disease, microscopic polyangntis, 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 the circulation through the extremities on exposure to cold Altered endothelial cell function has been shown to be important in this disease
  • Aneurysms are saccular or fusiform dilatations of the arterial or venous tree that are associated with altered endothelial cell and/or vascular smooth muscle cells
  • Arterial restenosis restenosis of the arterial wall
  • 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.
  • lymphedema is a condition involving impaired lymphatic vessels resulting from endothelial cell function.
  • lymphangiomas are benign tumors of the lymphatic system that are congenital, often cystic, malformations of the lymphatics that usually occur in newborns. Cystic tumors tend to grow into the adjacent tissue. Cystic tumors usually occur in the cervical and axillary region. They can also occur in the soft tissue of the extremities. The main symptoms are dilated, sometimes reticular, structured lymphatics and lymphocysts surrounded by connective tissue. Lymphangiomas are assumed to be caused by improperly connected embryonic lymphatics or their deficiency. The result is impaired local lymph drainage. Griener et al. , Lvmphology, 4: 140- 144 ( 1971 ).
  • tumor angiogenesis involves vascularization of a tumor to enable it to growth and or metastasize. This process is dependent on the growth of new blood vessels.
  • neoplasms and related conditions that involve tumor angiogenesis include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endometrial carcinoma, endometrial hype ⁇ lasia, endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas carcinoma
  • AMD Age-related macular degeneration
  • AMD Age-related macular degeneration
  • 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 antagonist thereto is expected to be useful in reducing the severity of AMD.
  • a PRO polypeptide or antagonist thereof that induces cartilage and/or bone growth in circumstances where bone is not normally formed has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a PRO polypeptide or antagonist thereof may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma-induced, or oncologic, resection-induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • PRO polypeptides or antagonists thereto may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • a PRO polypeptide or antagonist thereto may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, or endothelium), muscle (smooth, skeletal, or cardiac), and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.
  • organs including, for example, pancreas, liver, intestine, kidney, skin, or endothelium
  • muscle smooth, skeletal, or cardiac
  • vascular including vascular endothelium
  • a PRO polypeptide herein or antagonist thereto may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage. Also, the PRO polypeptide or antagonist thereto may be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells, or for inhibiting the growth of tissues described above.
  • a PRO polypeptide or antagonist thereto may also be used in the treatment of periodontal diseases and in other tooth-repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone- forming cells, or induce differentiation of progenitors of bone-forming cells.
  • a PRO polypeptide herein or an antagonist thereto may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes, since blood vessels play an important role in the regulation of bone turnover and growth.
  • tissue regeneration activity that may be attributable to the PRO polypeptide herein 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.
  • compositions herein may provide an environment to attract tendon- or ligament- forming cells, stimulate growth of tendon- or ligament- forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions herein may also be useful in the treatment of tendinitis, ca ⁇ al tunnel syndrome, and other tendon or ligament defects.
  • 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 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 antagonist may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson'sdisease,Huntington'sdisease, 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 antagonist thereto.
  • Ischemia-reperfusion injury is another indication. Endothelial cell dysfunction may be important in both the initiation of, and in regulation of the sequelae of events that occur following ischemia-reperfusion injury.
  • Rheumatoid arthritis is a further indication.
  • Blood vessel growth and targeting of inflammatory cells through the vasculature is an important component in the pathogenesis of rheumatoid and sero-negative forms of arthritis.
  • a PRO polypeptide or its antagonist may also be administered prophylactically to patients with cardiac hypertrophy, to prevent the progression of the condition, and avoid sudden death, including death of asymptomatic patients.
  • Such preventative therapy is particularly warranted in the case of patients diagnosed with massive left ventricular cardiac hypertrophy (a maximal wall thickness of 35 mm or more in adults, or a comparable value in children), or in instances when the hemodynamic burden on the heart is particularly strong.
  • a PRO polypeptide or its antagonist may also be useful in the management of atrial fibrillation, which develops in a substantial portion of patients diagnosed with hypertrophic cardiomyopathy.
  • Additional non-neoplastic conditions include psoriasis, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, thyroid hype ⁇ lasias (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.
  • 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 the disorders noted above, and as such can serve as therapeutic targets to augment or inhibit these processes or for vascular-related drug targeting in these disorders.
  • 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.
  • compositions of the PRO polypeptides or agonists or antagonists are prepared for storage by mixing the desired molecule having the appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the form of 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; antioxidantsincludingascorbic 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 immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • 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, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol.
  • 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, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrroli
  • Carriers for topical or gel-based forms of antagonist include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols.
  • conventional depot forms are suitably used.
  • Such forms include, for example, microcapsules, nano-capsules, hposomes, 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 inco ⁇ orating a PRO polypeptide or antagonist thereof into formed articles.
  • Such articles can be used in modulating endothelial cell growth and angiogenesis.
  • tumor invasion and metastasis may be modulated with these articles.
  • PRO polypeptide or antagonist to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
  • PRO polypeptide ordinarily will be stored in lyophilized form or in solution if administered systemically. If in lyophilized form, PRO polypeptide or antagonist thereto is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use.
  • An example of a liquid formulation of PRO polypeptide or antagonist is a sterile, clear, colorless unpreserved solution filled in a single- dose vial for subcutaneous injection.
  • Preserved pharmaceutical compositions suitable for repeated use may contain, for example, depending mainly on the indication and type of polypeptide: a) 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 the polypeptide or molecule against agitation-induced aggregation, d) an isotonifier, e) a preservative selected from the group of phenol, benzyl alcohol and a benzethonium hahde, e , chloride, and f) water
  • the detergent employed is non-ionic, it may, for example, be polysorbates (e g , POLYSORBATETM (TWEENTM) 20, 80, etc ) or poloxamers (e g , POLOXAMERTM 188)
  • non-ionic surfactants permits the formulation to be exposed to shear surface stresses without causing denaturation of the polypeptide
  • surfactant-containing formulations may be employed in aerosol devices such as those used in a pulmonary dosing, and needleless jet injector guns (see, e g , EP 257,956)
  • An isotonifier may be present to ensure isotonicity of a liquid composition of the PRO polypeptide or antagonist thereto, and includes polyhydric sugar alcohols, preferably t ⁇ hyd ⁇ c or higher sugar alcohols, such as glycerin, eryth ⁇ tol, arabitol, xy tol, 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 (l m ) injections, or as aerosol formulations suitable for intranasal or intrapulmonary delivery (for intrapulmonary delivery see, e g , EP 257,956)
  • PRO polypeptide 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 the form of shaped articles, e g , films, or microcapsules
  • 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(v ⁇ nylalcohol)), polylactides (U S Patent No 3,773,919, EP 58,481 ), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al , Biopolvmers, 22 547-556 (1983)), non-degradable ethylene-viny
  • Sustained-release PRO polypeptide compositions also include hposomally entrapped PRO polypeptides Liposomes containing the PRO polypeptide are prepared by methods known per se DE 3,218, 121 , Epstein et al , Proc Natl Acad Sci USA, 82 3688-3692 (1985), Hwang et al , Proc Natl Acad Sci USA.
  • the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy
  • the therapeutically effective dose of PRO polypeptide or antagonist thereto will, of course, vary depending on such factors as the pathological condition to be treated (including prevention), the method of administration, the type of compound being used for treatment, any co-therapy involved, the patient's age, weight, general medical condition, medical history, etc , and its determination is well within the skill of a practicing physician Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the maximal therapeutic effect If the 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 PRO polypeptide until a dosage is reached that achieves the desired effect for treatment of the condition in question For example, if the objective is the treatment of CHF, the amount would be one that inhibits the progressive cardiac hypertrophy associated with this condition The progress of this therapy is easily monitored by echo cardiography Similarly, in patients with hypertrophic cardiomyopathy, PRO polypeptide can
  • 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
  • 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
  • endotoxin contamination should be kept minimally at a safe level, for example, less than 0 5 ng/mg protein
  • 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 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, the severity of any infection, time of administration, and other clinical factors
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins m the pharmaceutical composition
  • 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, histomo ⁇ homet ⁇ c determinations, and tetracycline labeling
  • the route of PRO polypeptide or antagonist or agonist administration is in accord with known methods, e , by injection or infusion by intravenous, intramuscular, mtracerebral, intrapentoneai, intracerobrospinal, subcutaneous, intraocular, lntraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes, or by sustained-release systems as noted below
  • the PRO polypeptide or antagonists thereof also are suitably administered by intratumoral, pe ⁇ tumoral, mtralesional, or pe ⁇ lesional routes, to exert local as well as systemic therapeutic effects
  • the intrapentoneai route is expected to be particularly useful, for example, in the treatment of ovarian tumors
  • 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 , sodium salt, potassium salt), alkaline earth metal salts (e g , calcium salt, magnesium salt), ammonium salts, organic base salts (e g , py ⁇ dine salt, t ⁇ ethylamme salt), inorganic acid salts (e g , hydrochlo ⁇ de, sulfate, nitrate), and salts of organic acid (e g , acetate, oxalate, p-toluenesulfonate)
  • alkali metal salts e , sodium salt, potassium salt
  • alkaline earth metal salts e g , calcium salt, magnesium salt
  • ammonium salts e g , ammonium salts
  • organic base salts e g , py ⁇ dine salt, t ⁇ ethylamme salt
  • inorganic acid salts e
  • the therapeutic method includes administering the composition topically, system ically, 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 the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and preferably capable of being resorbed into the body Such matrices may be formed of materials presently in use for other implanted medical applications
  • compositions may be biodegradable and chemically defined calcium sulfate, t ⁇ calcium phosphate, hydroxyapatite, polylactic acid, polyglycohc acid, and polyanhyd ⁇ des
  • 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 of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and t ⁇ calcium phosphate
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing
  • sequestering agents include cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,ethylcellulose,hydoxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, one preferred being cationic salts of carboxymethylcellulose (CMC)
  • CMC carboxymethylcellulose
  • Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and poly(vmyl 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 deso ⁇ tion of the polypeptide (or its antagonist) from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide (or its antagonist) the
  • the effectiveness of the PRO230, PR0216 or PRO302 polypeptide or an agonist or antagonist thereof in preventing or treating the disorder m question may be improved by administering the active agent serially or in combination with another agent that is effective for those pu ⁇ oses, either in the same composition or as separate compositions
  • PRO polypeptide therapy can be combined with the administration of inhibitors of known cardiac myocyte hypertrophy factors, e g , inhibitors of ⁇ -adrenergic agonists such as phenyleph ⁇ ne, endothelin- 1 inhibitors such as BOSENTANTM and MOXONODINTM, inhibitors to CT- 1 (US 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
  • inhibitors of known cardiac myocyte hypertrophy factors e g
  • inhibitors of ⁇ -adrenergic agonists such as phenyleph ⁇ ne
  • endothelin- 1 inhibitors such as BOSENTANTM and MOXONODINTM
  • inhibitors to CT- 1 US Pat No 5,679,545
  • ACE inhibitors des-aspartate-angiotensin I inhibitors
  • the PRO polypeptide can be administered in combination with ⁇ -adrenergic receptor blocking agents, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol, ACE inhibitors, e g , quinap ⁇ l, captopril, enalap ⁇ l, ramip ⁇ l, benazep ⁇ l, fosmop ⁇ l, or lisinop ⁇ l, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlo ⁇ henamide, acetazolamide, or indapamide, and/or calcium
  • ⁇ - adrenergic-blocking drugs e , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol
  • verapamil difedipine
  • 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, ⁇ -adrenergic blocking agents, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazi
  • PRO polypeptides or their antagonists may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question
  • agents include various growth factors such as EGF, PDGF, TGF- ⁇ or TGF- ⁇ , IGF, FGF, and CTGF
  • PRO polypeptides or their antagonists used to treat cancer may be combined with cytotoxic, chemotherapeutic, or growth-inhibitory agents as identified above
  • cytotoxic, chemotherapeutic, or growth-inhibitory agents as identified above
  • the PRO polypeptide or antagonist thereof is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances
  • the effective amounts of the therapeutic agents administered in combination with the PRO polypeptide or antagonist thereof will be at the physician's or veterinarian's discretion Dosage administration and adjustment is done to achieve maximal management of the conditions to be treated For example, for treating hypertension, these amounts ideally take into account use of diuretics or digitalis, and conditions such as hyper- or hypotension, renal impairment, etc
  • the dose will additionally depend on such factors as the type of the therapeutic agent to be used and the specific patient being treated Typically, the amount employed will be the same dose as that used, if the given therapeutic agent is administered without the PRO polypeptide
  • An article of manufacture such as a kit containing PRO230, PR0216 or PRO302 polypeptide or antagonists thereof useful for the diagnosis or treatment of the disorders described above comprises at least a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes
  • the containers may be formed from a variety of materials such as glass or plastic
  • the container holds a composition that is effective for diagnosing or treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle)
  • the active agent in the composition is the PRO230, PR0216 or PRO302 polypeptide or an agonist or antagonist thereto
  • the label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice
  • the article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer'
  • Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments that may inhibit the production or the gene product of the genes identified herein and/or reduce the activity of the gene products
  • polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intrapentoneai injections
  • the immunizing agent may include the PRO230, PR0216 or PRO302 polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulm, and soybean trypsin inhibitor Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A or synthetic trehalose dicorynomycolate)
  • the immunization protocol may be selected by
  • the ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibodies may, alternatively, be monoclonal antibodies
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstem, Nature. 256 495 (1975)
  • a hybridoma method a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro
  • the immunizing agent will typically include the PRO230, PR0216 or PRO302 polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired
  • PBLs peripheral blood lymphocytes
  • 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 Godmg, 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
  • 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 heteromyelomacell lines also have been described forthe production of human monoclonal antibodies Kozbor, J Immunol , 133 3001 ( 1984), Brön et al , Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Ine New York, 1987) pp 51 -63
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the PRO230, PR0216 or PRO302 polypeptide
  • 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)
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem , 1_07 220 (1980)
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods Goding, supra Suitable culture media for this pu ⁇ ose 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 pu ⁇ ficationproceduressuch as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • immunoglobulin pu ⁇ ficationprocedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (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
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U S Patent No 4,816,567, Morrison
  • 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
  • the relevant cysteine residues are substituted with another ammo acid residue or are deleted so as to prevent crosslinking
  • the ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibodies may further comprise humanized antibodies or human antibodies
  • Humanized forms of non-human (e , murine) antibodies are chimenc immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity
  • donor antibody such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues
  • Humanized antibodies may also comprise residues that are found neither in the
  • 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).
  • humanized antibodies are chimenc antibodies (U S Patent No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries Hoogenboom and Winter, J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581 (1991) The techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1985) and Boerner et al , 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,
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for the PRO230, PR0216 or PRO302 polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
  • 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 of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J.. J O: 3655-3659 ( 1991 ).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant-domain sequences.
  • the fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH I) containing the site necessary for light-chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • 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 pu ⁇ ose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • cysteine res ⁇ due(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region
  • the homodime ⁇ c 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 1 191 - 1 195 ( 1992) and Shopes, J Immunol , 148 2918-2922 ( 1992)
  • Homodime ⁇ c antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al , Cancer Research. 53 2560-2565 (1993)
  • 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
  • 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 (/ e , a radioconjugate)
  • 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 (/ e , a radioconjugate)
  • 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, ab ⁇ n A chain, modeccin A chain, alpha-sarcin, Aleurites fordn proteins, dianthm proteins, Phvtolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaona ⁇ a officinalis inhibitor, gelonm, mitogel n, rest ⁇ ctocin, phenomycin, enomycin, and the t ⁇ cothecenes
  • radionuchdes are available for the production of radioconjugated antibodies Examples include 2l2 B ⁇ , l3 l I, ,3 'In, 90
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such asN-succ ⁇ n ⁇ m ⁇ dyl-3-(2-py ⁇ dyld ⁇ th ⁇ ol)prop ⁇ onate (SPDP), ⁇ m ⁇ noth ⁇ olane (IT), bifunctional derivatives of lmidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as b ⁇ s-(p-d ⁇ azon ⁇ umbenzoyl)-ethylened ⁇ am ⁇ ne), dnsocyanates (such as tolyene 2,6-dnsocyanate), and bis- active fluorine compounds (such as l ,5-d ⁇ fluoro-2,4
  • 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 , a radionucleotide)
  • a "receptor” such as streptavidin
  • a ligand e g avidin
  • cytotoxic agent e , a radionucleotide
  • 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, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising 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 of the antibody of the 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 Doxorubicin is optionally contained within the liposome. See, Gabizon et al , J. National Cancer Inst., 81(19): 1484 (1989).
  • Antibodies specifically binding a PRO230, PR0216 or PRO302 polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders as noted above and below in the form of pharmaceutical compositions.
  • PRO230, PR0216 or PRO302 polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred.
  • 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 of the target protein is preferred.
  • 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.
  • the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • cytotoxic agent 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 pu ⁇ ose 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.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (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.
  • encapsulated antibodies 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.
  • antibodies to a PRO230, PR0216 or PRO302 polypeptide may be used to treat various cardiovascular, endothelial, and angiogenic conditions as noted above.
  • the antibodies are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred.
  • chemotherapeutic agents may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service. Ed., M.C. Perry (Williams & Wilkins: Baltimore, MD, 1992). The chemotherapeutic agent may precede, or follow administration of the antibody, or may be given simultaneously therewith.
  • the antibody may be combined with an anti-estrogen compound such as tamoxifen or EVISTATM or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
  • the antibodies are used for treating cancer, it may be desirable also to administer antibodies against other tumor-associated antigens, such as antibodies that bind to one or more of the ErbB2, EGFR, ErbB3, ErbB4, or VEGF receptor(s). These also include the agents set forth above.
  • the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances.
  • two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to the patient.
  • the antibodies herein are co- administered with a growth-inhibitory agent.
  • the growth-inhibitory agent may be administered first, followed by an antibody of the present invention.
  • simultaneous administration or administration of the antibody of the present invention first is also contemplated.
  • Suitable dosages for the growth-inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth-inhibitory agent and the antibody herein.
  • vascularization of tumors is attacked in combination therapy.
  • the anti-PRO polypeptide antibody and another antibody e.g., anti-VEGF
  • 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.
  • 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
  • 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.
  • the anti-PRO polypeptide antibody is administered together with TNF and, optionally, auxiliary agent(s).
  • the therapeutic agents described herein are administered to the isolated tumor or organ.
  • 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.
  • an antibody herein will depend on the type of disorder to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic pu ⁇ oses, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • ⁇ g/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily or weekly dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is repeated or sustained until a desired suppression of disorder symptoms occurs.
  • 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.
  • 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 ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody
  • the indication for which the antibodies are used is cancer
  • cell-surface proteins such as growth receptors over expressed in certain tumors
  • growth receptors 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
  • antibodies directed against the PRO polypeptides may be used as tumor diagnostics or prognostics
  • 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 , fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluo ⁇ metry, or other techniques known in the art Such binding assays are performed essentially as described above
  • In situ detection of antibody binding to the marker gene products can be performed, for example, by lmmunofluorescence or immunoelectron microscopy
  • a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample
  • This procedure also allows for determining the distribution of the marker gene product in the tissue examined It will be apparent to those skilled in the art that a wide variety of histological methods are readily available for in situ detection
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases.
  • the EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA).
  • the search was performed using the computer program BLAST or BLAST2 [Altschul et al. Methods in Enzymology, 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington).
  • a consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA30857. An EST proprietary to Genentech was employed in the consensus assembly and is herein designated DNA20088. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above.
  • oligonucleotides were synthesized: 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO230.
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length.
  • the probe sequences are typically 40-55 bp in length.
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp.
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al. , Current Protocols in Molecular Biology, supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
  • hybridization probe 5'-GGCGGTATCTCTCTGGCCTCCC-3' (SEQ ID NO:4) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30857 sequence which had the following nucleotide sequence: hybridization probe:
  • DNA from the libraries was screened by PCR amplification with the PCR primers identified above. A positive library was then used to isolate clones encoding the PRO230 gene using the probe oligonucleotide and one of the PCR primers.
  • RNA for construction of the cDNA libraries was isolated from human fetal lung tissue.
  • the cDNA libraries used to isolate the cDN A 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
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 100-102 and a stop signal at nucleotide positions 592-594 ( Figure 1 , SEQ ID NO 1 )
  • the predicted polypeptide precursor is 164 amino acids long [Figure 2, (SEQ ID NO 2)] and has a calculated molecular weight of approximately 18,359 daltons and an estimated pi of about 7 45
  • Analysis of the full-length PRO230 sequence shown in Figure 2 evidences the presence of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis of the full-length PRO230 sequence (Figure 2, SEQ ID NO 2) evidences the following a signal peptide from about amino acid 1 to about a ino acid 21 , an N-linked glycosylation site from about amino acid 78 to about amino acid 82, casein kinase II phosphorylation sites from about ammo acid 80 to about amino acid 84, from
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss- Prot public database were used to search EST databases
  • the EST databases included public EST databases (e , GenBank) and a proprietary EST database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA)
  • the search was performed using the computer program BLAST or BLAST2 [Altschul et al. Methods in Enzymology, 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences.
  • BLAST or BLAST2 Altschul et al. Methods in Enzymology, 266:460-480 (1996)
  • a consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA28754. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above.
  • oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0216.
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length.
  • the probe sequences are typically 40-55 bp in length.
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1 -1.5 kbp.
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al. Current Protocols in Molecular Biology, supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
  • hybridization probe 5'-AATAATGAAGGTCAAAGTGCCCTT-3' (SEQ ID NO:9) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28754 sequence which had the following nucleotide sequence: hybridization probe:
  • DNA from the libraries was screened by PCR amplification with the PCR primers identified above. A positive library was then used to isolate clones encoding the PRO230 gene using the probe oligonucleotide and one of the PCR primers.
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue.
  • the cDN A libraries used to isolate the cDN A 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.
  • a suitable cloning vector such as pRKB or pRKD; pRK5B is a
  • DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PR0216 polypeptide (designated herein as DNA33087 [ Figure 3, SEQ ID NO:6]) and the derived protein sequence for that PR0216 polypeptide.
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 268-270 and a stop signal at nucleotide positions 1531 -1533 ( Figure 3, SEQ ID NO:6).
  • the predicted polypeptide precursor is 421 amino acids long [Figure 4; (SEQ ID NO:7)] and has a calulated molecular weight of approximately 49,492 daltons and an estimated pi of about 5.51.
  • Analysis of the full-length PR0216 sequence shown in Figure 4 (SEQ ID NO:7) evidences the presence of important polypeptide domains as shown in Figure 4, wherein the locations given for those important polypeptide domains are approximate as described above.
  • EXAMPLE 3 Isolation of cDNA Clones Encoding Human PRO302 (vitellogenic carboxypeptidase homolog)
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases.
  • the EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al.
  • a consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA35953. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above.
  • oligonucleotides were synthesized: 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO302.
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length.
  • the probe sequences are typically 40-55 bp in length.
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp.
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al. , Current Protocols in Molecular Biology, supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
  • PCR primers (forward and reverse) were synthesized: forward PCR primer 1 :
  • hybridization probe 5'-AGCTCTAGACCAATGCCAGCTTCC-3' (SEQ ID NO: 15) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA35953 sequence which had the following nucleotide sequence: hybridization probe:
  • DNA from the libraries was screened by PCR amplification with the PCR primers identified above. A positive library was then used to isolate clones encoding the PRO230 gene using the probe oligonucleotide and one of the PCR primers.
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228).
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA.
  • the cDN A was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see. Holmes et al. Science. 253: 1278-1280 ( 1991 )) in the unique Xhol and Notl sites.
  • a suitable cloning vector such as pRKB or pRKD; pRK5B is
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 34-36 and a stop signal at nucleotide positions 1390-1392 ( Figure 5, SEQ ID NO: l 1 ).
  • the predicted polypeptide precursor is 452 amino acids long [Figure 6; (SEQ ID NO: 12)] and has a calculated molecular weight of approximately 50,831 daltons and an estimated pi of about 5.74.
  • Analysis of the full-length PRO302 sequence shown in Figure 6 evidences the presence of important polypeptide domains as shown in Figure 6, wherein the locations given for those important polypeptide domains are approximate as described above.
  • EXAMPLE 4 Stimulation of Endothelial Tube Formation This assay follows the assay described in Davis and Camarillo, Experimental Cell Research. 224:39-51 (1996), or one modified from it as follows:
  • This assay will identify factors that facilitate cell survival in a 3-dimensional matrix in the presence of exogenous growth factors (VEGF, bFGF without PMA).
  • a positive result is equal to or less than 1.
  • 0 no apoptosis
  • 1 less than 20% cells are apoptotic
  • 2 less than 50% cells are apoptotic
  • 3 greater than 50% cells are apoptic.
  • Stimulators of apoptosis in this system are expected to be apoptotic factors, and inhibitors are expected to prevent or lessen apoptosis.
  • Vacuoles Assay This assay will identify factors that stimulate endothelial vacuole formation and lumen formation in the presence of bFGF and VEGF (40ng/ml).
  • a positive result is equal to or greater than 2.
  • 1 vacuoles present in less than 20% of cells
  • 2 vacuoles present in 20-50% of cells
  • 3 vacuoles present in greater than 50% of cells.
  • This assay is designed to identify factors that are involved in stimulating pinocytosis, ion pumping, permeability, and junction formation.
  • This assay is to identify factors that stimulate endothelial tube formation in a 3-dimensional matrix. This assay will identify factors that stimulate endothelial cells to differentiate into a tube-like structure in a 3-dimensional matrix in the presence of exogenous growth factors (VEGF, bFGF).
  • VEGF exogenous growth factors
  • a positive result is equal to or greater than 2.
  • This assay is designed to determine whether PR0216 (human osteomodulin) polypeptide shows the ability to induce c-fos in endothelial cells.
  • Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) in growth media (50% Ham's F12 w/o GHT: low glucose, and 50% DMEM without glycine: with NaHC03, 1 % glutamine, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF) were plated on 96-well microtiter plates at a cell density of lxl 0 4 cells/well.
  • the cells were starved by removing the growth media and treating the cells with 100 ⁇ l/well test samples and controls (positive control: growth media; negative control: 10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8).
  • positive control growth media
  • negative control 10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8
  • the cells were incubated for 30 minutes at 37°C, in 5% C0 2 .
  • the samples were removed, and the first part of the bDNA kit protocol (Chiron Diagnostics, cat. #6005-037) was followed, where each capitalized reagent/buffer listed below was available from the kit.
  • the amounts of the TM Lysis Buffer and Probes needed for the tests were calculated based on information provided by the manufacturer.
  • the appropriate amounts of thawed Probes were added to the TM Lysis Buffer.
  • the Capture Hybridization Buffer was warmed to room temperature.
  • the bDNA strips were set up in the metal strip holders, and 100 ⁇ l of Capture Hybridization Buffer was added to each b-DNA well needed, followed by incubation for at least 30 minutes.
  • the test plates with the cells were removed from the incubator, and the media was gently removed using the vacuum manifold. 100 ⁇ l of Lysis Hybridization Buffer with Probes were quickly pipetted into each well of the microtiter plates.
  • the plates were then incubated at 55 °C for 15 minutes. Upon removal from the incubator, the plates were placed on the vortex mixer with the microtiter adapter head and vortexed on the #2 setting for one minute. 80 ⁇ l of the lysate was removed and added to the bDNA wells containing the Capture Hybridization Buffer, and pipetted up and down to mix. The plates were incubated at 53 °C for at least 16 hours.
  • the second part of the bDNA kit protocol was followed. Specifically, the plates were removed from the incubator and placed on the bench to cool for 10 minutes. The volumes of additions needed were calculated based upon information provided by the manufacturer.
  • An Amplifier Working Solution was prepared by making a 1 : 100 dilution of the Amplifier Concentrate (20 fm/ ⁇ l) in AL Hybridization Buffer. The hybridization mixture was removed from the plates and washed twice with Wash A. 50 ⁇ l of Amplifier Working Solution was added to each well and the wells were incubated at 53 °C for 30 minutes. The plates were then removed from the incubator and allowed to cool for 10 minutes.
  • the Label Probe Working Solution was prepared by making a 1 : 100 dilution of Label Concentrate (40 pmoles/ ⁇ l) in AL Hybridization Buffer. After the 10-minute cool-down period, the amplifier hybridization mixture was removed and the plates were washed twice with Wash A. 50 ⁇ l of Label Probe Working Solution was added to each well and the wells were incubated at 53 °C for 15 minutes. After cooling for 10 minutes, the Substrate was warmed to room temperature. Upon addition of 3 ⁇ l of Substrate Enhancer to each ml of Substrate needed for the assay, the plates were allowed to cool for 10 minutes, the label hybridization mixture was removed, and the plates were washed twice with Wash A and three times with Wash D. 50 ⁇ l of the Substrate Solution with Enhancer was added to each well. The plates were incubated for 30 minutes at 37°C and RLU was read in an appropriate luminometer.
  • EXAMPLE 6 Guinea Pig Vascular Leak This assay is designed to determine whether PRO302 polypeptide shows the ability to induce vascular permeability.
  • Test samples containing the PRO302 polypeptide or a physiological buffer without the test polypeptide are injected into skin on the back of the test animals with 100 ⁇ l per injection site intradermally. There were approximately 16-24 injection sites per animal.
  • One ml of Evans blue dye (1% in PBS) is then injected intracardially. Skin vascular permeability responses to the compounds (i.e., blemishes at the injection sites of injection) are visually scored by measuring the diameter (in mm) of blue-colored leaks from the site of injection at 1 and 6 hours post administration of the test materials.
  • the mm diameter of blueness at the site of injection is observed and recorded as well as the severity of the vascular leakage. Blemishes of at least 5 mm in diameter are considered positive for the assay when testing purified proteins, being indicative of the ability to induce vascular leakage or permeability. A response greater than 7 mm diameter is considered positive for conditioned media samples.
  • Human VEGF at 0.1 ⁇ g/100 ⁇ l is used as a positive control, inducing a response of 15-23 mm diameter.
  • 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 33 P-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 NTB2TM nuclear track emulsion and exposed for 4 weeks.
  • the tubes were incubated at 37°C for one hour A total of 1 0 ⁇ l RQ1 DNase was added, followed by incubation at 37 °C for 15 minutes
  • a total of 90 ⁇ l TE (10 M T ⁇ s 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-50TM 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 ⁇ l TE was added, then 1 ⁇ l of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR IITM
  • the probe was run on a TBE/urea gel A total of 1-3 ⁇ l of the probe or 5 ⁇ l of RNA Mrk III was added to 3 ⁇ l 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 (SARANTM brand) and exposed to XAR film with an intensifying screen in a -70 °C freezer one hour to overnight
  • 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 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 H,0)
  • 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
  • the slides were deparaffinized, placed in SQ H,0, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time
  • the sections were deproteinated in 20 ⁇ g/ml protemase K (500 ⁇ l of 10 mg/ml in 250 ml RNase-free RNase buffer, 37 °C, 15 minutes) for human embryo tissue, or 8 x protemase K ( 100 ⁇ l 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
  • the slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper
  • the tissue was covered with 50 ⁇ l of hybridization buffer (3 75 g dextran sulfate + 6 ml SQ H 2 0), 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 H,0 were added, and the tissue was vortexed well and incubated at 42 °C for 1-4 hours
  • Sections showed an intense signal associated with arterial and venous vessels in the fetus In arteries, the signal appeared to be confined to smooth-muscle/pe ⁇ cyte cells The signal was also seen in capillary vessels and in glomeru It was not clear whether endothelial cells were expressing this mRNA Expression was also observed in epithelial cells in the fetal lens Strong expression was also seen in cells within placental trophoblastic villi, these cells lie between the trophoblast and the fibroblast-like cells that express HGF-uncertain histogenesis In the adult, there was no evidence of expression and the wall of the aorta and most vessels appeared to be negative However, expression was seen over vascular channels in the normal prostate and in the epithelium lining the gallbladder Insurers expression was seen in the vessels of the soft-tissue sarcoma and a renal cell carcinoma
  • PRO230 is a molecule that shows relatively specific vascular expression in the fetus as well as in some adult organs Expression was also observed in the fetal lens and the adult gallbladder
  • Sections showed strong specific expression in osteoblasts at all sites of enchondral and pe ⁇ osteal new bone formation Additional sites of expression included the developing pulmonary arterial and aortic trunks Otherwise, all fetal tissues tested negative
  • the fetal tissues examined included placenta, umbilical cord, brain, spinal cord, eye, optic nerve, trachea, lung, heart, thymus, liver, spleen, esophagus, small intestine, pancreas, adrenal, thyroid, body wall, and lower limb All of the adult tissues were negative
  • the adult tissues examined included liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung and skin PR0216 has a probable role in control of bone matrix deposition and/or osteoblast growth All adult tissues in the multiblock were positive for beta-actin
  • the following method describes use of a nucleotide sequence encoding PRO230, PR0216 or PRO302 as a hybridization probe
  • DNA comprising the coding sequence of full-length or mature PRO230, PR0216 or PRO302 (as shown in Figures 1 , 3 and 5, respectively, SEQ ID NOS 1 , 6 and 1 1 , respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO230, PR0216 or PRO302) in human tissue cDNA libraries or human tissue genomic libraries
  • Hybridization and washing of filters containing either library DNAs is performed under the following high- stringency conditions
  • Hybridization of radiolabeled probe derived from the gene encoding a PRO230, PR0216 or PRO302 polypeptide to the filters is performed in a solution of 50% formamide, 5x SSC, 0 1% SDS, 0 1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6 8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20 hours
  • Washing of the filters is performed in an aqueous solution of 0 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
  • EXAMPLE 9 Expression of Nucleic Acid Encoding PRO230, PRQ216 or PRO302 in E colt This Example illustrates preparation of an unglycosylated form of PRO230, PR0216 or PRO302 by recombinant expression in E colt
  • the DNA sequence encoding PRO230, PR0216 or PRO302 (SEQ ID NOS 1 , 6 and 1 1 , respectively) is initially amplified using selected PCR primers
  • the primers should contain restriction enzyme sites that correspond to the restriction enzyme sites on the selected expression vector
  • a variety of expression vectors may be employed
  • An example of a suitable vector is pBR322 (derived from E coli, see Bolivar et al , Gene, 2 95 (1977)), which contains genes for ampicil n 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 that encode an antibiotic-resistance gene, a ftp promoter, a poly-His leader (including the first six STII codons, poly-His sequence, and enterok ase cleavage site), the region encoding PRO230, PR0216 or PRO302, lambda transcription
  • the hgation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic-resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing
  • Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics
  • the overnight culture may subsequently be used to inoculate a larger-scale culture
  • the cells are then grown to a desired optical density, during which the expression promoter is turned on
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation
  • the cell pellet obtained by the centrifugation can be solubi zed using various agents known in the art, and the solubi zed PRO230, PR0216 or PRO302 polypeptide can then be purified using a metal-chelating column under conditions that allow tight binding of the polypeptide
  • This Example illustrates preparation of a potentially glycosylated form of PRO230, PR0216 or PRO302 by recombinant expression in mammalian cells
  • the vector, pRK5 (see, EP 307,247, published March 15, 1989), is employed as the expression vector
  • the PRO230, PR0216 or PRO302 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the DNA encoding PRO230, PR0216 or PRO302 using ligation methods such as described in Sambrook et al , supra
  • the resulting vector is called pRK5-(DNA encoding PRO230, PR0216 or PRO302)
  • the selected host cells are 293 cells
  • Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and or antibiotics
  • About 10 ⁇ g DNA of pRK5-(DNA encoding PRO230, PR0216 or PRO302) is mixed with about 1 ⁇ g DNA encoding the VA RNA gene (Thimmappaya et al , Cell, 3J_ 543 ( 1982)) and dissolved in 500 ⁇ l of 1 M T ⁇ s-HCl, 0 1 mM EDTA, 0 227 M CaCl, To this mixture is added, dropwise, 500 ⁇ l of 50 mM HEPES (pH 7 35), 280 mM NaCl, 1 5 mM NaP0 4 , 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
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 35 S-cyste ⁇ ne and 200 ⁇ Ci/ml 35 S-meth ⁇ on ⁇ ne
  • the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel
  • the processed gel may be dried and exposed to film for a selected period of time to reveal the presence of the PRO230, PR0216 or PRO302 polypeptide
  • the cultures containing transfected cells may undergo further incubation (in serum-free medium) and the medium is tested in selected bioassays
  • the gene encoding PRO230, PR0216 or PRO302 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Natl Acad Sci . 12 7575 (1981 ) 293 cells are grown to maximal density in a spinner flask and 700 ⁇ g pRK5-(DNA encoding PRO230, PR0216 or PRO302) 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 culture medium, 5 ⁇ g/ml bovine insulin, and 0 1 ⁇ g/ml bovine transfernn After about four days, the conditioned media is centnfuged and filtered to remove cells and debris The sample containing the
  • the gene encoding PRO230, PR0216 or PRO302 can be expressed in CHO cells
  • the pRK5-(DNA encoding PRO230, PR0216 or PRO302) nucleic acid can be transfected into CHO cells using known reagents such as CaP0 4 or DEAE-dextran
  • the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35 S-meth ⁇ on ⁇ ne
  • the culture medium may be replaced with serum-free medium
  • the cultures are incubated for about 6 days, and then the conditioned medium is harvested
  • the medium containing the expressed PRO230, PR0216 or PRO302 can then be concentrated and purified by any selected method
  • Epitope-tagged gene encoding the PRO230, PR0216 or PRO302 polypeptide may also be expressed in host CHO cells
  • the gene encoding PRO230, PR0216 or PRO302 may be subcloned out of the pRK5 vector
  • the subclone insert can undergo PCR amplification to fuse in frame with a selected epitope tag such as a poly-His tag into a baculovirus expression vector
  • the gene insert encoding the poly-H ⁇ s-tagged-PRO230, -PR0216 or -PRO230 can then be subcloned into a SV40- driven vector containing a selection marker such as DHFR for selection of stable clones
  • the CHO cells can be transfected (as described above) with the SV40-d ⁇ ven vector Labeling may be performed, as described above, to verify expression
  • the culture medium containing the expressed gene encoding the poly-H ⁇ s-tagged-PRO230, -PR0216 or -PRO302 can then
  • PRO230 and PR0216 were stably expressed in CHO cells by the above described method
  • EXAMPLE 1 Expression of Nucleic Acid Encoding PRO230, PRQ216 or PRO302 in Yeast The following method describes recombinant expression of the gene encoding PRO230, PR0216 or PRO302 in yeast
  • yeast expression vectors are constructed for intracellular production or secretion of PRO230, PR0216 or PRO302 from the ADH2/GAPDH promoter.
  • DNA encoding PRO230, PR0216 or PRO302 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the gene encoding PRO230, PR0216 or PRO302.
  • DNA encoding PRO230, PR0216 or PRO302 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO230, PR0216 or PRO302 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of the gene encoding PRO230, PR0216 or PRO302.
  • yeast cells such as yeast strain AB 1 10
  • yeast cells can then be transformed with the expression plasmids described above and cultured in selected fermentation media.
  • the transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
  • Recombinant PRO230, PR0216 or PRO302 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters.
  • the concentrate containing PRO230, PR0216 or PRO302 may further be purified using selected column- chromatography resins.
  • the following method describes recombinant expression in Baculovirus-infected insect cells.
  • sequence coding for PRO230, PR0216 or PRO302 is fused upstream of an epitope tag contained within a baculovirus expression vector.
  • 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).
  • sequence encoding PRO230, PR0216 or PRO302 or the desired portion of the coding sequence of PRO230, PR0216 or PRO302 is amplified by PCR with primers complementary to the 5' and 3' regions.
  • the 5' primer may inco ⁇ orate flanking (selected) restriction enzyme sites.
  • the product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9") cells (ATCC CRL 171 1 )using lipofectin (commercially available from GIBCO-BRL). After4 - 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)).
  • Expressed poly-His tagged- PRO230, -PR0216or-PRO302 can then be purified, for example, by Ni 2 "-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).
  • Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCl,; 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 ⁇ m filter
  • loading buffer 50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7 8) and filtered through a 0 45 ⁇ m filter
  • a Ni 2+ -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
  • purification of the IgG-tagged (or Fc tagged)-PRO230, -PR0216 or -PRO302 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography
  • PRO230 and PR0216 were successfully expressed in baculovirus infected Sf9 insect cells While expression was actually performed in a 0 5-2 L scale, it can be readily scaled up for larger (e g , 8 L) preparations
  • the proteins were expressed as an IgG construct (lmmunoadhesin), in which the protein extracellular region was fused to an IgG 1 constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms
  • baculovirus expression vector pb PH IgG for IgG fusions and pb PH His c for poly-His tagged proteins
  • vector and Baculogold® baculovirus DNA were co-transfected into 105 Spodoptera frugtperda ("Sf9") cells (ATCC CRL 171 1 ), using Lipofectm (Gibco BRL)
  • pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL 1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences
  • the cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells were incubated for 5 days at 28 °C The supernatant was harvested and subsequently used for the first viral amplification by infecting S
  • the first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOl of 0 1 Cells were incubated for 3 days at 28°C The supernatant was harvested and filtered Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed
  • the conditioned medium from the transfected cells (0 5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0 22 micron filters
  • the protein construct were purified using a Ni 2+ -NTA column (Qiagen) Before purification, lmidazole was added to the conditioned media to a concentration of 5 mM
  • the conditioned media were 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 lmidazole at a flow rate of 4-5 ml/mm at 4°C
  • the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M lmidazole
  • the highly purified protein was subsequently desalted into a storage buffer containing 1 O mM Hepes, 0 14 M NaCl and 4% mannito
  • a modified baculovirus procedure may be used inco ⁇ orating h ⁇ gh-5 cells
  • the DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector
  • epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG)
  • a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIE 1 - 1 (Novagen)
  • the pIE 1 - 1 and pIE 1 -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus lei promoter in stably-transformed insect cells
  • the plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lei -mediated gene expression in unmfected insect cells as well as the
  • H ⁇ gh-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no C02, NO pen/strep
  • 30 ⁇ g of pIE based vector containing the sequence was mixed with 1 ml Ex-Cell medium (Media Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401 -78P (note this media is light sensitive)), and in a separate tube, 100 ⁇ l of CellFectin (CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium The two solutions were combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex-Cell media was added to the 2 ml of DNA/CellFECTIN mix and this is layered on h ⁇ gh-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/Cell medium
  • the supernatant was harvested and the expression of the sequence in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 ml of Ni 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) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters.
  • the protein comprising the sequence is purified using a Ni 2+ -NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni 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. at48°C.
  • the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein was then subsequently desalted into a storage buffer containing 10 M Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80 °C.
  • lmmunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows.
  • the conditioned media was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8.
  • the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
  • the eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9.
  • the highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins.
  • the homogeneity of the sequence was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary.
  • PR0216and PRO302 were successfully expressed by the above modified baculovirus procedure inco ⁇ orating high-5 cells.
  • This Example illustrates preparation of monoclonal antibodies that can specifically bind PRO230, PR0216 or PRO302.
  • Immunogens that may be employed include purified PRO230, PR0216 or PRO302 fusion proteins containing PRO230, PR0216 or PRO302, and cells expressing the gene encoding PRO230, PR0216 or PRO302 on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
  • mice such as Balb/c are immunized with the PRO230, PR0216 or PRO302 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1 to 100 micrograms.
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads.
  • mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional immunization injections Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibodies
  • the animals "positive" for antibodies can be injected with a final intravenous injection of PRO230, PR0216 or PRO302
  • the mice are sacrificed and the spleen cells are harvested
  • the spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU 1, available from ATCC, No CRL 1597
  • the fusions generate hybridoma cells that can then be plated in 96-well tissue culture plates containing HAT medium to inhibit proliferation of non- fused cells, myeloma hybrids, and spleen cell hybrids
  • hybridoma cells will be screened in an ELISA for reactivity against PRO230, PR0216 or PRO302 Determination of "positive" hybridoma cells secreting the desired monoclonal antibodiesagainst PRO230, PR0216 or PRO302 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 ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 monoclonal antibodies
  • the hybridoma cells can be grown in tissue-culture flasks or roller bottles Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium-sulfateprecipitation, followed by gel-exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed

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Abstract

L'invention concerne des compositions et des procédés permettant de stimuler ou d'inhiber l'angiogénèse et/ou la vascularisation cardiaque chez les mammifères, y compris les humains. Les compositions pharmaceutiques sont basées sur des polypeptides ou des antagonistes de ces derniers, ayant été identifiés pour une ou plusieurs des utilisations précitées. Parmi les troubles pouvant être diagnostiqués, prévenus ou traités par cette composition, figurent des traumatismes, tels que des plaies, divers cancers, et des troubles vasculaires dont l'athérosclérose et l'hypertrophie cardiaque. L'invention concerne également des nouveaux polypeptides et des molécules d'acide nucléique les codant, ainsi que des vecteurs et des cellules hôtes renfermant ces séquences d'acide nucléique, des molécules de polypeptides chimériques renfermant les polypeptides de la présente invention fusionnés à des séquences de polypeptides hétérologues, des anticorps qui se lient aux polypeptides de la présente invention et des procédés de préparation de ces polypeptides.
PCT/US1999/028214 1994-09-08 1999-11-29 Promotion ou inhibition de l'angiogenese et de la vascularisation cardiaque WO2001019987A1 (fr)

Priority Applications (217)

Application Number Priority Date Filing Date Title
AU17471/00A AU1747100A (en) 1999-09-13 1999-11-29 Promotion or inhibition of angiogenesis and cardiovascularization
PCT/US1999/030999 WO2001005836A1 (fr) 1999-07-20 1999-12-20 Compositions polypeptidiques et methodes de traitement des tumeurs
AU23907/00A AU2390700A (en) 1999-07-20 1999-12-20 Polypeptidic compositions and methods for the treatment of tumors
PCT/US2000/004414 WO2001004311A1 (fr) 1999-07-07 2000-02-22 Polypeptides secretes et transmembranaires et acides nucleiques codant pour ces polypeptides
AU28839/00A AU2883900A (en) 1999-07-07 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/005841 WO2000053758A2 (fr) 1999-03-08 2000-03-02 Compositions et methodes de traitement des maladies immunitaires
JP2000603379A JP2004516227A (ja) 1999-03-08 2000-03-02 免疫関連疾患を治療するための組成物と方法
EP00913764A EP1220905A2 (fr) 1999-03-08 2000-03-02 Compositions et methodes pour le traitement de maladies immunitaires
CA002362427A CA2362427A1 (fr) 1999-03-08 2000-03-02 Compositions et methodes de traitement des maladies immunitaires
AU35144/00A AU3514400A (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
KR1020017011406A KR20010103046A (ko) 1999-03-08 2000-03-02 면역 관련 질환 치료용 조성물 및 치료 방법
AU63910/00A AU6391000A (en) 1999-07-28 2000-07-28 Method of preventing the injury or death of retinal cells and treating ocular diseases
PCT/US2000/020710 WO2001009327A2 (fr) 1999-07-28 2000-07-28 Procede de prevention de la deterioration ou de la mort des cellules de la retine et de traitement des troubles oculaires
US09/902,572 US20030108983A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,634 US20030082540A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,759 US20030077654A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,979 US20030113718A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,903 US20030044839A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,713 US20030082541A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,736 US20030049676A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,853 US20020192659A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,692 US20030054400A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,615 US20030092002A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,011 US20030003530A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,943 US20030054349A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,562 US6965015B2 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,925 US20030096233A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,520 US20030054401A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,823 US20030104381A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,640 US7208308B2 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,119 US20030049621A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids enconding the same
US09/903,786 US20030044793A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,749 US7147853B2 (en) 1997-09-17 2001-07-11 Anti-pro211 polypeptide antibodies
US09/903,806 US20030130489A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,603 US20030148419A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,088 US20030073077A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,877 US20030186358A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,766 US20030152999A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,786 US7557192B2 (en) 1997-09-17 2001-07-12 Anti-PRO335 antibodies
US09/905,291 US20020160374A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,859 US20030036060A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,992 US20030135025A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,056 US20030054441A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,125 US6664376B2 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,938 US20030211569A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,485 US20030064367A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,532 US7151160B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,838 US20030148370A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,820 US20030036094A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,075 US7169906B2 (en) 1997-09-17 2001-07-13 PRO211 polypeptides
US09/905,449 US6965011B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,920 US6806352B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,381 US6818746B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,553 US20030059828A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,348 US20030064923A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,462 US6878807B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,956 US20030049622A1 (en) 1995-12-01 2001-07-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,742 US20030023054A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,700 US6723535B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,815 US7094567B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,722 US6946262B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,646 US6852848B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,777 US20030148371A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,760 US20030096340A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,618 US6828146B2 (en) 1997-09-17 2001-07-16 Nucleic acid encoding PRO229 polypeptides
US09/906,838 US7070979B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/908,093 US20030017498A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,613 US20030027145A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,728 US20030190611A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,979 US20030082542A1 (en) 1994-09-08 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,575 US20030073079A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,942 US7087738B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,925 US20030054352A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,841 US7033825B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,824 US20020197671A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,652 US20030104469A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,794 US6635468B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,320 US7074592B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides nucleic acid encoding
US09/908,576 US20040005553A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,064 US6818449B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,204 US20030036061A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,088 US20020146709A1 (en) 1997-09-17 2001-07-18 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
US10/028,072 US20030004311A1 (en) 1997-06-18 2001-12-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/081,056 US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/121,044 US20030190717A1 (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
US10/121,045 US20030073210A1 (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
US10/121,046 US20030194791A1 (en) 1997-03-31 2002-04-11 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
US10/121,041 US20030077776A1 (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
US10/121,059 US20030190721A1 (en) 1997-03-31 2002-04-11 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
US10/121,048 US20030199051A1 (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
US10/121,063 US20030199055A1 (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
US10/121,062 US20030077779A1 (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
US10/121,054 US20030199054A1 (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
US10/121,060 US20030190722A1 (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
US10/121,061 US20030082761A1 (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
US10/121,058 US20030190720A1 (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
US10/123,213 US7193048B2 (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
US10/123,322 US20030199059A1 (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
US10/123,235 US20030082762A1 (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
US10/123,155 US20030068794A1 (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
US10/123,154 US20030190724A1 (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
US10/123,292 US20030073211A1 (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
US10/123,236 US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,108 US7635478B2 (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
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,261 US20030068796A1 (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
US10/123,262 US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,906 US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US20030087344A1 (en) 1997-06-18 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,912 US20030100087A1 (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
US10/123,904 US20030022328A1 (en) 1997-03-31 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
US10/123,902 US20030077781A1 (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
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 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
US10/124,816 US20030190728A1 (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
US10/124,819 US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,820 US20030190729A1 (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
US10/125,805 US20030194794A1 (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
US10/124,813 US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/124,822 US7109305B2 (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
US10/124,817 US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,927 US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 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
US10/140,020 US20030207415A1 (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
US10/140,023 US20030207416A1 (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
US10/140,024 US20040058424A1 (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
US10/140,928 US20030068798A1 (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
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,864 US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,865 US20030207420A1 (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
US10/140,809 US20030207418A1 (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
US10/140,860 US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 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
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/143,114 US20030036180A1 (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
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/146,730 US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,792 US20030207428A1 (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
US10/147,492 US20030082765A1 (en) 1997-03-31 2002-05-17 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
US10/152,395 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
US10/160,498 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
US10/158,791 US20030207429A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,913 US20030022298A1 (en) 1997-09-15 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/771,187 US7355002B2 (en) 1997-11-12 2004-02-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/970,823 US7307152B2 (en) 1997-10-17 2004-10-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
US11/518,609 US20070077623A1 (en) 1997-09-17 2006-09-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
USPCT/US99/20944 1999-09-13
PCT/US1999/020944 WO2000015792A2 (fr) 1998-09-14 1999-09-13 Promotion ou inhibition d'angiogenese et de cardiovascularisation
USPCT/US99/21090 1999-09-15
PCT/US1999/021090 WO2000015796A2 (fr) 1998-09-16 1999-09-15 Polypeptides secretes et transmembranaires et acides nucleiques codant pour ces polypeptides

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
PCT/US1999/021547 Continuation-In-Part WO2000015797A2 (fr) 1994-09-08 1999-09-15 Compositions et methodes de traitement des maladies relatives au syteme immunitaire
PCT/US1999/021090 Continuation-In-Part WO2000015796A2 (fr) 1994-09-08 1999-09-15 Polypeptides secretes et transmembranaires et acides nucleiques codant pour ces polypeptides
PCT/US1999/023089 Continuation-In-Part WO2000021996A2 (fr) 1994-09-08 1999-10-05 Procedes et compositions inhibant la croissance des cellules neoplasiques

Related Child Applications (3)

Application Number Title Priority Date Filing Date
PCT/US1999/028313 Continuation-In-Part WO2000032221A2 (fr) 1994-09-08 1999-11-30 Promotion et inhibition de l'angiogenese et de la vascularisation cardiaque
US09/903,749 Continuation US7147853B2 (en) 1997-09-17 2001-07-11 Anti-pro211 polypeptide antibodies
US10/081,056 Continuation US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis

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WO2003041652A2 (fr) * 2001-11-13 2003-05-22 Millennium Pharmaceuticals, Inc. Methode d'utilisation de 18080, element de la famille de la serine carboxypeptidase humaine

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EP0335243A2 (fr) * 1988-03-28 1989-10-04 The President And Fellows Of Harvard College Angiogénine mutante humaine (facteur d'angiogénèse avec une activité d'angiogénine supérieure), gènes pour celle-ci et méthode d'expression
WO1995029242A1 (fr) * 1994-04-26 1995-11-02 The Children's Medical Center Corporation Angiostatine et procede d'utilisation de ladite substance pour inhiber l'angiogenese
WO1999014234A2 (fr) * 1997-09-17 1999-03-25 Genentech, Inc. Stimulation ou inhibition de l'angiogenese et de la cardiovascularisation
WO1999014327A2 (fr) * 1997-09-17 1999-03-25 Genentech, Inc. Genes amplifies dans des tumeurs, anticorps diriges contre les proteines codees par ces genes, et leur utilisation dans le diagnostic et le traitement du cancer

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EP0335243A2 (fr) * 1988-03-28 1989-10-04 The President And Fellows Of Harvard College Angiogénine mutante humaine (facteur d'angiogénèse avec une activité d'angiogénine supérieure), gènes pour celle-ci et méthode d'expression
WO1995029242A1 (fr) * 1994-04-26 1995-11-02 The Children's Medical Center Corporation Angiostatine et procede d'utilisation de ladite substance pour inhiber l'angiogenese
WO1999014234A2 (fr) * 1997-09-17 1999-03-25 Genentech, Inc. Stimulation ou inhibition de l'angiogenese et de la cardiovascularisation
WO1999014327A2 (fr) * 1997-09-17 1999-03-25 Genentech, Inc. Genes amplifies dans des tumeurs, anticorps diriges contre les proteines codees par ces genes, et leur utilisation dans le diagnostic et le traitement du cancer

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Publication number Priority date Publication date Assignee Title
WO2003041652A2 (fr) * 2001-11-13 2003-05-22 Millennium Pharmaceuticals, Inc. Methode d'utilisation de 18080, element de la famille de la serine carboxypeptidase humaine
WO2003041652A3 (fr) * 2001-11-13 2003-08-14 Millennium Pharm Inc Methode d'utilisation de 18080, element de la famille de la serine carboxypeptidase humaine

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