WO2001019987A1 - Promotion or inhibition of angiogenesis and cardiovascularization - Google Patents

Promotion or inhibition of angiogenesis and cardiovascularization

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
French (fr)
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/en
Priority claimed from PCT/US1999/021090 external-priority patent/WO2000015796A2/en
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU17471/00A priority Critical patent/AU1747100A/en
Priority to AU23907/00A priority patent/AU2390700A/en
Priority to PCT/US1999/030999 priority patent/WO2001005836A1/en
Priority to PCT/US2000/004414 priority patent/WO2001004311A1/en
Priority to AU28839/00A priority patent/AU2883900A/en
Priority to CA002362427A priority patent/CA2362427A1/en
Priority to PCT/US2000/005841 priority patent/WO2000053758A2/en
Priority to AU35144/00A priority patent/AU3514400A/en
Priority to EP00913764A priority patent/EP1220905A2/en
Priority to KR1020017011406A priority patent/KR20010103046A/en
Priority to JP2000603379A priority patent/JP2004516227A/en
Priority to AU63910/00A priority patent/AU6391000A/en
Priority to PCT/US2000/020710 priority patent/WO2001009327A2/en
Publication of WO2001019987A1 publication Critical patent/WO2001019987A1/en
Priority to US09/902,713 priority patent/US20030082541A1/en
Priority to US09/902,692 priority patent/US20030054400A1/en
Priority to US09/902,853 priority patent/US20020192659A1/en
Priority to US09/902,979 priority patent/US20030113718A1/en
Priority to US09/902,615 priority patent/US20030092002A1/en
Priority to US09/902,759 priority patent/US20030077654A1/en
Priority to US09/902,634 priority patent/US20030082540A1/en
Priority to US09/902,572 priority patent/US20030108983A1/en
Priority to US09/902,903 priority patent/US20030044839A1/en
Priority to US09/902,736 priority patent/US20030049676A1/en
Priority to US09/903,640 priority patent/US7208308B2/en
Priority to US09/903,823 priority patent/US20030104381A1/en
Priority to US09/904,011 priority patent/US20030003530A1/en
Priority to US09/903,520 priority patent/US20030054401A1/en
Priority to US09/903,749 priority patent/US7147853B2/en
Priority to US09/904,119 priority patent/US20030049621A1/en
Priority to US09/903,786 priority patent/US20030044793A1/en
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Priority to US09/903,603 priority patent/US20030148419A1/en
Priority to US09/903,806 priority patent/US20030130489A1/en
Priority to US09/903,562 priority patent/US6965015B2/en
Priority to US09/903,925 priority patent/US20030096233A1/en
Priority to US09/905,291 priority patent/US20020160374A1/en
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Priority to US09/904,766 priority patent/US20030152999A1/en
Priority to US09/904,877 priority patent/US20030186358A1/en
Priority to US09/905,088 priority patent/US20030073077A1/en
Priority to US09/904,786 priority patent/US7557192B2/en
Priority to US09/905,125 priority patent/US6664376B2/en
Priority to US09/904,992 priority patent/US20030135025A1/en
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Priority to US09/906,722 priority patent/US6946262B2/en
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Priority to US09/906,815 priority patent/US7094567B2/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 US09/907,575 priority patent/US20030073079A1/en
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Priority to US09/907,613 priority patent/US20030027145A1/en
Priority to US09/909,320 priority patent/US7074592B2/en
Priority to US09/909,064 priority patent/US6818449B2/en
Priority to US09/908,576 priority patent/US20040005553A1/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 US10/121,041 priority patent/US20030077776A1/en
Priority to US10/121,046 priority patent/US20030194791A1/en
Priority to US10/121,040 priority patent/US20030082759A1/en
Priority to US10/121,047 priority patent/US20030077778A1/en
Priority to US10/121,059 priority patent/US20030190721A1/en
Priority to US10/121,042 priority patent/US20030096386A1/en
Priority to US10/121,051 priority patent/US20030092147A1/en
Priority to US10/121,045 priority patent/US20030073210A1/en
Priority to US10/121,044 priority patent/US20030190717A1/en
Priority to US10/121,054 priority patent/US20030199054A1/en
Priority to US10/121,063 priority patent/US20030199055A1/en
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Priority to US10/121,056 priority patent/US20030082760A1/en
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Priority to US10/121,062 priority patent/US20030077779A1/en
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Priority to US10/121,049 priority patent/US20030022239A1/en
Priority to US10/121,043 priority patent/US7220831B2/en
Priority to US10/121,048 priority patent/US20030199051A1/en
Priority to US10/121,058 priority patent/US20030190720A1/en
Priority to US10/121,057 priority patent/US20030190719A1/en
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Priority to US10/123,156 priority patent/US20030194792A1/en
Priority to US10/123,154 priority patent/US20030190724A1/en
Priority to US10/123,215 priority patent/US7291329B2/en
Priority to US10/123,212 priority patent/US7276577B2/en
Priority to US10/123,214 priority patent/US7343721B2/en
Priority to US10/123,291 priority patent/US20030199058A1/en
Priority to US10/123,235 priority patent/US20030082762A1/en
Priority to US10/123,261 priority patent/US20030068796A1/en
Priority to US10/123,771 priority patent/US20030199060A1/en
Priority to US10/123,292 priority patent/US20030073211A1/en
Priority to US10/123,108 priority patent/US7635478B2/en
Priority to US10/123,262 priority patent/US20030049816A1/en
Priority to US10/123,109 priority patent/US20030190723A1/en
Priority to US10/123,236 priority patent/US20030068795A1/en
Priority to US10/123,155 priority patent/US20030068794A1/en
Priority to US10/123,322 priority patent/US20030199059A1/en
Priority to US10/123,213 priority patent/US20030199057A1/en
Priority to US10/123,157 priority patent/US20030190725A1/en
Priority to US10/123,213 priority patent/US7193048B2/en
Priority to US10/123,905 priority patent/US20030087344A1/en
Priority to US10/123,903 priority patent/US20030073212A1/en
Priority to US10/123,912 priority patent/US20030100087A1/en
Priority to US10/123,911 priority patent/US7408032B2/en
Priority to US10/123,904 priority patent/US20030022328A1/en
Priority to US10/123,908 priority patent/US7335728B2/en
Priority to US10/123,906 priority patent/US20030190726A1/en
Priority to US10/123,910 priority patent/US7329404B2/en
Priority to US10/123,902 priority patent/US20030077781A1/en
Priority to US10/123,909 priority patent/US7193049B2/en
Priority to US10/123,907 priority patent/US7084258B2/en
Priority to US10/123,913 priority patent/US20030203462A1/en
Priority to US10/123,905 priority patent/US7285625B2/en
Priority to US10/125,795 priority patent/US7304131B2/en
Priority to US10/124,822 priority patent/US7109305B2/en
Priority to US10/124,819 priority patent/US7285626B2/en
Priority to US10/125,704 priority patent/US7357926B2/en
Priority to US10/124,817 priority patent/US20030077786A1/en
Priority to US10/124,813 priority patent/US7312307B2/en
Priority to US10/124,823 priority patent/US20030199062A1/en
Priority to US10/124,816 priority patent/US20030190728A1/en
Priority to US10/124,824 priority patent/US20030077659A1/en
Priority to US10/124,814 priority patent/US7105335B2/en
Priority to US10/124,821 priority patent/US20030199023A1/en
Priority to US10/124,820 priority patent/US20030190729A1/en
Priority to US10/124,818 priority patent/US20030082763A1/en
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Priority to US10/125,922 priority patent/US7309762B2/en
Priority to US10/125,924 priority patent/US7342097B2/en
Priority to US10/127,831 priority patent/US20030082689A1/en
Priority to US10/128,689 priority patent/US20030087365A1/en
Priority to US10/131,825 priority patent/US7282566B2/en
Priority to US10/131,823 priority patent/US7304132B2/en
Priority to US10/131,817 priority patent/US7291701B2/en
Priority to US10/137,865 priority patent/US20030032155A1/en
Priority to US10/137,868 priority patent/US20030082764A1/en
Priority to US10/137,867 priority patent/US20030207349A1/en
Priority to US10/139,963 priority patent/US7288625B2/en
Priority to US10/140,024 priority patent/US20040058424A1/en
Priority to US10/140,023 priority patent/US20030207416A1/en
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Priority to US10/140,474 priority patent/US20030032156A1/en
Priority to US10/139,980 priority patent/US7247710B2/en
Priority to US10/140,470 priority patent/US20030022331A1/en
Priority to US10/140,925 priority patent/US20030073215A1/en
Priority to US10/140,865 priority patent/US20030207420A1/en
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Priority to US10/140,921 priority patent/US7317080B2/en
Priority to US10/140,860 priority patent/US7307151B2/en
Priority to US10/140,808 priority patent/US7425621B2/en
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Priority to US10/140,928 priority patent/US20030068798A1/en
Priority to US10/141,754 priority patent/US7361732B2/en
Priority to US10/141,760 priority patent/US7342104B2/en
Priority to US10/141,701 priority patent/US20030207421A1/en
Priority to US10/141,756 priority patent/US7488586B2/en
Priority to US10/141,755 priority patent/US7297764B2/en
Priority to US10/142,417 priority patent/US7304133B2/en
Priority to US10/143,114 priority patent/US20030036180A1/en
Priority to US10/142,430 priority patent/US7309766B2/en
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Priority to US10/142,419 priority patent/US7153941B2/en
Priority to US10/142,431 priority patent/US7285629B2/en
Priority to US10/146,792 priority patent/US20030207428A1/en
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Priority to US10/147,528 priority patent/US20030219885A1/en
Priority to US10/147,519 priority patent/US20030077791A1/en
Priority to US10/147,492 priority patent/US20030082765A1/en
Priority to US10/147,536 priority patent/US20040077064A1/en
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
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Priority to US10/160,498 priority patent/US20030073216A1/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

Abstract

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

Description

PROMOTION OR INHIBITION OF ANGIOGENESIS AND CARDIOVASCULARIZATION
Background of the Invention Field of the Invention
The present invention relates to compositions and methods useful for promoting or inhibiting angiogenesis and/or cardiovascuiarization in mammals in need of such biological effect. This includes the diagnosis and treatment of cardiovascular disorders as well as oncological disorders.
Description of Background
A. Cardiac Disorders and Factors
Heart failure affects approximately five million Americans, and new cases of heart failure number about 400,000 each year. It is the single most frequent cause of 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 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. See, review by Brown and Vaughan, Circulation, 97: 141 1-1420 (1998). As noted below, angiotensin II may also have directly deleterious effects on the heart by promoting myocyte necrosis (impairing systolic function) and intracardiac fibrosis (impairing diastolic and in some cases systolic function). See, Weber, Circulation, 96: 4065-4082 (1998).
A consistent feature of congestive heart failure (CHF) is cardiac hypertrophy, an enlargement of the heart that is activated by both mechanical and hormonal stimuli and enables the heart to adapt to demands for increased cardiac output. Morgan and Baker, Circulation, 83: 13-25 (1991 ). This hypertrophic response is frequently associated with a variety of distinct pathological conditions such as hypertension, aortic stenosis, myocardial infarction, 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.
Adult myocyte hypertrophy is initially beneficial as a short term response to impaired cardiac function by permitting a decrease in the load on individual muscle fibers. With severe, long-standing overload, however, the hypertrophied cells begin to deteriorate and die. Katz, "Heart Failure", in: Katz A.M. ed., Physiology of the Heart (New York: Raven Press, 1992) pp. 638-668. Cardiac hypertrophy is a significant risk factor for both mortality and morbidity in the clinical course of heart failure. Katz, Trends Cardiovasc. Med.. 5: 37-44 (1995). For further details of the causes and pathology of cardiac hypertrophy see, e.g., Heart Disease, A Textbook of Cardiovascular Medicine, Braunwald, E. ed. (W.B. Saunders Co., 1988), Chapter 14, "Pathophysiology of Heart Failure."
On a cellular level, the heart is composed of myocytes and surrounding support cells, 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.,
Annu. Rev. Physiol., 55: 77-95 (1993). An increment in myocardial mass as a result of an increase in myocyte size that is associated with an accumulation of interstitial collagen within the extracellular matrix and around intramyocardial coronary arteries has been described in left ventricular hypertrophy secondary to pressure overload in humans. Caspari et al., Cardiovasc. Res.. U : 554-558 (1977); Schwarz et al., Am. J. Cardiol., 42: 895-903 (1978); Hess et al.. Circulation, 63: 360-371 (1981 ; Pearlman et al.. Lab. Invest., 46: 158-164 (1982).
It has also been suggested that paracrine factors produced by non-myocyte supporting cells may additionally be involved in the development of cardiac hypertrophy, and various non-myocyte derived hypertrophic factors, such as, leukocyte inhibitory factor (LIF) and endothelin, have been identified. Metcalf, Growth Factors, 7: 169-173 (1992); Kurzrock et al., Endocrine Reviews. J2: 208-217 (1991); Inoue et al., Proc. Natl. Acad. Sci. USA. 86: 2863-2867 (1989); Yanagisawa and Masaki, Trends Pharm. Sci., ]0: 374-378 (1989); U.S. Patent No.
5,573,762 (issued November 12, 1996). Further exemplary factors that have been identified as potential mediators of cardiac hypertrophy include cardiotrophin-1 (CT-1) (Pennica et al., Proc. Nat. Acad. Sci. USA. 92: 1 142-1 146 (1995)), catecholamines, adrenocorticosteroids, angiotensin, and prostaglandins
At present, the treatment of cardiac hypertrophy varies depending on the underlying cardiac disease Catecholamines, adrenocorticosteroids, angiotensin, prostaglandins, LIF, endothelin (including endothelin- 1, -2, and -3 and big endothelin), and CT-1 are among the factors identified as potential mediators of hypertrophy For example, beta-adrenergic receptor blocking drugs (beta-blockers, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, carvedilol, etc ) and verapamil have been used extensively in the treatment of hypertrophic cardiomyopathy The beneficial effects of beta-blockers on symptoms (e g , chest pain) and exercise tolerance are largely due to a decrease in the heart rate with a consequent prolongation of diastole and increased passive ventricular filling Thompson etal , Br Heart J , 44 488-98 (1980), Harrison et al , Circulation, 29 84-98 (1964) Verapamil has been described to improve ventricular filling and probably reducing myocardial ischemia Bonow et al , Circulation, 72 853-64 (1985)
Nifedipine and diltiazem have also been used occasionally in the treatment of hypertrophic cardiomyopathy Lorell et al , Circulation. 65 499-507 (1982), Betocchi et al , Am J Cardiol , 78 451-457 (1996) However, because of its potent vasodilating properties, nifedipine may be harmful, especially in patients with outflow obstruction Disopyramide has been used to relieve symptoms by virtue of its negative inotropic properties Pollick, N Engl J Med , 307 997-999 ( 1982) In many patients, however, the initial benefits decrease with time Wigle et al , Circulation, 92 1680-1692 (1995) Antihypertensive drug therapy has been reported to have beneficial effects on cardiac hypertrophy associated with elevated blood pressure Examples of drugs used in antihypertensive therapy, alone or in combination, are calcium antagonists, e g , nitrendipine, adrenergic receptor blocking agents, e g , those listed above, angiotensin converting enzyme (ACE) inhibitors such as quinapπl, captopπl, enalapπl, ramipπl, benazepπl, fosinopπl, and lisinopπl, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothιazιde,benzthιazιde,dιchloφhenamιde, acetazolamιde, and indapamide, and calcium channel blockers, e g , diltiazem, nifedipine, verapamil, and nicardipine
For example, treatment of hypertension with diltiazem and captopπl showed a decrease in left ventricular muscle mass, but the Doppler indices of diastolic function did not normalize Szlachcic et al , Am J Cardiol , 63
198-201 (1989), Shahi et al , Lancet, 336 458-461 (1990) These findings were interpreted to indicate that excessive amounts of interstitial collagen may remain after regression of left ventricular hypertrophy Rossi et al , Am Heart J . 124 700-709 (1992) Rossi et al , supra, investigated the effect of captopπl on the prevention and regression of myocardial cell hypertrophy and interstitial fibrosis in pressure overload cardiac hypertrophy, in experimental rats
Agents that increase cardiac contractility directly (lontropic agents) were initially thought to benefit patients with heart failure because they improved cardiac output in the short term However, all positive inotropic agents except digoxigenin have been found to result in increased long-term mortality, in spite of short-term improvements in cardiac performance Massie, Curr Op in Cardiology, \2 209-217 (1997), Reddy et al , Curr Opin Cardiol , 12 233-241 (1997) Beta-adrenergic receptor blockers have recently been advocated for use in heart failure Evidence from clinical trials suggests that improvements in cardiac function can be achieved without increased mortality, though documented improvements patient survival have not yet been demonstrated See also, U.S. Pat. Nos. 5,935,924, 5,624,806; 5,661 ,122; and 5,610,134 and WO 95/28173 regarding the use of cardiotropin-1 or antagonists thereof, or growth hormone and/or insulin-like growth factor-I in the treatment of CHF. Another treatment modality is heart transplantation, but this is limited by the availability of donor hearts. 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.
Current therapy for heart failure is primarily directed to using angiotensin-converting enzyme (ACE) inhibitors, such as captopril, and diuretics. These drugs improve hemodynamic profile and exercise tolerance and reduce the incidence of morbidity and mortality in patients with CHF. Kramer et al.. Circulation, 67(4): 807-816
(1983); Captopril Multicenter Research Group, J.A.C.C. 2(4): 755-763 (1983); The CONSENSUS Trial Study Group, N. Engl. J. Med., 316(23): 1429-1435 (1987); The SOLVD Investigators, N. Engl. J. Med..325(5): 293-302 (1991). Further, they are useful in treating hypertension, left ventricular dysfunction, atherosclerotic vascular disease, and diabetic nephropathy. Brown and Vaughan, supra. However, despite proven efficacy, response to ACE inhibitors has been limited. For example, while prolonging survival in the setting of heart failure, ACE inhibitors appear to slow the progression towards end-stage heart failure, and substantial numbers of patients on ACE inhibitors have functional class III heart failure.
Moreover, improvement of functional capacity and exercise time is only small and mortality, although reduced, continues to be high. The CONSENSUS Trial Study Group, N. Engl. J. Med., 316(23): 1429-1453 (1987); The SOLVD Investigators, N. Engl. J. Med., 325(5): 293-302 (1991 ); Cohn et al., N. Engl. J. Med., 325(5):
303-310 (1991); The Captopril-Digoxin Multicenter Research Group, JAMA, 259(4): 539-544 (1988). Hence, ACE inhibitors consistently appear unable to relieve symptoms in more than 60% of heart failure patients and reduce mortality of heart failure only by approximately 15-20%. For further adverse effects, see Brown and Vaughan, supra. An alternative to ACE inhibitors is represented by specific ATI receptor antagonists. Clinical studies are planned to compare the efficacy of these two modalities in the treatment of cardiovascular and renal disease. However, animal model data suggests that the ACE/Ang II pathway, while clearly involved in cardiac hypertrophy, is not the only, or even the primary pathway active in this role. Mouse genetic "knockout" models have been made to test individual components of the pathway. In one such model, the primary cardiac receptor for Ang II, AT sub IA, has been genetically deleted; these mice do not develop hypertrophy when Ang II is given experimentally
(confirming the basic success of the model in eliminating hypertrophy secondary to Ang II). However, when the aorta is constricted in these animals (a model of hypertensive cardiac stress), the hearts still become hypertrophic. This suggests that alternative signaling pathways, not depending on this receptor (AT sub 1 A), are activated in hypertension ACE inhibitors would presumably not be able to inhibit these pathways See, Harada et al , Circulation, 97 1952-1959 (1998) See also. Homey, Circulation, 97 1890-1892 (1998) regarding the enigma associated with the process and mechanism of cardiac hypertrophy About 750,000 patients suffer from acute myocardial infarction (AMI) annually, and approximately one-fourth of all deaths in the United States are due to AMI In recent years, thrombolytic agents, e g , streptokinase, urokinase, and in particular tissue plasminogen activator (t-PA) have significantly increased the survival of patients who suffered myocardial infarction When administered as a continuous intravenous infusion over 1 5 to 4 hours, t-PA produces coronary patency at 90 minutes in 69% to 90% of the treated patients Topol et al , km J Cardiol , 61 , 723-728 (1988), euhaus e α/ , J Am Coll Cardiol . 12 581-587 (1988), Neuhaus et al , J Am Coll Cardiol , J_4 1566-1569 (1989) The highest patency rates have been reported with high dose or accelerated dosing regimens Topol, J Am Coll Cardiol , 15 922-924 (1990) t-PA may also be administered as a single bolus, although due to its relatively short half-life, it is better suited for infusion therapy Tebbe et al , Am J Cardiol , 64 448-453 (1989) A t-PA variant, specifically designed to have longer half-life and very high fibrin specificity, TNK t-PA (a T103N, N 1 17Q, KHRR(296-299)AAAA t-PA variant, Keyt etal , ?roc Natl Acad
Sci USA, 91 3670-3674 (1994)) is particularly suitable for bolus administration However, despite all these advances, the long-term prognosis of patient survival depends greatly on the post-infarction monitoring and treatment of the patients, which should include monitoring and treatment of cardiac hypertrophy
B Growth Factors
Various naturally occurring polypeptides reportedly induce the proliferation of endothelial cells Among those polypeptides are the basic and acidic fibroblast growth factors (FGF) (Burgess and Maciag, Annual Rev Biochem , 58 575 (1989)), platelet-derived endothelial cell growth factor (PD-ECGF) (Ishikawa et al , Nature, 338 557 ( 1989)), and vascular endothelial growth factor (VEGF) Leung et al , Science, 246 1306 ( 1989), Ferrara and Henzel, Biochem Biophys Res Commun . 161 851 (1989), Tischer et al , Biochem Biophvs Res Commun ,
165 1 198 ( 1989), EP 471 ,754B granted July 31 , 1996
Media conditioned by cells transfected with the human VEGF (hVEGF) cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not Leung et al , Science, 246 1306 ( 1989) Several additional cDNAs were identified in human cDNA libraries that encode 121 -, 189-, and 206-amιno acid isoforms of hVEGF (also collectively referred to as hVEGF-related proteins) The 121-amιno acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 1 16 and 159 in hVEGF The 189-amιno acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 1 16 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF) The 206-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 , Mol Endocπn . 5 1806 (1991 ), Ferrara et al . 5 Cell Biochem . 47 21 1 (1991), Ferrara et al . Endocrine Reviews, 13 18 (1992), Keck et al , Science, 246 1309 ( 1989), Connolly et al , J Biol Chem . 264 20017 ( 1989), EP 370,989 published May 30, 1990 It is now well established that angiogenesis, which involves the formation of new blood vessels from preexisting endothelium, is implicated in the pathogenesis of a variety of disorders These include solid tumors and metastasis, atherosclerosis, retrolental 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 . J Biol Chem , 267 10931-10934 (1992), Klagsbrun gf α/ , Annu Rev Phvsiol , 53 217- 239 (1991), and Garner A , "Vascular diseases" In Pathobiology of Ocular Disease A Dynamic Approach. Garner A , K ntworth GK, eds , 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710
In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasm, and for providing nourishment to the growing solid tumor Folkman et al . Nature, 339 58 (1989) The neovasculaπzation allows the tumor cells to acquire a growth advantage and 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), Macchiaπm et al , Lancet, 340 145-146 (1992) The search for positive regulators of angiogenesis has yielded many candidates, including aFGF, bFGF, TGF- α, TGF-β, HGF, TNF-α, angiogenm, IL-8, etc Folkman et al , J B C , supra, and Klagsbrun et al , supra The negative regulators so far identified include thrombospondin (Good et αl , Proc Natl Acad Sci USA . 87 6624- 6628 (1990)), the 16-kilodalton N-terminal fragment of prolactin (Clapp et αl , Endocrinology, 133 1292-1299 (1993)). angiostatin (O'Reilly et αl . Cell, 79 315-328 (1994)), and endostatin O'Reilly et αl , Ceh, 88 277-285 (1996)
Work done over the last several years has established the key role of VEGF, not only in stimulating vascular endothelial cell proliferation, but also in inducing vascular permeability and angiogenesis Ferrara et αl , Endocr Rev , 18 4-25 ( 1997) The finding that the loss of even a single VEGF allele results in embryonic lethality points to an irreplaceable role played by this factor in the development and differentiation of the vascular system Furthermore, VEGF has been shown to be a key mediator of neovasculaπzation associated with tumors and intraocular disorders Ferrara et αl , Endocr Rev . supra The VEGF mRNA is overexpressed by the majority of human tumors examined Berkman et al , J Clin Invest , 91 153-159 (1993), Brown et al , Human Pathol , 26 86-91 (1995). Brown et al . Cancer Res , 53 4727-4735 (1993). Mattern e/ α/ , Brit J Cancer, 73 931-934 (1996), Dvorak et al . Am J Pathol , 146 1029-1039 (1995) Also, the concentration levels of VEGF in eye fluids are highly correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies Aiello et al , N Engl J Med , 331 1480-1487 (1994) Furthermore, recent studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by AMD Lopez et al , Invest Ophthalmol Vis Sci , 37 855-868 (1996) Anti-VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in nude mice
(Kim et l Nature, 362 841 -844 (1993), Warren et al . J Clin Invest , 95 1789-1797 (1995), Borgstrom et al Cancer Res , 56 4032-4039 ( 1996), Melnyk et al , Cancer Res , 56 921 -924 ( 1996)) and also inhibit intraocular angiogenesis in models of ischemic retinal disorders. Adamis et al., Arch. Ophthalmol., 1 14: 66-71 (1996). Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of solid tumors and various intraocular neovascular disorders. Such antibodies are described, for example, in EP 817,648 published January 14, 1998 and in PCT/US 98/06724 filed April 3, 1998. There exist several other growth factors and mitogens, including transforming oncogenes, that are capable of rapidly inducing a complex set of genes to be expressed by certain cells. Lau and Nathans, Molecular Aspects of Cellular Regulation. 6: 165-202 ( 1991 ). These genes, which have been named immediate-early- or early-response genes, are transcriptionally activated within minutes after contact with a growth factor or mitogen, independent of de novo protein synthesis. A group of these intermediate-early genes encodes secreted, extracellular proteins that are needed for coordination of complex biological processes such as differentiation and proliferation, regeneration, and wound healing. Ryseck et al, Cell Growth Differ., 2: 235-233 (1991).
Highly-related proteins that belong to this group include ceflO (Simmons et al. , Proc. Natl. Acad. Sci. USA, 86: 1 178-1 182 (1989)), cyr 61, which is rapidly activated by serum- or platelet-derived growth factor (PDGF) (O'Brien et al., Mol. Cell Biol., 10: 3569-3577 (1990), human connective tissue growth factor (CTGF) (Bradham et al, J. Cell. Biol.. 1 14: 1285-1294 (1991 )), which is secreted by human vascular endothelial cells in high levels after activation with transforming growth factor beta (TGF-β), exhibits PDGF-like biological and immunological activities, and competes with PDGF for a particular cell surface receptor, fιsp-12 Ryseck et al, Cell Growth Differ., 2: 235-233 (1991 )), human vascular IBP-like growth factor (VIGF) (WO 96/17931), and nov, normally arrested in adult kidney cells, which was found to be overexpressed in myeloblastosis-associated-virus-type-1- induced nephroblastomas. Joloit et al, Mol. Cell. Biol., 12: 10-21 ( 1992).
The expression of these immediate-early genes acts as "third messengers" in the cascade of events triggered by growth factors. It is also thought that they are needed to integrate and coordinate complex biological processes, such as differentiation and wound healing in which cell proliferation is a common event.
As additional mitogens, insulin-like growth factor binding proteins (IGFBPs) have been shown, in complex with insulin-like growth factor (IGF), to stimulate increased binding of IGF to fibroblast and smooth muscle cell surface receptors. Clemmons et l, }. Clin. Invest., 77: 1548 (1986). Inhibitory effects of IGFBP on various IGF actions in vitro include stimulation of glucose transport by adipocytes, sulfate incoφoration by chondrocytes, and thymidine incoφoration in fibroblast. Zapf et al, J. Clin. Invest., 63: 1077 ( 1979). In addition, inhibitory effects of IGFBPs on growth factor-mediated mitogen activity in normal cells have been shown.
C. Need for Further Treatments
In view of the role of vascular endothelial cell growth and angiogenesis in 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.
Summary of the Invention A. Embodiments
Accordingly, 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. In one embodiment, the present invention provides a composition comprising a PRO polypeptide in admixture with a pharmaceutically acceptable carrier. In one aspect, the composition comprises a therapeutically effective amount of the polypeptide. In another aspect, the composition comprises a further active ingredient, namely, a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent. Preferably, the composition is sterile. The 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.
In a further embodiment, the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of a PRO polypeptide with a pharmaceutically acceptable carrier.
In another embodiment, the present invention provides a composition comprising an agonist or antagonist of a PRO polypeptide in admixture with a pharmaceutically acceptable carrier. In one aspect, the composition comprises a therapeutically effective amount of the agonist or antagonist. In another aspect, the composition comprises a further active ingredient, namely, a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent. Preferably, the composition is sterile. The 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.
In a further embodiment, the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of a PRO polypeptide agonist or antagonist with a pharmaceutically acceptable carrier.
In yet another embodiment, the present invention concerns a composition comprising an anti-PRO antibody in admixture with a pharmaceutically acceptable carrier. In one aspect, the composition comprises a therapeutically effective amount of the antibody. In another aspect, the composition comprises a further active ingredient, namely, a cardiovascular, endothelial or angiogenic agent or an angiostatic agent, preferably an angiogenic or angiostatic agent. Preferably, the composition is sterile. The 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. In preferred embodiments, the antibody is a monoclonal antibody, an antibody fragment, a humanized antibody, or a single-chain antibody.
In a further embodiment, the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of an anti-PRO antibody with a pharmaceutically acceptable carrier.
In a still further aspect, the present invention provides an article of manufacture comprising:
(a) a composition of matter comprising a PRO polypeptide or agonist or antagonist thereof;
(b) a container containing said composition; and (c) a label affixed to said container, or a package insert included in said container referring to the use of said
PRO polypeptide or agonist or antagonist thereof in the treatment of a cardiovascular, endothelial or angiogenic disorder, wherein the agonist or antagonist may be an antibody which binds to the PRO polypeptide. The composition may comprise a therapeutically effective amount of the PRO polypeptide or the agonist or antagonist thereof. In another embodiment, the present invention provides a method for identifying an agonist of a PRO polypeptide comprising:
(a) contacting cells and a test compound to be screened under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide; and
(b) determining the induction of said cellular response to determine if the test compound is an effective agonist, wherein the induction of said cellular response is indicative of said test compound being an effective agonist.
In another embodiment, the present invention provides a method for identifying an agonist of a PRO polypeptide comprising:
(a) contacting cells and a test compound to be screened under conditions suitable for the stimulation of cell proliferation by a PRO polypeptide; and
(b) measuring the proliferation of said cells to determine if the test compound is an effective agonist, wherein the stimulation of cell proliferation is indicative of said test compound being an effective agonist.
In another embodiment, the invention provides a method for identifying a compound that inhibits the activity of a PRO polypeptide comprising contacting a test compound with a PRO polypeptide under conditions and for a time sufficient to allow the test compound and polypeptide to interact and determining whether the activity of the
PRO polypeptide is inhibited. In a specific preferred aspect, either the test compound or the PRO polypeptide is immobilized on a solid support. In another preferred aspect, the non-immobilized component carries a detectable label. In a preferred aspect, this method comprises the steps of:
(a) contacting cells and a test compound to be screened in the presence of a PRO polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide; and
(b) determining the induction of said cellular response to determine if the test compound is an effective antagonist.
In another preferred aspect, this process comprises the steps of:
(a) contacting cells and a test compound to be screened in the presence of a PRO polypeptide under conditions suitable for the stimulation of cell proliferation by a PRO polypeptide; and
(b) measuring the proliferation of the cells to determine if the test compound is an effective antagonist. In another embodiment, the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that normally expresses the polypeptide, wherein the method comprises contacting the cells with a test compound and determining whether the expression of the PRO polypeptide is inhibited. In a preferred aspect, this method comprises the steps of:
(a) contacting cells and a test compound to be screened under conditions suitable for allowing expression of the PRO polypeptide; and
(b) determining the inhibition of expression of said polypeptide.
In a still further embodiment, the invention provides a compound that inhibits the expression of a PRO polypeptide, such as a compound that is identified by the methods set forth above.
Another aspect 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.
One type of antagonist of a PRO polypeptide that inhibits one or more of the functions or activities of the PRO polypeptide is an antibody. Hence, in another aspect, the invention provides an isolated antibody that binds a PRO polypeptide. In a preferred aspect, the antibody is a monoclonal antibody, which preferably has non-human complementarity-determining-region (CDR) residues and human framework-region (FR) residues. The antibody may be labeled and may be immobilized on a solid support. In a further aspect, the antibody is an antibody fragment, a single-chain antibody, or a humanized antibody. Preferably, the antibody specifically binds to the polypeptide.
In a still further aspect, the present invention provides a method for diagnosing a disease or susceptibility to a disease which is related to a mutation in a PRO polypeptide-encoding nucleic acid sequence comprising determining the presence or absence of said mutation in the PRO polypeptide nucleic acid sequence, wherein the presence or absence of said mutation is indicative of the presence of said disease or susceptibility to said disease.
In a still further aspect, the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises analyzing the level of expression of a gene encoding a 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.
In a still further aspect, the present invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises detecting the presence or absence of a PRO polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of said PRO polypeptide in said test sample is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in said mammal.
In a still further embodiment, the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal comprising (a) contacting an anti-PRO antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the PRO polypeptide in the test sample, wherein the formation of said complex is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in the mammal. The detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells 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.
In another embodiment, the invention provides a method for determining the presence of a PRO polypeptide in a sample comprising exposing a sample suspected of containing the PRO polypeptide to an anti-PRO antibody and determining binding of said antibody to a component of said sample. In a specific aspect, the sample comprises a cell suspected of containing the PRO polypeptide and the antibody binds to the cell. The antibody is preferably detectably labeled and/or bound to a solid support.
In further aspects, the invention provides a cardiovascular, endothelial or angiogenic disorder diagnostic kit comprising an anti-PRO antibody and a carrier in suitable packaging. Preferably, such kit further comprises instructions for using said antibody to detect the presence of the PRO polypeptide. Preferably, the carrier is a buffer, for example. Preferably, the cardiovascular, endothelial or angiogenic disorder is cancer.
In yet another embodiment, the present invention provides a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of a PRO polypeptide. Preferably, the disorder is cardiac hypertrophy, trauma such as wounds or burns, or a type of cancer. In a further aspect, the mammal is further exposed to angioplasty or a drug that treats cardiovascular, endothelial or angiogenic disorders such as ACE inhibitors or chemotherapeutic agents if the cardiovascular, endothelial or angiogenic disorder is a type of cancer. Preferably, the mammal is human, preferably one who is at risk of developing cardiac hypertrophy and more preferably has suffered myocardial infarction.
In another preferred aspect, the cardiac hypertrophy is characterized by the presence of an elevated level of PGF2α. Alternatively, 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. In another preferred embodiment, the cardiovascular, endothelial or angiogenicdisorderis 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 puφose 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.
In another preferred aspect, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy and the PRO polypeptide is administered following primary angioplasty for the treatment of acute myocardial infarction, preferably wherein the mammal is further exposed to angioplasty or a cardiovascular, endothelial, or angiogenic agent.
In another preferred embodiment, the cardiovascular, endothelial or angiogenic disorder is a cancer and the PRO polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent or a cytotoxic agent.
In a further embodiment, the invention 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. Preferably, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration. Also preferred is where the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the agonist.
In a further embodiment, the invention concerns a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of an antagonist of a PRO polypeptide. Preferably, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration. Also preferred is where the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the antagonist.
In a further embodiment, the invention concerns a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of an anti-PRO antibody. Preferably, the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration. Also preferred is where the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the antibody.
In still further embodiments, the invention provides a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal that suffers therefrom comprising administering to the mammal a nucleic acid molecule that codes for either (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide or (c) an antagonist of a PRO polypeptide, wherein said agonist or antagonist may be an anti-PRO antibody. In a preferred embodiment, the mammal is human. In another preferred embodiment, the gene is administered via ex vivo gene therapy. In a further preferred embodiment, the gene is comprised within a vector, more preferably an adenoviral, adeno-associated viral, lentiviral, or retroviral vector.
In yet another aspect, the invention 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 Preferably, the signal sequence is from a mammal, such as from a native PRO polypeptide
In a still further embodiment, the invention supplies an ex vivo producer cell comprising a nucleic acid construct that expresses retroviral structural proteins and also comprises a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptide of a PRO polypeptide or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein said producer cell packages the retroviral vector in association with the structural proteins to produce recombinant retroviral particles In yet another embodiment, the invention provides a method for inhibiting endothelial cell growth in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein endothelial cell growth in said mammal is inhibited, and wherein said agonist or antagonist may be an anti-PRO antibody Preferably, the mammal is human and the endothelial cell growth is associated with a tumor or a retinal disorder In yet another embodiment, the invention provides a method for stimulating endothelial cell growth in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein endothelial cell growth in said mammal is stimulated, and wherein said agonist or antagonist may be an anti-PRO antibody Preferably, the mammal is human
In yet another embodiment, the invention provides a method for inhibiting cardiac hypertrophy in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is inhibited, and wherein said agonist or antagonist may be an anti-PRO antibody Preferably, the mammal is human and the cardiac hypertrophy has been induced by myocardial infarction
In yet another embodiment, the invention provides a method for stimulating cardiac hypertrophy in a mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is stimulated, and wherein said agonist or antagonist may be an anti-PRO antibody Preferably, the mammal is human who suffers from congestive heart failure
In yet another embodiment, the invention provides a method for inhibiting angiogenesis induced by a PRO polypeptide in a mammal comprising administering a therapeutically effective amount of an anti-PRO antibody to the mammal Preferably, the mammal is a human, and more preferably the mammal has a tumor or a retinal disorder
In yet another embodiment, the invention provides a method for stimulating angiogenesis induced by a PRO polypeptide in a mammal comprising administering a therapeutically effective amount of a PRO polypeptide to the mammal Preferably, the mammal is a human, and more preferably angiogeneisis would promote tissue regeneration or wound healing B. Additional Embodiments
In other embodiments of the present invention, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% 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 ldueenπtuitiyy,, y yeeti mmoυrree prreefiecriaabDliyy a ati l iecaassti a abuouuuti 8 o7 /% vo s seeqquueennccee i ldueeniitiintyy,, y yeetu miiourree prreefieerraabDliyy a ati l leeaassti abυoouuti 8 o8a%/o s seeqquueennccee iiddeennttiittyy,, yyeett mmoorree pprreeffeerraabbllyy aatt lleeaasstt aabboouutt 8899%% sseeqquueennccee iiddeennttiittyy,, yyeett mmoorree pprreeffeerraabbllyy aatt lleeaasstt aabboouutt 9900%% sseeqquueennccee iiddeennttiittyy,, yyeett mmoorree 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% sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
In other aspects, 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% sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PRO polypeptide cDNA as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a). In a further aspect, 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 least about 99% sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a).
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. Such nucleic acid fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, 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 nucleotides in length, yet more preferably at least about 180 nucleotides in length, yet more preferably at least about 190 nucleotides in length, yet more preferably at least about 200 nucleotides in length, yet more preferably at least about 250 nucleotides in length, yet more preferably at least about 300 nucleotides in length, yet more preferably at least about 350 nucleotides in length, yet more preferably at least about 400 nucleotides in length, yet more preferably at least about 450 nucleotides in length, yet more preferably at least about 500 nucleotides in length, yet more preferably at least about 600 nucleotides in length, yet more preferably at least about 700 nucleotides in length, yet more preferably at least about 800 nucleotides in length, yet more preferably at least about 900 nucleotides in length and yet more preferably at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence 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.
In another embodiment, the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
In a certain aspect, 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% sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein.
In a further aspect, 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% sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein. In a further aspect, 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 compared with the amino acid sequence of a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an ammo acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein
In a specific aspect, the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and 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
In yet another embodiment, the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein In a particular embodiment, the agonist or antagonist is an anti-PRO antibody or a small molecule In a further embodiment, the invention 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 Preferably, the PRO polypeptide is a native PRO polypeptide
In a still further embodiment, 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 Optionally, 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 In additional embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides Host cell comprising any such vector are also provided By way of example, 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.
In other embodiments, 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.
In yet another embodiment, the invention provides an antibody which specifically binds to any of the above orbelow described polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences.
Brief Description of the Drawings
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.
Detailed Description of the Invention I. Definitions
The phrases "cardiovascular, endothelial and angiogenic disorder", "cardiovascular, endothelial and angiogenic dysfunction", "cardiovascular, endothelial or angiogenic disorder" and "cardiovascular, endothelial or angiogenic disfunction" are used interchangeably and refer in part to systemic disorders that affect vessels, such as diabetes 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, and lymphangiosarcoma, tumor angiogenesis, trauma such as wounds, burns, and other injured tissue, implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. This would also include angina, myocardial infarctions such as acute myocardial infarctions, cardiac hypertrophy, and heart failure such as CHF.
"Hypertrophy", as used herein, is defined as an increase in mass of an organ or structure independent of natural growth that does not involve tumor formation. Hypertrophy of an organ or tissue is due either to an increase in the mass of the individual cells (true hypertrophy), or to an increase in the number of cells making up the tissue (hypeφlasia), 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 moφhologically by increases in the size of myofibrils and mitochondria, as well as by enlargement of mitochondria and nuclei. At this stage, while muscle cells are larger than normal, cellular organization is largely preserved. At a more advanced stage of cardiac hypertrophy, there are preferential increases in the size or number of specific organelles, such as mitochondria, and new contractile elements are added in localized areas of the cells, in an irregular manner. Cells subjected to long-standing hypertrophy show more obviousdisruptions in cellular organization, including markedly enlarged nuclei with highly lobulated membranes, which displace adjacent myofibrils and cause breakdown of normal Z-band registration. The phrase "cardiac hypertrophy" is used to include all stages 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.
"Congestive heart failure" (CHF) is a progressive pathologic state where the heart is increasingly unable to supply adequate cardiac output (the volume of blood pumped by the heart over time) to deliver the oxygenated blood to peripheral tissues. As CHF progresses, structural and hemodynamic damages occur. While these damages have a variety of manifestations, one characteristic symptom is ventricular hypertrophy. CHF is a common end result of a number of various cardiac disorders.
"Myocardial infarction" generally results from atherosclerosis of the coronary arteries, often with superimposed coronary thrombosis. It may be divided into two major types: transmural infarcts, in which myocardial necrosis involves the full thickness of the ventricular wall, and subendocardial (nontransmural) infarcts, in which the necrosis involves the subendocardium, the intramural myocardium, or both, without extending all the way through the ventricular wall to the epicardium Myocardial infarction is known to cause both a change in hemodynamic effects and an alteration in structure in the damaged and healthy zones of the heart Thus, for example, myocardial infarction reduces the maximum cardiac output and the stroke volume of the heart Also associated with myocardial infarction is a stimulation of the DNA synthesis occurring in the interstice as well as an increase in the formation of collagen in the areas of the heart not affected
As a result of the increased stress or strain placed on the heart in prolonged hypertension due, for example, to the increased total peripheral resistance, cardiac hypertrophy has long been associated with "hypertension" A characteristic of the ventricle that becomes hypertrophic as a result of chronic pressure overload is an impaired diasto c performance Fouad e/a/ . J Am Coll Cardiol . 4 1500-1506 (1984), Smith et al , J Am Coll Cardiol , 5 869-874 ( 1985) A prolonged left ventricular relaxation has been detected in early essential hypertension, in spite of normal or supranormal systolic function Hartford et al . Hypertension. 6 329-338 (1984) However, there is no close parallelism between blood pressure levels and cardiac hypertrophy Although improvement in left ventricular function in response to antihypertensive therapy has been reported in humans, patients variously treated with a diuretic (hydrochlorothiazide), a β-blocker (propranolol), or a calcium channel blocker (diltiazem), have shown reversal of left ventricular hypertrophy, without improvement in diastohc function Inouye et al , Am J Cardiol , 53 1583-7 ( 1984)
Another complex cardiac disease associated with cardiac hypertrophy is "hypertrophic cardiomyopathy" This condition is characterized by a great diversity of moφhologic, functional, and clinical features (Maron et al ,
N Engl J Med , 316 780-789 ( 1987). Spiπto e/ / . N Engl J Med , 320 749-755 ( 1989), Louie and Edwards, Prog Cardiovasc Dis , 36 275-308 ( 1994), Wigle et al , Circulation, 92 1680- 1692 ( 1995)), the heterogeneity of which is accentuated by the fact that it afflicts patients of all ages Spiπto et al , N Engl J Med . 336 775-785 ( 1997) The causative factors of hypertrophic cardiomyopathy are also diverse and little understood In general, mutations in genes encoding sarcomeπc proteins are associated with hypertrophic cardiomyopathy Recent data suggest that β-myosin heavy chain mutations may account for approximately 30 to 40 percent of cases of familial hypertrophic cardiomyopathy Watkins e/ a/ , N Engl J Med , 326 1 108-1 1 14 (1992), Schwartz et al. Circulation, 9 532-540 (1995), Marian and Roberts. Circulation, 92 1336-1347 (1995), Thierfelder e/ / , Cell, 77 701 -712 (1994), Watkins et al , Nat Gen , 1 1 434-437 ( 1995) Besides β-myosin heavy chain, other locations of genetic mutations include cardiac troponin T, alpha topomyosin, cardiac myosin binding protein C, essential myosin light chain, and regulatory myosin light chain See, Malik and Watkins, Curr Opin Cardiol , V2 295-302 ( 1997)
Supravalvular "aortic stenosis" is an inherited vascular disorder characterized by narrowing of the ascending aorta, but other arteries, including the pulmonary arteries, may also be affected Untreated aortic stenosis may lead to increased intracardiac pressure resulting in myocardial hypertrophy and eventually heart failure and death The pathogenesis of this disorder is not fully understood, but hypertrophy and possibly hypeφlasia 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 atπo ventricular orifices and operation of the heart Defects such as the narrowing of the valve stenosis or the defective closing of the valve result in an accumulation of blood in the heart cavity or regurgitation of blood past the valve If uncorrected, prolonged valvular stenosis or insufficiency may result in cardiac hypertrophy and associated damage to the heart muscle, which may eventually necessitate valve replacement
The treatment of all these, and other cardiovascular, endothelial and angiogenic disorders, which may or may not be accompanied by cardiac hypertrophy, is encompassed by the present invention The terms "cancer", "cancerous", and "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth Examples of cancer include but are not limited to, carcinoma including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's 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, endometπal carcinoma, salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer The preferred cancers for treatment herein are breast, colon, lung, melanoma, ovarian, and others involving vascular tumors as noted above The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells The term is intended to include radioactive isotopes (e g , nιI, 12SI, 90Y, and l86Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer Examples of chemotherapeutic agents include alkylating agents, folic acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics, pyπmidine analogs, 5-fluorouracιl, cisplatin, puπne nucleosides, amines, amino acids, triazol nucleosides, or corticosteroids Specific examples include Adπamycin, Doxorubicin, 5-Fluorouracιl, Cytos e arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopteπn, Dactinomycin, Mitomycins,
Esperamicins (see U S Pat No 4,675,187), Melphalan, and other related nitrogen mustards Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors, such as tamoxifen and onapπstone
A "growth-inhibitory agent" when used herein refers to a compound or composition that inhibits growth of a cell, such as an Wnt-overexpressing cancer cell, either in vitro or in vivo Thus, the growth-inhibitory agent is one which significantly reduces the percentage of malignant cells in S phase Examples of growth-inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G 1 arrest and M-phase arrest Classical M-phase blockers include the vincas (vincπstine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, daunorubicin, etoposide, and bleomycin Those agents that arrest Gl also spill over mto S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracιl, and ara-C Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds , Chapter 1 , entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al (WB Saunders Philadelphia, 1995), especially p 13 Additional examples include tumor necrosis factor (TNF), an antibody capable of inhibiting or neutralizing the angiogenic activity of acidic or basic FGF or 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)
"Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a cardiovascular, endothelial, and angiogenic disorder The concept of treatment is used in the broadest sense, and specifically includes the prevention (prophylaxis), moderation, reduction, and curing of cardiovascular, endothelial, and angiogenic disorders of any stage Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a cardiovascular, endothelial, and angiogenic disorder such as hypertrophy Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented The disorder may result from any cause, including ldiopathic, cardiotrophic, or myotrophic causes, or ischemia or 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 puφoses of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, pigs, etc Preferably, the mammal is human
Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order
The phrase "cardiovascular, endothelial or angiogenic agents" refers geneπcally to any drug that acts in treating cardiovascular, endothelial, and angiogenic disorders Examples of cardiovascular agents are those that promote vascular homeostasis by modulating blood pressure, heart rate, heart contractility, and endothelial and smooth muscle biology, all of which factors have a role in cardiovascular disease Specific examples of these include angιotensιn-II receptor antagonists, endothelin receptor antagonists such as, for example, BOSENTAN™ and MOXONODIN™, interferon-gamma (IFN-γ), 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-PA variant, Keyt et al , Proc Natl Acad Sci
USA 91 , 3670-3674 (1994)), inotropic or hypertensive agents such as digoxigenin and β-adrenergic receptor blocking agents, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, and carvedilol; angiotensin converting enzyme (ACE) inhibitors, e.g., quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, and lisinopril; diuretics, e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichloφhenamide, acetazolamide, and indapamide; and calcium channel blockers, e.g., diltiazem, nifedipine, verapamil, nicardipine. One preferred category of this type is a therapeutic agent used for the treatment of cardiac hypertrophy or of a physiological condition instrumental in the development of cardiac hypertrophy, such as elevated blood pressure, aortic stenosis, or myocardial infarction. "Angiogenic agents" and "endothelial agents" are active agents that promote angiogenesis and/or endothelial cell growth, or, if applicable, vasculogenesis. This would include factors that accelerate wound healing, such as growth hormone, insulin-like growth factor-I (IGF-I), VEGF, VIGF, PDGF, epidermal growth factor (EGF), CTGF and members of its family, FGF, and TGF-α and TGF-β.
"Angiostatic agents" are active agents that inhibit angiogenesis or vasculogenesis or otherwise inhibit or prevent growth of cancer cells. Examples include antibodies or other antagonists to angiogenic agents as defined above, such as antibodies to VEGF. They additionally include cytotherapeutic agents such as cytotoxic agents, chemotherapeutic agents, growth-inhibitory agents, apoptotic agents, and other agents to treat cancer, such as anti- HER-2, anti-CD20, and other bioactive and organic chemical agents.
In a pharmacological sense, in the context of the present invention, a "therapeutically effective amount" of an active agent such as a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto or an anti- PRO230, 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. As used herein, an "effective amount" of an active agent such as a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto or an anti-PRO230, anti-PR0216 or anti-PRO302 antibody, refers to an amount effective for carrying out a stated puφose, wherein such amounts may be determined empirically for the desired effect.
The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (i.e., PRO/number) refers to specific polypeptide sequences as described herein. The terms "PRO/number polypeptide" and "PRO/number" wherein the term "number" is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein). The PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence PRO polypeptide" specifically encompasses naturally-occurring truncated or secreted forms 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. In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons are shown in bold font and underlined in the figures. However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the PRO polypeptides.
The PRO polypeptide "extracellular domain" or "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. Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are contemplated by the present invention.
The approximate location of the "signal peptides" of the various PRO polypeptides disclosed herein are shown in the present specification and or the accompanying figures. It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al, Prot. Eng., JO: 1 -6 (1997) and von Heinje et al., Nucl. Acids Res.. 14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side 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
26 of Figure 2 (SEQ ID NO:2), or (c) another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO:2).
"PR0216 variant polypeptide" means an active PR0216 polypeptide (other than a native sequence PR0216 polypeptide) as defined below having at least about 80% amino acid sequence identity with the 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).
Such PRO230, PR0216 or PRO302 variant polypeptides include, for instance, PRO230, PR0216 or PRO302 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C-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. Ordinarily, a PRO230 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about
89% 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 sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any amino acid residue from 17 to 26 of Figure 2 (SEQ ID NO:2), or (c) another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO:2).
Ordinarily, a PR0216 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% 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 sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO:7) or (b) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7).
Ordinarily, a PRO302 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% 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 sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12), (b) X to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12), wherein X is any amino acid residue from 21 to 30 of Figure 6 (SEQ ID NO: 12), or (c) another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO: 12). Variants do not encompass the native PRO230, PR0216 or PRO302 polypeptide sequence. Ordinarily,
PRO230, PR0216 and PRO302 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 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.
As shown below, 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.
In addition, 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.
Table 1
/* *
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is _M; stop-stop = 0; J (joker) match = 0
*/
^define M /* value of a match with a stop */ int day[26][26] {
/* A B C D F G H I J K L M N O P Q R S T U V W X Y Z*/
/* A*/ 2 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0},
/*B*/ 0 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1},
/*C */ -2 -4,15, -5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5},
/*D*/ 0.3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2},
/*E */ 0.2,-5, 3,4,-5,0, 1,-2, 0, 0,-3,-2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3},
/* p */ -4 ■5,-4, -6,-5,9,-5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5}, l*G*l 1.0,-3, 1,0,-5,5,-2,-3, 0,-2,-4,-3, 0,_M,-l,-l,-3, 1, 0, 0,-1,-7, 0,-5, 0}, /*H*/ 1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0, 3, 2,-1,-1, 0,-2,-3, 0.0, 2}, 1*1*1 1,-2,-2, -2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2,_M,-2,-2,-2,-l, 0, 0, 4,-5, 0,-1,-2}, /*J */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, l*K*l 1,0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-4, 0}, /*L*/ 2,-3,-6, -4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3,-3.-l.0.2,-2, 0.-1,-2}, /*M */ 1,-2,-5, -3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-l, 0,-2,-1, 0, 2,-4, 0,-2,-1}, /*N*/ 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0.-2, 1}, ι*o*ι M,_M, M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M, 0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M},
I* p */ 1,-1,-3, -1, -1, -5, ,-1. .0, -2. , 0, .-1, ,-3. ,-2. ,-L , M , 6 , 0 , 0, . i. , 0. , 0, -1,-6, .0,-5, 0}, /*Q */ 1,-5, 2, 2, -5, -1, 3, -2, 0, 1, -2, -1, 1, M, 0, 4, 1, -1, -1, o, -2,-5, 0,-4, 3},
/*R*/ 0,-4, -1, -1, .-4, ,-3, .2, -2. , 0, 3, .-3, 0, 0, M, 0, 1, 6, 0, -1, 0, -2,2, 0,-4, 0},
/*S */ 0,0, 0, 0, -3, 1, -1, -1, 0, 0, -3, -2, 1, M, 1, -1, 0, 2, 1, 0, -1,-2, 0,-3,0}, I* Ύ */ 0,-2, 0, 0, -3, o, -1, 0, o, 0, -1, -1, 0, M, 0, -1, -1, 1, 3, 0, 0,-5, 0,-3, 0}, ι*υ*ι 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, M, 0, 0, 0, 0, 0, 0, 0,0,ι 0, 0, 0}, /* v*/ 0,-2,-2, -2, -2, ,-1, ,-1. rl- ,4. , 0, rl. ,2, .2, -2. "M, rl, ,-2. ,-2. ,-1 ,0 , 0 , 4,-6 , 0,-2,-2}, /* w */ 6,-5,-8, -1, rl. , 0, rl. r5. ,-5 , 0, ,-3 ,-2 ,-4 ,-4 , M ,-6 ,-5 ,2 ,-2 ,-5 .0 ,-6,1" ', 0, 0,-6} /*x*/ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, M, 0, 0, 0. 0, 0, 0. 0,0,ι 0, 0, 0}, ι*γ */ 3,-3, 0, -4. -4, ,7, -5, , o, -1. .0, -4. ,-1, .-2, ,-2~ " M. ,-5 ,-4 ,-4 ,-3 ,-3 .0 ,-2,0 ,0,10,-4} ι*ι*ι 0, 1,-5, 2, 3, -5, 0, 2, -2, 0, 0, -2, -1, 1, M, 0, 3, 0, 0, 0, 0, -2,-6, 0,-4, 4}
Page 1 of day. h /* */
^include < stdio h > ^include < ctype h >
#define MAXJMP 16 /* max jumps in a diag */
#define MAXGAP 24 /* don't continue to penalize gaps larger than this */
#define JMPS 1024 /* max jmps in an path */
#define MX 4 /* save if there's at least MX-1 bases since last jmp */
#define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty tor mismatched bases */
#define DINSO 8 /* penalty for a gap */
#define DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap */
#defϊne PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP], /* size of jmp (neg for dely) */ unsigned short x[MAXJMP], /* base no of jmp in seq x */ }. /* limits seq to 2Λ16 -1 */ struct diag { int score, /* score at last jmp */ long offset, /* offset of prev block */ short ljmp, /* current jmp index */ struct jmp JP> /* list of jmps */ }. struct path { int spc, /* number of leading spaces */ short n[JMPS], /* size of jmp (gap) */ int x[JMPS], /* loc of jmp (last elem before gap) */
}. char *ofile, /* output file name */ char *namex[2], /* seq names getseqs() */ char *prog, /* prog name tor err msgs */ char *seqx[2], /* seqs getseqs() */ int dmax, /* best diag nw() */ int dmaxO, /* final diag */ int dna /* set it dna maιn() */ int endgaps, /* set if penalizing end gaps */ int gapx, gapy, /* total gaps in seqs */ int lenO, lenl , /* seq lens */ int ngapx, ngapy, /* total size of gaps */ int smax, /* max score nw() */ int *xbm, /* bitmap for matching */ long offset, /* current offset in jmp file */ struct diag *dx, /* holds diagonals */ struct path pp[2], /* holds path for seqs char *calloc(), *malloc(), *mdex(), *strcpy(), char *getseq(), *g_calloc(),
Page 1 of nw.h /* Needleman-Wunsch alignment program
*
* usage- progs filel fιle2
* where filel and file2 are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ',', '>' or ' < ' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align. out" *
* The program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650 */
^include "nw.h" ^include "day.h" static _dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
}; static _pbval[26] = {
1, 2|(1< <('D'-'A'))|(1< <('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1< < 10, 1<<11, 1< < 12, 1< < 13, 1< < 14,
1< < 15, 1< < 16, 1< < 17, 1< < 18, 1< < 19, 1< <20, 1 < <21, 1 < <22,
1< <23, 1< <24, 1< <25|(1< <('E'-'A'))|(1< <('Q'-'A'))
};
Figure imgf000032_0001
int ac; char *av[];
{ prog = av[0]; if (ac ' = 3) { fpπntf(stderr, "usage: %s filel file2\n", prog); fpπntf(stderr, "where filel and fιle2 are two dna or two protein sequences. \n"), fpπntf(stderr,"The sequences can be in upper- or lower-case\n"), fpπntf(stderr,"Any lines beginning with ',' or ' < ' are ιgnored\n"), fpπntf(stderr, "Output is in the file \"ahgn.out\"\n"), exιt(l);
} namex[0] = av[l], namex[l] = av[2], seqx[0] = getseq(namex[0], &len0); seqx[l] = getseq(namex[l], &lenl), xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ ofile = "align. out"; /* output file */ nw(); /* fill in the matrix, get the possible jmps */ readjmps(), /* get the actual jmps */ print(), /* print stats, alignment */ cleanup(O), /* unlink any tmp files */
}
Page 1 of nw.c /* do the alignment, return best score: maιn()
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
Figure imgf000033_0001
nw() n
{ char *px, *; py; /* seqs and ptrs */ int *ndely, *dely; /* keep track of dely */ int ndelx, delx; /* keep track of delx */ int *tmp; /* for swapping rowO, rowl */ int mis; /* score for each type */ int insO, insi ; /* insertion penalties */ register id; /* diagonal index */ register 'J- /* jmp index */ register *col0, *col l ; /* score for curr, last row */ register xx, yy ; /* index into seqs */ dx = (struct diag *)g_calloc("to get diags", lenO+lenl + 1 , sizeof(struct diag)), ndely = (int *)g_calloc("to get ndely", lenl + 1 , sizeof(int)): dely = (int *)g_calloc("to get dely", lenl + 1. sizeof(int)); colO = (int *)g_calloc("to get colO", lenl + 1 , sizeof(int)), col l = (int *)g_calloc("to get coll ", lenl + 1 , sizeof(int)); msO = (dna)? DINSO : PINSO; msl = (dna)? DINS 1 : PINS1 ; smax = -10000; if (endgaps) { for (colO[0] = dely[0] = -insO, yy = 1 ; yy < = lenl ; yy + +) { col0[yy] = dely[yy] = col0[yy-l] - msl ; ndely[yy] = yy,
} colO[0] = 0, /* Waterman Bull Math Biol 84 */
} else for (yy = 1 ; yy < = lenl ; yy + +) dely[yy] = -insO,
/* fill in match matrix */ for (px = seqx[0], xx = 1 ; xx < = lenO; px+ + , xx+ +) { /* initialize first entry in col */ if (endgaps) { if (xx = = 1) coll[0] = delx = -(msO + insl), else coll [0] = delx = colO[0] - msl ; ndelx = xx;
} else {
Figure imgf000033_0002
ndelx = 0,
Page 2 of nw.c ...nw seqx[l], yy = 1 , yy < = lenl , py+ + , yy+ +) {
Figure imgf000034_0001
mis + = (xbm[*px-'A']&xbm[*py-'A'])^ DMAT DMIS, else mis + = _day[*px-'A'][*py-'A'],
/* update penalty for del m x seq,
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps | | ndely [yy] < MAXGAP) { if (col0[yy] - msO > = dely[yy]) { dely[yy] = col0[yy] - (insO+insl), ndely [yy] = 1 , } else { dely[yy] -= msl , ndely[yy] + + ,
} } else { if (col0[yy] - (insO+insl) > = dely[yy]) { dely[yy] = col0[yy] - (insO+insl), ndely[yy] = 1 , } else ndely [yy] + + ,
}
/* update penalty for del in y seq,
* favor new del over ongong del */ if (endgaps | | ndelx < MAXGAP) { if (coll [yy-l] - insO > = delx) { delx = coll [yy-l] - (insO+insl), ndelx = 1 , } else { delx -= insi , ndelx+ + , }
} else { if (col l [yy-l] - (insO + insl) > = delx) { delx = coll [yy-l] - (insO+ insl), ndelx = 1 ,
} else ndelx+ + ,
}
/* pick the maximum score, we're favoring * mis over any del and delx over dely
*/
Page 3 of nw.c ...nw id = xx - yy + lenl - 1, if (mis > = delx && mis > = dely[yy])
Figure imgf000035_0001
else if (delx > = dely[yy]) { collfyy] = delx; ij = dx[ιd].ιjmp; if (dx[ιd].jp.n[0] && (!dna 11 (ndelx > = MAXJMP && xx > dx[ιd].jp.x[ιj]+MX) | | mis > dx[ιd]. score + DINS0)) { dx[ιd] ιjmp+ + ; if(++ιj >= MAXJMP) { wπtejmps(ιd); ij = dx[ιd].ιjmp = 0; dx[ιd]. offset = offset; offset += sizeof(struct jmp) + sizeof(offset); } } dx[ιd].jp.n[ιj] = ndelx; dx[ιd].jp.x[ιj] = xx; dx[ιd]. score = delx;
} else { coll[yy] = dely[yy], ij = dx[ιd].ιjmp, if (dx[ιd].jp.n[0] && (!dna | | (ndely [yy] > = MAXJMP
&& xx > dx[ιd] jp.x[ιj]+MX) | | mis > dx[ιd]. score + DINS0)) { dx[ιd] ιjmp+ + ; if(++ιj >= MAXJMP) { wπtejmps(ιd); ij = dx[ιd].ιjmp = 0; dx[ιd]. offset = offset, offset += sizeof (struct jmp) + sizeof(ottset); } } dx[ιd].jp.n[ιj] = -ndely [yy]; dx[ιd] jp.x[ιj] = xx, dx[ιd]. score = dely[yy],
} if (xx = = lenO && yy < lenl) { /* last col */ if (endgaps) coll[yy] -= ιnsO + msl*(lenl-yy); if (coll [yy] > smax) { smax = coll[yy], dmax = id,
} } } if (endgaps && xx < lenO) coll[yy-l] -= ιnsO + msl*(lenO-xx); if (coll[yy-l] > smax) { smax = coll[yy-l], dmax = id,
} tmp = colO; colO = coll, coll = tmp;
}
(void) free((char *)ndely); (void) free((char *)dely); (void) free((char *)col0) (void) free((char *)coll)
Page 4 of nw.c * prιnt() — only routine visible outside this module
*
* static:
* getmat() — trace back best path, count matches: pπnt()
* pr_align() — print alignment of described in array p[]: pπnt()
* dumpblockO — dump a block of lines with numbers, stars: pr_alιgn()
* numsO - put out a number line: dumpblockO
* pufhneO ~ put out a line (name, [num], seq, [num]): dumpblockO
* stars() - -put a line of stars: dumpblockO
* stπpnameO — strip any path and prefix from a seqname */
#include "nw.h"
#define SPC 3
#defιne PJLINE 256 /* maximum output line */
^define P_SPC 3 /* space between name or num and seq */ extern _day[26][26]; int olen; /* set output line length */
FILE *fx, /* output file */ pπnto print
{ int Ix, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, "w")) = = 0) { fpπntf(stderr," %s: can't write %s\n" , prog, ofile); cleanup(l);
} fpπntf(fx, " < first sequence: %s (length = %d)\n" , namex[0], lenO), fprιntf(fx, " < second sequence %s (length = %d)\n" , namex[l], lenl), olen = 60, lx = lenO, ly = lenl , firstgap = lastgap = 0, if (dmax < lenl - 1) { /* leading gap in x */ pp[0].spc = firstgap = lenl - dmax - 1 ; ly -= pp[0].spc,
} else if (dmax > lenl - 1) { /* leading gap in y */ pp[l].spc = firstgap = dmax - (lenl - 1); lx -= pp[l].spc;
} if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = lenO - dmaxO -1 ; lx -= lastgap,
} else if (dmaxO > lenO - 1) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1), ly -= lastgap;
} getmat(lx, ly. firstgap, lastgap); pr ahgnO;
Page 1 of nwprint.c /* * trace back the best path, count matches
*/ static getmat(lx, ly, firstgap, last; gap) getmat int lx, ly, /* ' core" (minus endgaps) */ int firstgap, lastgap, /* leading ; trailing ; overlap */
I int nm , ι0, il , sizO, sizl , char outx[32], double pet, register nO, nl , register char *p0, *pl ,
/* get total matches, score */
Figure imgf000037_0001
pO = seqx[0] + pp[l] spc, pi = seqxfl] + pp[0] spc, nO = pp[l] spc + 1 , nl = pp[0] spc + 1 , nm = 0, while ( *p0 && *pl ) {
Figure imgf000037_0002
if (xbm[*pO-'A']&xbm[*pl-'A']) nm+ + , if (n0+ + = = pp[0] x[ιO]) sizO = pp[0] n[ιO+ +] if (nl + + = = pp[l] x[ιl]) sizl = pp[l] n[ι l + +], pθ+ + , pl + + ,
}
/* pet homology
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core */ if (endgaps) lx = (lenO < lenl)'' lenO lenl , else lx = (lx < ly)1? lx ly, pet = 100 *(double)nm/(double)lx, fpπntf(fx, "\n"), fpπntf(fx " < %d match%s in an overlap of %d % 2f percent sιmιlaπty\n ' nm, (nm = = l) " " "es", lx, pet),
Page 2 of nwprint.c fpπntf(fx, " < gaps in first sequence: %d" , gapx), .getmat if (gapx) {
(void) spπntf(outx, " (%d %s%s)" , ngapx, (dna)? "base": "residue", (ngapx = = l) "" : "s"); fpπntf (fx , " % s " , outx) ; fpπntf(fx, ", gaps in second sequence: %d" , gapy); i (gapy) {
(void) spπntf(outx, " (%d %s%s)", ngapy, (dna)? "base" - "resιdue", (ngapy = = l) " " : "s"); fpπntf(f x , " % s " , outx) ;
} if (dna) fpπntf(fx,
"\n < score: d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n" smax, DMAT, DMIS, DINSO, DINS1); else fpπntf(fx,
"\n < score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per resιdue)\n", smax, PINSO, PINS1); if (endgaps) fpπntf(fx,
" < endgaps penalized, left endgap: %d %s%s, right endgap %d %s%s\n", firstgap, (dna)? "base" : "residue" , (firstgap = = \f " " • "s" , lastgap, (dna)? "base" : "residue", (lastgap = = 1)? " " : "s"); else fpπntf(fx, " < endgaps not penal ιzed\n").
static nm; /* matches in core — for checking */ static Imax; /* lengths of stripped file names */ static ιj[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static nι[2]; /* current elem number — for gapping */ static sιz[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() */
/*
* print alignment of described in struct path pp[]
*/ static pr. ahgn() pr align
{ int nn, /* char count */ int more; register 1; for (i = 0, Imax = 0. , i < 2; . + +) { nn = stπpname(namex[ι]) , if (nn > Imax) Imax = i nn; nc[ι] = l ; m[ι] = 1 , sιz[ι] = ij[ι] = 0; ps[ι] = seqx[ι], po[ι] = out[ι];
Page 3 of nwprint.c for (nn = nm = 0, more = 1; more; ) { ...pr align for (l = more = 0; l < 2; ι+ +) { /*
* do we have more of this sequence? */ if(!*ps[ι]) continue; more+ + ; if (pp[ι].spc) { /* leading space */ *po[ι] + + = ' '; pp[ι].spc-;
} else if (sιz[ι]) { /* in a gap */
*po[ι] + + = '-'; sιz[ι]-,
} else { /* we're putting a seq element
*/ *po[ι] = *ps[ι]; if (ιslower(*ps[ι]))
*ps[ι] = toupper(*ps[ι]); po[ι]+ + ; ps[ι] + + ,
/* are we at next gap for this seq?
*/ if(nι[>] ==ppιι].x[ij[ι]]){ /*
* we need to merge all gaps
* at this location */ sιz[ι] = pp[ι].n[ιj[ι] + +]; while (nι[ι] == pp[ι].x[ιj[ι]]) sιz[ι] += pp[ι].n[ιj[ι] + +],
} nι[ι] + + ,
}
} if (+ +nn = = olen | | !more && nn) { dumpblockO, for (l = 0, l < 2; ι++) po[ι] = out[ι], nn = 0, }
* dump a block of lines, including numbers, stars' pr_alιgn() */ static dumpblockO dumpblock
{ register I, for (ι = 0; l < 2, ι++) *po[ι]- = '\0',
Page 4 of nwprint.c ...dumpblock
(void) putc('\n', fx), for (l = 0; i < 2, ι + +) { if (*out[ι] && (*out[ι] ! = ' ' I I *(po[ι]) != ' ')){ if(ι ==0) nums(ι); if (l == 0&& *out[l]) stars(); putlme(ι); if (i == 0&& *out[l]) fpπntf(fx, star), if (i == 1) nums(ι);
}
}
* put out a number h nc dumpblockO */ static nums(ιx) nums int lx, /* index in ou t[] holding seq line */
{ char nhne[P_LINE]; register i.j: register char *pn, *px, *py; for(pn = nline, i = 0; l < lmax + P_SPC; ι++, pn++)
*pn = ' '; for (i = nc[ιx], py = out[ιx]; *py; py+ + , pn++) { if(*py == ' ' j I *py == '-')
*pn = ' '; else { if (ι%10 == 0 || (l == 1 &&nc[ιx] ' = 1)) { j = (l < 0)? -i : l, for (px = pn, j, j /= 10, px-)
*px =j%10 + '0', if (l < 0)
} else
*pn ι + + ;
}
}
*pn = '\0', nc[ιx] = i; for (pn = nline; *pn; pn++)
(void) putc(*pn, fx); (void) putc('\n', fx),
* put out a line (name, [num], seq, [num]): dumpblockO
*/ static puthne(ιx) putline
Figure imgf000040_0001
Page 5 of nwprint.c ...putline int 1; register char px; for (px = namexfix], i = 0; *px && *px != ':'; px+ + , ι + +)
(void) putc(*px, fx); for (; l < lmax+P_SPC; ι++)
(void) putc(' ', fx);
/* these count from 1 :
* nι[] is current element (from 1)
* nc[] is number at start of current line */ for (px = out[ιx]; *px; px++) (void) putc(*px&0x7F, fx); (void) putc('\n', fx);
/*
* put a line of stars (seqs always in out[0], out[l]): dumpblockO
*/ static stars() stars
{ int i; register char *p0, *pl, ex, *px; if (!*out[0] 11 (*out[0] == ' •&& *(po[0]) == ' ') 11 !*out[l] 11 (*out[l] == ' '&& *(po[l]) == ' ')) return; px = star; for (i = lmax + P SPC; I; i-)
*px++ = ' '; for(p0 = out[0], pi = out[l]; *p0 && *pl; p0+ + , pl + +) { if (ιsalpha(*pO) && ιsalpha(*pl)) { if (xbm[*pO-'A']&xbm[*pl-'A']) { ex = '*'; nm+ + ;
} else if (!dna && _day[*ρO-'A'][*pl-'A'] > 0) ex = '.'; else ex = ' ';
} else ex = ' '; *px++ = ex; }
*px++ = '\n'; *px = '\0';
Page 6 of nwprint.c /*
* strip path or prefix from pn, return len: pr_align()
*/ static stripname(pn) Stripname char *pn; /* file name (may be path) */
{
py = 0; for (px = pn; *px; px+ +) if (*pχ = = '/') py = px + 1; if (py)
(void) strcpy(pn, py); return(strlen(pn));
Page 7 of nwprint.c * cleanupO — cleanup any tmp file
* getseqO — read in seq, set dna, len, maxlen
* g_calloc() — callocO with error checkin
* readjmpsO — get the good jmps, from tmp file if necessary
* writejmpsO — write a filled array of jmps to a tmp file nw() */
^include "nw h" ^include < sys/file h > char *jname = "/tmp/homgXXXXXX", /* tmp file for jmps */ FILE *fj, int cleanupO, /* cleanup tmp file */ long lseek(),
/* * remove any tmp file if we blow
*/ cleanup(ι) cleanup int l,
{ if(j)
(void) unhnk(jname), exιt(ι),
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with ',', '<', or ' > '
* seq in upper or lower case */ char * getseq(fιle, len) getseq char *fιle, /* file name */ int *len, /* seq len */
{ char lιne[1024], *pseq, register char *px, *py, int natgc, tlen,
FILE *tp, if ((fp = topen(fιle,"r')) == 0) { fpπntf(stderr,"%s can t read %s\n' , prog, file), exιt(l),
} tlen = natgc = 0, while (fgets(lιne, 1024, fp)) { if(*hne == ',' | | *lme == < || *lιne == ' > ') continue, for (px = line, *px '= '\n', px++) if (ιsupper(*px) | | ιslower(*px)) tlen+ + ,
} if ((pseq = malloc((unsigned)(tlen + 6))) == 0) { fpπntf(stderr,"%s malloc() failed to get %d bytes for %s\n' , prog, tlen+6, file), exιt(l),
} pseq[0] = pseq[l] = pseq[2] = pseq[3] = '\0',
Page 1 of nwsubr.c ...getseq py = pseq + 4; *len = tlen; rewιnd(fp); while (fgetsfline, 1024, fp)) { if (*lme = = ', ' | | *hne = = ' < ' | | *lιne = = ' > ') continue, for (px = line; *px ! = '\n' ; px+ +) { if (ιsupper(*px))
*py+ + = *px; else if (ιslower(*px))
*py+ + = toupper(*px); if (ιndex("ATGCU" ,*(py-l))) natgc + + ; } } *py + + = '\0' ;
*py = '\0' ; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq +4), } char * g_calloc(msg, nx, sz) g_CallθC char *msg; /* program, calling routine */ int nx, sz, /* number and size of elements */
{ char *px, *calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) { tpπntf(stderr, " %s: g_calloc() failed s (n= %d, sz= %d)\n" , prog, msg, nx, sz); exιt(l); } } return(px),
* get final jmps from dx[] or tmp file, set pp[], reset dmax: maιn()
*/ readjmpsO readjmps
{ int fd = -1 ;
Figure imgf000044_0001
register 1, J , XX, if (fj) {
(void) fclose(tj); if ((fd = open(jname, 0_RDONLY, 0)) < 0) { tpπntf(stderr, " %s: can't open() %s\n", prog, jname), cleanup(l); } } for (i = IO = i l = 0, dmaxO = dmax, xx = lenO; ; ι+ +) { while (1) { for (j = dx[dmax].ιjmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j--)
Page 2 of nwsubr.c ...readjmps if < 0 && dx[dmax]. offset && fj) {
(void) lseek(fd, dx[dmax]. offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof (struct jmp));
(void) read(fd, (char *)&dx[dmax]. offset, sizeof (dx [dmax]. offset)), dx[dmax].ιjmp = MAXJMP-1 ;
} else break;
} if 0 > = = JMPS) { fpπntf(stderr, " %s: too many gaps in ahgnmenfΛn prog); cleanup(l);
} if > = = 0) { siz = dx[dmax].jp.n|j], xx = dx[dmax] .jp.x[j];
Figure imgf000045_0001
if (siz < 0) { /* gap in second seq
Figure imgf000045_0002
/* id = xx - yy + lenl - 1 */ pp[l].x[ιl] = xx - dmax + lenl - 1 ; gapy+ + ; ngapy -= siz, /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP | | endgaps)? -siz : MAXGAP; ιl + + ;
} else if (siz > 0) { /* gap in first seq */ pp[0].n[ι0] = siz, pp[0].x[ι0] = xx; gapx+ + ; ngapx + = siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP | | endgaps)? siz : MAXGAP; ι0+ + ,
} } else break; }
/* reverse the order of jmps */ for 0 = 0, ι0~; j < lO; j + + , ι0-) { i = pp[0].n[j]; pp[0].n[j] = pp[0].n[ι0]; pp[0] .n[ι0] = ι, i = pp[0].x[)]; pp[0].x[j] = pp[0].x[ι0]; pp[0] x[ι0] = i,
} for (j = 0, ιl-; j < ιl ; j + + , il--) { i = ppM.nu]; PP[l].nQ] = pp[l}.n[ιl], pp[l].n[ιl] = I; i = pp[l].x[j]; pp[l].xϋl = PPtl].x[il], pp[ l].x[ιl] = i,
} if (fd > = 0)
(void) close(fd); if (fj) {
(void) unlιnk(jname); fj = θ;offset = 0;}} Page 3 of nwsubr.c /* * write a filled jmp struct offset of the prev one (if any): nw()
*/ wπtejmps(ιx) ritejmpS int IX;
{ char *mktemp(); if (!f)) { if (mktemp(jname) < 0) { fpπntf(stderr, " %s: can't mktempO %s\n", prog, jname); cleanup(l);
} if ((f) = fopen(jname, "w")) = = 0) { fpπntf(stderr, "%s: can't write %s\n", prog, jname); exιt(l); } }
(void) fwπte((char *)&dx[ιx].jp, sizeof(struct jmp), 1 , fj), (void) fwπte((char *)&dx[ιx]. offset, sizeof(dx[ιx]. offset), 1, fj),
Page 4 of nwsubr.c Table 2A
PRO xxxxxxxxxxxxxxx (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =
5 divided by 15 = 33.3%
Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =
5 divided by 10 = 50%
Table 2C
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
6 divided by 14 = 42.9%
Table 2D
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
4 divided by 12 = 33.3%
"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. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code shown in Table 1 has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
For puφoses herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations, Tables 2A-2B demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "PRO".
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al, Nucleic Acids Res.. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0 01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % ammo acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of ammo acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A
In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzvmology, 266 460-480 ( 1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values, ; e , the adjustable parameters, are set with the following values overlap span = 1 , overlap fraction = 0 125, word threshold (T) = 1 1 , and scoring matrix = BLOSUM62 For purposes herein, 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 polypeptide of interest For example, in the statement "a polypeptide comprising an am o acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B", the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest
"PRO230 variant polynucleotide" or "PRO230 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO230 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 164 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) Ordinarily, a PRO230 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO:2), (b) a nucleic acid sequence which encodes amino acids X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO:2), wherein X is any 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). PRO230 polynucleotide variants do not encompass the native PRO230 nucleotide sequence.
"PR0216 variant polynucleotide" or "PR0216 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PR0216 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO:7) or (b) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7). Ordinarily, a PR0216 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91 % nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO:7) or (b) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO:7). PR0216 polynucleotide variants do not encompass the native PR0216 nucleotide sequence. "PRO302 variant polynucleotide" or "PRO302 variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO302 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12), (b) a nucleic acid sequence which encodes amino acids X to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12), wherein X is any 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). Ordinarily, a PRO302 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91 % nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12), (b) a nucleic acid sequence which encodes amino acids X to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO: 12). wherein X is any amino acid residue from 21 to 30 of Figure 6 (SEQ ID NO: 12), or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO: 12). PRO302 polynucleotide variants do not encompass the native PRO302 nucleotide sequence.
Ordinarily, PRO230, PR0216 and PRO302 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more.
"Percent (%) nucleic acid sequence identity" with respect to the PRO230, PR0216 and PRO302 polypeptide- encoding nucleic acid sequences identified herein is defined as the percentage 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 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 OD All sequence comparison parameters are set by the ALIGN-2 program and do not vary
For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C As examples of % nucleic acid sequence identity calculations, Tables 2C-2D demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison D A" to the nucleic acid sequence designated "PRO- DNA"
Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res , 25 3389-3402 ( 1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0 01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or compπses a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI- BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST-2 computer program (Altschul et al, Methods in Enzvmology. 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 1 1 , and scoring matrix = BLOSUM62. For purposes herein, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide- encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide- encoding nucleic acid and the comparison nucleic acid molecule of interest (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. For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.
In other embodiments, PRO230, PR0216 and PRO302 variant polynucleotides are nucleic acid molecules that encode an active PRO230, PR0216 or PRO302 polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO230, PR0216 or PRO302 polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO: 12), respectively. PRO230, PR0216 and PRO302 variant polypeptides may be those that are encoded by a PRO230, PR0216 or PRO302 variant polynucleotide.
The term "positives", in the context of the amino acid sequence identity comparisons performed as described above, includes amino acid residues in the sequences compared that are not only identical, but also those that have similar properties. Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) of the amino acid residue of interest.
For purposes herein, the % value of positives of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value by the sequence alignment program ALIGN- 2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % positives of A to B will not equal the % positives of B to A.
"Isolated", when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by 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. Preferably, 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. However, 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.
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a 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. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
"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 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42 °C, with washes at 42 °C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.
"Moderately-stringent conditions" may be identified as described by Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Press, 1989), and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength, and % SDS) less stringent than those described above. An example of 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 37-50cC. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
The modifier "epitope-tagged" when used herein refers to a chimeric polypeptide comprising a 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.
"Biological activity" in the context of a molecule that antagonizes a PRO polypeptide that can be identified by the screening assays disclosed herein (e.g. , an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to bind or complex with the PRO polypeptide identified herein, or otherwise interfere with the interaction of the PRO 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.
The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes one or more of the biological activities of a native 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. 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. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments, or amino acid sequence variants of native PRO polypeptides, peptides, small organic molecules, etc.
A "small molecule" is defined herein to have a molecular weight below about 500 daltons.
The term "PRO polypeptide receptor" as used herein refers to a cellular receptor for a PRO polypeptide, ordinarily a cell-surface receptor found on vascular endothelial cells, as well as variants thereof that retain the ability to bind a PRO polypeptide.
"Antibodies" ( Abs) and "immunoglobulins" (Igs) 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. The term "antibody" is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.
"Native antibodies" and "native 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 (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody to and for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. See, 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. Examples of 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 VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (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 (λ), based on the amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy chains, 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 α, δ, e, γ, and μ, 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. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, Nature, 256: 495 ( 1975), or may be made by recombinant DNA methods (see, e.g., U.S. 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 U S Patent No 4,816,567, Morrison et al , Proc Natl Acad Sci USA, 81 6851 - 6855 (1984)
"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 For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity In some instances, Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences These modifications are made to further refine and maximize antibody performance In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence The humanized antibody preferably also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin For further details, see Jones et al Nature, 321 522-525 (1986), Reichmann et al , Nature, 332 323-329 ( 1988), and Presta, Curr Op Struct Biol , 2 593-596 (1992) The humanized antibody includes a PRIMATIZED™ antibody wherein the antigen-bind g region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain Preferably, the F v polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding For a review of sFv see, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 1 13, Rosenburg and Moore, eds (Springer- Verlag New York, 1994), pp 269-315
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites Diabodies are described more fully in, for example, EP 404,097, WO 93/1 1 161 , and Holhnger ef α/, Proc Natl Acad Sci USA, 90 6444-6448 (1993)
An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step
The word "label" when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody The label may be detectable by itself (e g , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable 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, Bι-212, and Pd- 109 The label may also be a non- detectable entity such as a toxin
By "solid phase" is meant a non-aqueous matrix to which an antibody of the present invention can adhere Examples of solid phases encompassed herein include those formed partially or entirely of glass (e g controlled pore glass), polysacchaπdes (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and si cones In certain embodiments, depending on the context, 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 The components of the hposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes
As used herein, the term "lmmunoadhesin" designates antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains Structurally, the immunoadhesins comprise a fusion 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, or IgM
II Compositions and Methods of the Invention
A PRO230, PRQ216 and PRO302 Variants In addition to the full-length native sequence PRO230, PR0216 and PRO302 polypeptides described herein, it is contemplated that PRO230, PR0216 and PRO302 variants can be prepared PRO230, PR0216 and PRO302 variants can be prepared by introducing appropriate nucleotide changes into the PRO230, PR0216 or PRO302 DNA, and/or by synthesis of the desired PRO230, PR0216 or PRO302 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO230, PR0216 or PRO302, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
Variations in the native full-length sequence PRO230, PR0216 or PRO302 or in various domains of the PRO230, PR0216 or PRO302 described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U S Patent No 5,364,934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO230, PR0216 or PRO302 that results in a change in the amino acid sequence of the PRO230, PR0216 or PRO302 as compared with the native sequence PRO230, PR0216 or PRO302 Optionally the variation is 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 the number of amino acid sequence changes made in regions of high homology Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a seπne, / e conservative amino acid replacements Insertions or deletions may optionally be in the range of about 1 to 5 amino acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence
In particular embodiments, conservative substitutions of interest are shown in Table 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
Table 3
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val, leu, lie val Arg (R) lys, gin, asn lys Asn (N) gin, his, lys, arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro, ala ala His (H) asn, gin, lys, arg arg He (I) leu, val, met, ala, phe, norleucine leu
Leu (L) norleucine, lie, val, met, ala, phe lie
Lys (K) arg, gin, asn arg Met (M) leu, phe, lie leu Phe (F) leu, val, lie, ala, tyr leu Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe; ala; norleucine leu
Substantial modifications in function or immunological identity of the 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:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in 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. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation 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.
B. Modifications of PRO230, PRQ216 and PRO302 Covalent modifications of PRO230, PR0216 and PRO302 are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a PRO230, PR0216 or PRO302 polypeptide with an organic 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. Commonly used crosslinkingagentsinclude, 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.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of 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. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
Addition of glycosylation sites to the PRO230, PR0216 or PRO302 polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO230, PR0216 or PRO302 (for O-linked glycosylation sites). The PRO230, PR0216 or PRO302 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO230, PR0216 or PRO302 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the PRO230, PR0216 or PRO302 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 1 1 September 1987, and in 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.
The PRO230, PR0216 or PRO302 of the present invention may also be modified in a way to form a chimeric molecule comprising PRO230, PR0216 or PRO302 fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PRO230, PR0216 or PRO302 with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO230, PR0216 or PRO302. The presence of such epitope-tagged forms of the PRO230, PR0216 or PRO302 can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO230, PR0216 or PRO302 to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-His) or poly-histidine-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. 5:3610-3616 (1985)]; and the Heφes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al, Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al, BioTechnology. 6: 1204- 1210 ( 1988)]; the K.T3 epitope peptide [Martin et al, Science. 255: 192-194 (1992)]; an α-tubulin epitope peptide [Skinner et al, J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al. Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO230, PR0216 or PRO302 with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO230, PR0216 or PRO302 polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI , CH2 and CH3 regions of an IgG l molecule. For the production of immunoglobulin fusions see also, US Patent No. 5,428,130 issued June 27, 1995.
C. Preparation of the PRO230, PRQ216 and PRO302 polypeptides
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO230, PR0216 or PRO302. In particular, cDNAs encoding PRO230, PR0216 or PRO302 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below. It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the 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
The description below relates primarily to production of PRO230, PR0216 or PRO302 polypeptides by culturing cells transformed or transfected with a vector containing nucleic acid encoding PRO230, PR0216 or PRO302 polypeptides It is, of course, contemplated that alternative methods that are well known in the art may be employed to prepare PRO230, PR0216 or PRO302 For instance, the PRO230, PR0216 or PRO302 polypeptide sequence, or portions thereof, 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 Various portions of PRO230, PR0216 or PRO302 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO230, PR0216 or PRO302 polypeptide
l Isolation of DNA Encoding PRO230, PRQ216 or PRO302
DNA encoding PRO230, PR0216 or PRO302 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA encoding PRO230, PR0216 or PRO302 and to express it at a detectable level Accordingly, DNAs encoding human PRO230, PR0216 or PRO302 can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in the Examples The gene encoding PRO230, PR0216 or PRO302 polypeptide may also be obtained from a genomic library or by oligonucleotide synthesis
Libraries can be screened with probes (such as antibodies to the PRO230, PR0216 or PRO302 polypeptide or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , supra An alternative means to isolate the gene encoding PRO230, PR0216 or PRO302 is to use PCR methodology Sambrook et al , supra, Dieffenbach et al , PCR Primer A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1995)
The Examples below describe techniques for screening a cDNA library The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened Methods of labeling are well known in the art, and include the use of radiolabels like 32P-labeled ATP, biotinylation, or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supra
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as 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
11 Selection and Transformation of Host Cells
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 In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology A Practical Approach, M Butler, ed (IRL Press, 1991 ) and Sambrook et al , supra
Methods of transfection are known to the ordinarily skilled artisan, for example, CaP04 treatment and electroporation Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells The calcium treatment employing calcium chloride, as described in Sambrook et al , supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al , Gene, 23 315 (1983) and WO 89/05859 published 29 June 1989 For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52 456-457 ( 1978) can be employed General aspects of mammalian cell host system transformations have been described in U S Patent No 4,399,216 Transformations into yeast are typically carried out according to the method of Van Solingen et al , J Bact , 130 946 (1977) and Hsiao et al , Proc Natl Acad Sci (USA). 76 3829 (1979) However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e g , polybrene or polyornithme, may also be used For various techniques for transforming mammalian cells, see, Keown et al , Methods in Enzymology, 185 527-537 (1990) and Mansour et al , Nature, 336 348-352 (1988)
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells Suitable prokaryotes include, but are not limited to, eubacteπa, such as Gram-negative or Gram- positive organisms, for example, Enterobacteπaceae such as E coli Various 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 Enterobacteπaceae such as Eschenchia, e g , E coli, Enterobacter, Erwima, Klebstella, Proteus, Salmonella, e g , Salmonella typhtmurium, Serratta, e g , Serratta marcescans, and Shigella, as well as Bacilli such as B subtihs and B lichentformis (e g , B lichentformis 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P aerugmosa, and Streptomyces These examples are illustrative rather than limiting Strain W31 10 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations Preferably, the host cell secretes minimal amounts of proteolytic enzymes For example, strain W31 10 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E coli W31 10 strain 1 A2, which has the complete genotype tonA , E coli W31 10 strain 9E4, which has the complete genotype tonA ptr3, E coli W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan', E colt W31 10 strain 37D6, which has the complete genotype tonA ptr3 phoA El 5 (argF-lac) 169 degP ompT rbs7 ilvG kari ', E colt W 1 10 strain 40B4, which is strain 37D6 with a non- kanamycin resistant degP deletion mutation, and an E coli strain having mutant peπplasmic protease disclosed in U S Patent No 4,946,783 issued 7 August 1990 Alternatively, in vitro methods of cloning, e g , PCR or other nucleic acid polymerase reactions, are suitable
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding 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 Bacteπol . 737 119831). 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, Sreekπshna et al , J Basic Microbiol . 28 265-278 [1988]), 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), and 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. 81 1470-1474 [ 1984]) and A ger (Kelly and Hynes, EMBO J . 4 475-479 [1985]) Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida Kloeckera, 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 Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ), human 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 . 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51 ). The selection of the appropriate host cell is deemed to be within the skill in the art.
iii. Selection and Use of a Replicable Vector
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. 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. In general, the signal sequence may be a component of the vector, or it may be a part of the DNA encoding PRO230, PR0216 or PRO302 that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g. , the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362, 179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., 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.
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the nucleic acid encoding PRO230, PR0216 or PRO302, such as DHFR or 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. , Nature, 282: 39 (1979); Kingsman et al, Gene, 7: 141 (1979); Tschemper ef at. Gene. 10: 157 (1980). Ihe trpλ 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 β-lactamase and lactose promoter systems (Chang et al, Nature, 275: 615 ( 1978); Goeddel et al, Nature, 281 : 544 (1979)), alkaline phosphatase, a tryptophan (tφ) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP 36,776), and hybrid promoters such as the tac promoter. deBoer et al , Proc. Natl. Acad. Sci. USA. 80: 21 -25 ( 1983). Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO230, PR0216 or PRO302.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase (Hitzeman et al, J. Biol. Chem.. 255: 2073 ( 1980)) or other glycolytic enzymes (Hess et al, J. Adv. Enzyme Reg., 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.
Other yeast promoters that are inducible promoters having the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
PRO230, PR0216 or PRO302 nucleic acid transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1 ,504 published 5 July 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.
Transcription of a DNA encoding the PRO230, PR0216 or PRO302 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers The enhancer may be spliced into the vector at a position 5' or 3' to the sequence coding for PRO230, PR0216 or PRO302, but is preferably located at a site 5' from the promoter
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary 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
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO230, PR0216 or PRO302 in recombinant vertebrate ceil culture are described in Gething et al , Nature.293 620-625 (1981 ), Mantei et al . Nature, 281 40-46 (1979), EP 1 17,060, and EP 1 17,058
iv Detecting Gene Amplification/Expression
Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc 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, alternatively, 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
v Purification of Polypeptide
Forms of PRO230, PR0216 or PRO302 polypeptides may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e g TRITON-X™ 100) or by enzymatic cleavage Cells employed in expression of nucleic acid encoding the PRO230, PR0216 or PRO302 polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell-lysing agents
It may be desired to purify the PRO230, PR0216 or PRO302 polypeptide from recombinant cell proteins or polypeptides The following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation- exchange resin such as DEAE, chromatofocusιng,SDS-PAGE,ammonιum 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) The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO230, PR0216 or PRO302 produced
D Uses of the PRO230. PRQ216 or PRO302 polypeptides l Assays for Cardiovascular, Endothelial, and Angiogenic Activity
Various assays can be used to test the polypeptide herein for cardiovascular, endothelial, and angiogenic activity Such assays include those provided in the Examples below
Assays for testing for endothelin antagonist activity, as disclosed in U S Pat No 5,773,414, include a rat heart ventricle binding assay where the polypeptide is tested for its ability to inhibit 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. Maιbach, 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
There are several cardiac hypertrophy assays n vitro assays include induction of spreading of adult rat cardiac myocytes In this assay, ventricular myocytes are isolated from a single (male Sprague-Dawley) rat, essentially following a modification of the procedure described in detail by Piper et al , "Adult ventricular rat heart muscle cells" in Cell Culture Techniques in Heart and Vessel Research, H M Piper, ed (Berlin 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 Phenylephπne and Prostaglandin F2α (PGF2α) have been shown to induce a spreading response in these adult cells The inhibition of myocyte spreading induced by PGF2α or PGF2α analogs (e g fluprostenol) and phenylephπne by various potential inhibitors of cardiac hypertrophy is then tested
One example of an in vivo assay is a test for inhibiting cardiac hypertrophy induced by fluprostenol in vivo This pharmacological model tests the ability 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 PGF2„) It is known that rats with pathologic cardiac hypertrophy induced by myocardial infarction have chronically elevated levels of extractable PGF2α in their myocardium Lai et al , Am J Physiol (Heart Circ Physiol ), 271 H2197- H2208 (1996) Accordingly, factors that can inhibit the effects of fluprostenol on myocardial growth in vivo are potentially useful for treating cardiac hypertrophy The effects of the 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 not receiving the PRO polypeptide
Another example of an in vivo assay is the pressure-overload cardiac hypertrophy assay For in vivo testing it is common to induce pressure-overload cardiac hypertrophy by constriction of the abdominal aorta of test animals In a typical protocol, rats (e g , male Wistar or Sprague-Dawley) are treated under anesthesia, and the abdominal aorta of each rat is narrowed down just below the diaphragm Beznak M , Can J Biochem Physiol . 33 985-94 (1955) The aorta is exposed through a surgical incision, and a blunted needle is placed next to the vessel The aorta is constricted with a ligature of silk thread around the needle, which is immediately removed and which reduces the lumen of the aorta to the diameter of the needle This approach is described, for example, in Rossi et al Am Heart J , 124 700-709 ( 1992) and O' Rourke and Reibel, P S E M B , 200 95- 100 ( 1992)
In yet another in v vo assay, the effect on cardiac hypertrophy following experimentally induced myocardial infarction (MI) is measured Acute MI is induced in rats by left coronary artery ligation and confirmed by electrocardiographic examination A sham-operated group of animals is also prepared as control animals Earlier data have shown that cardiac hypertrophy is present in the group of animals with MI, as evidenced by an 18% increase in heart weight-to-body weight ratio Lai et al supra Treatment of these animals with candidate blockers of cardiac hypertrophy, e g , PRO polypeptide, provides valuable information about the therapeutic potential of the candidates tested One further such assay test for induction of cardiac hypertrophy is disclosed in U S Pat No 5,773,415, using Sprague-Dawley rats
For cancer, a variety of well-known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies and other antagonists of the native PRO polypeptides, such as small-molecule antagonists The in vivo nature of such models makes them particularly predictive of responses in human patients Animal models of tumors and cancers (e g , breast cancer, colon cancer, prostate cancer, lung cancer, etc ) include both non-recombinant and recombinant (transgenic) animals Non-recombinant animal models include, for example, rodent, e g , murine models Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e , 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 , published September 18, 1997 Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice The observation that the nude mouse with thymic hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this puφose The autosomal recessive nu gene has been introduced into a very large number of distinct congemc strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, BIO LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII, and SJL In addition, a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts For further details see, e g , The Nude Mouse in Oncology Research. E Boven and B Winograd, eds (CRC Press, Ine , 1991 )
The cells introduced into such animals can be derived from known tumor/cancer cell lines, such as any of the above-listed tumor cell lines, and, for example, the 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 For solid-block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s c space Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously Tumor cells can also be injected as subdermal implants In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s c tissue
Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogene was initially isolated), or rtew-transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al Proc Nat Acad Sci USA, 83 9129-9133 (1986)
Similarly, animal models of colon cancer can be generated by passaging colon cancer cells in animals, e g , nude mice, leading to the appearance of tumors in these animals An orthotopic transplant model of human colon cancer in nude mice has been described, for example, by Wang e/ α/ , Cancer Research. 54 4726-4728 (1994) and Too et al , Cancer Research. 55 681 -684 (1995) This model is based on the so-called "METAMOUSE"™ sold by AntiCancer, 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
For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al , J Exp Med . 146 720 ( 1977)), which provide a highly controllable model system for studying 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 OxlO6 to 10xl07 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 diagnosed with small-cell carcinoma of the lung (SCCL) This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture Zupi et al , Br J Cancer, 41 suppl 4, 30 (1980) Evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive For further information about this tumor model see, Zacharski, Haemostasis, 16 300-320 (1986)
One way of evaluating the efficacy of a test compound in an animal model with an implanted tumor is to measure the size of the tumor before and after treatment Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula However, the measurement of tumor size is very inaccurate The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay Another important variable in the description of tumor growth is the tumor volume doubling time Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc 6th Int Workshop on Immune-Deficient Animals. Wu and Shengeds (Basel, 1989), p 301 It is noted, however, that necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially Therefore, these changes need to be carefully monitored, by a combination of a moφhometπc method and flow cytometπc analysis
Further, recombinant (transgenic) animal models can be engineered by introducing the coding portion of the PRO230, PR0216 or PRO302 genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g baboons, chimpanzees and monkeys Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (U S Patent No 4,873, 191 ), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al , Proc Natl Acad Sci USA. 82 6148-615 ( 1985)), gene targeting in embryonic stem cells (Thompson et al , Ce]], 56 313-321 (1989)), electroporation of embryos (Lo, Mol Cell Biol , 3 1803- 1814 (1983)), and sperm-mediated gene transfer Lavitrano et al , Cell, 57 717-73 (1989) For a review, see for example, U S Patent No 4,736,866
For the puφose of the present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals") The transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems Selective introduction 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)
The expression of the transgene in transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene The level of 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. For example, cDNA encoding a particular PRO230, PR0216 or PRO302 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques. A portion of the genomic DNA encoding a particular PRO230, PR0216 or PRO302 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector. See, e.g., Thomas and Capecchi, Cell, 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.
The efficacy of antibodies specifically binding the PRO polypeptides identified herein, and other drug candidates, can be tested also in the treatment of spontaneous animal tumors. A suitable target for such studies is the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species. It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor. These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity. At present, there is no effective treatment for this tumor. Prior to entry into the study, each cat undergoes complete clinical examination and biopsy, and is scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves. Each cat is treated repeatedly, over a longer period of time. Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck. After treatment, each cat undergoes another CT scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter. The data are evaluated for differences in survival, response, and toxicity as compared to control groups. Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span.
In addition, other spontaneousanimaltumors,such as fibrosarcoma, adenocarcinoma, lymphoma, chondroma, or leiomyosarcoma of dogs, cats, and baboons can also be tested. Of these, mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans. However, the use of this model is limited by the rare occurrence of this type of tumor in animals.
Other in vitro and in vivo cardiovascular, endothelial, and angiogenic tests known in the art are also suitable herein.
ii. Tissue Distribution
The results of the cardiovascular, endothelial, and angiogenic assays herein can be verified by further studies, such as by determining mRNA expression in various human tissues.
As noted before, gene amplification and/or gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201 -5205 ( 1980)),dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
Gene expression in various tissues, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/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.
iii. Antibody Binding Studies
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.
Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of target protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte that remain unbound
Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected In a sandwich assay, the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex See, e g , US Pat No 4,376,1 10 The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobu n antibody that is labeled with a detectable moiety (indirect sandwich assay) For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme
For immunohistochemistry, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example
iv Cell-Based Tumor Assays
Cell-based assays and animal models for cardiovascular, endothelial, and angiogenic disorders, such as tumors, can be used to verify the findings of a cardiovascular, endothelial, and angiogenic assay herein, and further to understand the relationship between the genes identified herein and the development and pathogenesis of undesirable cardiovascular, endothelial, and angiogenic cell growth The role of gene products identified herein in the development and pathology of undesirable cardiovascular, endothelial, and angiogenic cell growth, e g , tumor cells, can be tested by using cells or cells lines that have been identified as being stimulated or inhibited by the PRO230, PR0216 or PRO302 polypeptide herein Such cells include, for example, those set forth in the Examples below
In a different approach, cells of a cell type known to be involved in a particular cardiovascular, endothelial, and angiogenic disorder are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessive growth or inhibit growth is analyzed If the cardiovascular, endothelial, and angiogenic disorder is cancer, suitable tumor cells include, for example, stable tumor cells lines such as the B104- 1- 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and rαs-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumoπgenic growth Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumoπgenic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC) Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of cardiovascular, endothelial, and angiogenic disorders such as cancer
In addition, primary cultures derived from tumors in transgenic animals (as described above) can be used in the cell-based assays herein, although stable cell lines are preferred Techniques to derive continuous cell lines from transgenic animals are well known in the art See, e g , Small el al , Mol Cell Biol , 5 642-648 (1985)
v Gene Therapy The 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
There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells in vivo and ex vivo For in vivo delivery the nucleic acid is injected directly into the patient, usually at the sites where the PRO230, PR0216 or PRO302 polypeptide is required, / e , the site of synthesis of the PRO230, PR0216 or PRO302 polypeptide, if known, and the site (e g , wound) where biological activity of PRO230, PR0216 or PRO302 polypeptide is needed For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated withm porous membranes that are implanted into the patient (see, e g , U S Pat Nos 4,892,538 and 5,283,187) There are a variety of techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or transferred in vivo in the cells of the intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of hposomes, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc Transduction involves the association of a replication-defective, recombinant viral (preferably retroviral) particle with a cellular receptor, followed by introduction of the nucleic acids contained by the particle into the cell A commonly used vector for ex vivo delivery of the gene is a retrovirus
The currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral vectors (such as adenovirus, lentivirus, Heφes simplex I virus, or adeno-associated virus (AAV)) and 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 In addition, a viral vector such as a retroviral vector includes a nucleic acid molecule that, when transcribed in the presence of a gene encoding PRO230, PR0216 or PRO302 polypeptide, is operably linked thereto and acts as a translation initiation sequence Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used (if these are not already present in the viral vector) In addition, such vector typically includes a signal sequence for secretion of the PRO230, PR0216 or PRO302 polypeptide from a host cell m which it is placed Preferably the signal sequence for this puφose is a mammalian signal sequence, most preferably the native signal sequence for the PRO230, PR0216 or PRO302 polypeptide Optionally, the vector construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence By way of example, such vectors will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendπmers
In some situations, it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc Where hposomes are employed, proteins that bind to a cell-surface membrane protein associated with endocytosis may be used for targeting and or to facilitate uptake, e g , capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life The technique of receptor-mediated endocytosis is described, for example, by Wu et al , J Biol Chem , 262 4429-4432 ( 1987), and Wagner et al , Proc Natl Acad Sci USA, 87 3410-3414 (1990) For a review of the currently known gene marking and gene therapy protocols, see, Anderson et al , Science, 256 808-813 (1992) See also WO 93/25673 and the references cited therein
Suitable gene therapy and methods for making retroviral particles and structural proteins can be found in, e g , U S Pat No 5,681,746
vi Use of Gene as Diagnostic
This invention is also related to the use of the gene encoding the PRO230, PR0216 or PRO302 polypeptide as a diagnostic Detection of a mutated form of the PRO230, PR0216 or PRO302 polypeptide will allow a diagnosis of a cardiovascular, endothelial, and angiogenic disease or a susceptibility to a cardιovascular,endothelιal, and angiogenic disease, such as a tumor, since mutations in the PRO230, PR0216 or PRO302 polypeptide may cause tumors
Individuals carrying mutations in the genes encoding a human PRO230, PR0216 or PRO302 polypeptide may be detected at the DNA level by a variety of techniques Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy, and autopsy material The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al , Nature, 324 163-166 (1986)) prior to analysis RNA or cDNA may also be used for the same puφose As an example, PCR primers complementary to the nucleic acid encoding the PRO230, PR0216 or PRO302 polypeptide can be used to identify and analyze PRO230, PR0216 or PRO302 polypeptide mutations For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA encoding the PRO230, PR0216 or PRO302 polypeptide, or alternatively, radiolabeled antisense DNA sequences encoding the PRO230, PR0216 or PRO302 polypeptide Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis DNA fragments of different sequences may be distinguished on denaturing formamidine gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures See, e g , 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)
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes, e g , restriction fragment length polymoφhisms (RFLP), and Southern blotting of genomic DNA
vii Use to Detect PRO Polypeptide Levels
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis
Expression of nucleic acid encoding the PRO polypeptide may be linked to vascular disease or neovasculaπzation 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 neovasculaπzation associated with tumor formation, since an altered level of this PRO polypeptide may be indicative of such disorders
A competition assay may be employed wherein antibodies specific to the PRO polypeptide are attached to a solid support and the labeled PRO polypeptide and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of the PRO polypeptide in the sample
vin Chromosome Mapping
The sequences of the present invention are also valuable for chromosome identification The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome Moreover, there is a current need for identifying particular sites on the chromosome Few chromosome marking reagents based on actual sequence data (repeat polymoφhisms) are presently available for marking chromosomal location The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA Computer analysis for the 3'- untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process These primers are then 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
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner Other mapping strategies that can similarly be used to map to its chromosome include 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 For a review of this technique, see, Verma et al , Human Chromosomes a Manual of Basic Techniques (Pergamon Press, New York, 1988)
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data are found, for example, in V McKusick, Mende an Inheritance in Man (available online through Johns Hopkins University Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region is then identified through linkage analysis (coinheπtance of physically adjacent genes)
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes (This assumes 1 megabase mapping resolution and one gene per 20 kb)
lx Screening Assays for Drug Candidates
This invention encompasses methods of screening compounds to identify those that mimic the PRO230, PR0216 or PRO302 polypeptide (agonists) or prevent the effect of the PRO230, PR0216 or PRO302 polypeptide (antagonists) Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO230, PR0216 or PRO302 polypeptide encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates
The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art
All assays for antagonists are common in that they call for contacting the drug candidate with a PRO230, PR0216 or PRO302 polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture In a particular embodiment, 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 Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the PRO230, PR0216 or PRO302 polypeptide and drying Alternatively, an immobilized antibody, 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. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
If the candidate compound interacts with but does not bind to a particular PRO230, PR0216 or PRO302 polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London), 340: 245-246 ( 1989); Chien et al. , Proc. Natl. Acad. Sci. USA, 88: 9578-9582 ( 1991 )) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 ( 1991 ). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA- binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-/αcZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid 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.
Compounds that interfere with the interaction of a gene encoding a PRO230, PR0216 or PRO302 polypeptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control 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.
If the PRO polypeptide has the ability to stimulate the proliferation of endothelial cells in the presence of the co-mitogen ConA, then one example of a screening method takes advantage of this ability. Specifically, in the proliferation assay, human umbilical vein endothelial cells are obtained and cultured in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) and supplemented with a reaction mixture appropriate for facilitating proliferation of the cells, the mixture containing Con-A (Calbiochem, La Jolla, CA). Con-A and the compound to be screened are added and after incubation at 37°C, cultures are pulsed with 3Η-thymidine and harvested onto glass fiber filters (phD; Cambridge Technology, Watertown, MA). Mean 3Η- thymidine incoφoration (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA). Significant " (H)thymidine incoφoration indicates stimulation of endothelial cell proliferation.
To assay for antagonists, the assay described above is performed; however, in this assay the PRO polypeptide is added along with the compound to be screened and the ability of the compound to inhibit 3~(H)thymidine incoφoration in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide. Alternatively, antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of 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). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the PRO polypeptide. Transfected cells that are grown on glass slides are exposed to the labeled PRO polypeptide. The PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub- pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, 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.
In another assay for antagonists, 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.
The compositions useful in the treatment of cardiovascular, endothelial, and angiogenic disorders include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple-helix molecules, etc., that inhibit the expression and/or activity of the target gene product. More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with a PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the PRO polypeptide that recognizes the receptor but imparts no effect, thereby competitively 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. For example, 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. When 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. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biology, 4: 469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997).
Nucleic acid molecules in 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. For further details see, e.g., PCT publication No. WO 97/33551 , supra.
These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.
x. Types of Cardiovascular, Endothelial, and Angiogenic Disorders to be Treated
The PRO230, PR0216 or PRO302 polypeptides, or agonists or antagonists thereto, that have activity in the cardiovascular, angiogenic, and endothelial assays described herein, and/or whose gene product has been found to be localized to the cardiovascular system, are likely to have therapeutic uses in a variety of cardiovascular, endothelial, and angiogenic disorders, including systemic disorders that affect vessels, such as diabetes 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. 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.
Moreover, 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. If the objective is the treatment of human patients, the PRO polypeptide preferably is recombinant human PRO polypeptide (rhPRO polypeptide). The treatment for cardiac hypertrophy can be performed at any of its various stages, which may result from a variety of diverse pathologic conditions, including myocardial infarction, hypertension, hypertrophic cardiomyopathy, and valvular regurgitation. The treatment extends to all stages of the progression of cardiac hypertrophy, with or without structural damage of the heart muscle, regardless of the underlying cardiac disorder.
The decision of whether to use the molecule itself or an agonist thereof for any particular indication, as opposed to an antagonist to the molecule, would depend mainly on whether the molecule herein promotes cardiovasculanzation, genesis of endothelial cells, or angiogenesis or inhibits these conditions For example, if the molecule promotes angiogenesis, an antagonist thereof would be useful for treatment of disorders where it is desired to limit or prevent angiogenesis Examples of such disorders include vascular tumors such as haemangioma, tumor angiogenesis, neovasculaπzation in the retina, choroid, or cornea, associated with diabetic retinopathy or premature infant retinopathy or macular degeneration and proliferative vitreoretmopathy, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation, psoriasis, endometπosis associated with neovasculaπzation, restenosis subsequent to balloon angioplasty, scar tissue oveφroduction, for example, that seen in a keloid that forms after surgery, fibrosis after myocardial infarction, or fibrotic lesions associated with pulmonary fibrosis
If, however, the molecule inhibits angiogenesis, it would be expected to be used directly for treatment of the above conditions
On the other hand, if the molecule stimulates angiogenesis it would be used itself (or an agonist thereof) for indications where angiogenesis is desired such as peripheral vascular disease, hypertension, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, arterial restenosis, thrombophlebitis, lymphangitis, lymphedema, wound healing and tissue repair, ischemia reperfusion injury, angina, myocardial infarctions such as acute myocardial infarctions, chronic heart conditions, heart failure such as congestive heart failure, and osteoporosis
If, however, the molecule inhibits angiogenesis, an antagonist thereof would be used for treatment of those conditions where angiogenesis is desired
Specific types of diseases are described below, where the PRO polypeptide herein or antagonists thereof may serve as useful for vascular-related drug targeting or as therapeutic targets for the treatment or prevention of the disorders Atherosclerosis is a disease characterized by accumulation of plaques of intimal thickening in arteries, due to accumulation of lipids, proliferation 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 arteπtis, Takayasu's arteπtis, polyarteπtis 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) may occur following angioplasty as a result of alteration in the function and proliferation of endothelial and vascular smooth muscle cells.
Thrombophlebitis and lymphangitis are inflammatory disorders of veins and lymphatics, respectively, that may result from, and/or in, altered endothelial cell function. Similarly, lymphedema is a condition involving impaired lymphatic vessels resulting from endothelial cell function.
The family of benign and malignant vascular tumors are characterized by abnormal proliferation and growth of cellular elements of the vascular system. For example, lymphangiomas are benign tumors of the lymphatic system that are congenital, often cystic, malformations of the lymphatics that usually occur in newborns. Cystic tumors tend to grow into the adjacent tissue. Cystic tumors usually occur in the cervical and axillary region. They can also occur in the soft tissue of the extremities. The main symptoms are dilated, sometimes reticular, structured lymphatics and lymphocysts surrounded by connective tissue. Lymphangiomas are assumed to be caused by improperly connected embryonic lymphatics or their deficiency. The result is impaired local lymph drainage. Griener et al. , Lvmphology, 4: 140- 144 ( 1971 ).
Another use for the PRO polypeptides herein or antagonists thereto is in the prevention of tumor angiogenesis, which involves vascularization of a tumor to enable it to growth and or metastasize. This process is dependent on the growth of new blood vessels. Examples of neoplasms and related conditions that involve tumor angiogenesis include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endometrial carcinoma, endometrial hypeφlasia, endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma, abnormal vascular proliferation associated with phakomatoses, edema (such as that associatedwith brain tumors), and Meigs' syndrome.
Age-related macular degeneration (AMD) is a leading cause of severe visual loss in the elderly population. The exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment. Because choroidal neovascularization is associated with a dramatic worsening in prognosis, the PRO polypeptide or antagonist thereto is expected to be useful in reducing the severity of AMD.
Healing of trauma such as wound healing and tissue repair is also a targeted use for the PRO polypeptides herein or their antagonists. Formation and regression of new blood vessels is essential for tissue healing and repair. This category includes bone, cartilage, tendon, ligament, and/or nerve tissue growth or regeneration, as well as wound healing and tissue repair and replacement, and in the treatment of burns, incisions, and ulcers. A 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.
It is expected that 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. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate.
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.
Another category of 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. De novo tendon/ligament-like tissue formation induced by a composition of the PRO polypeptide herein or antagonist thereto contributes to the repair of congenital, trauma-induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions herein may provide an environment to attract tendon- or ligament- forming cells, stimulate growth of tendon- or ligament- forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions herein may also be useful in the treatment of tendinitis, caφal tunnel syndrome, and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art. The 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.
Further indications include angina, myocardial infarctions such as acute myocardial infarctions, and heart failure such as congestive heart failure. Additional non-neoplastic conditions include psoriasis, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, thyroid hypeφlasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.
In view of the above, the PRO polypeptides or agonists or antagonists thereof described herein, which are shown to alter or impact endothelial cell function, proliferation, and/or form, are likely to play an important role in the etiology and pathogenesis of many or all of the disorders noted above, and as such can serve as therapeutic targets to augment or inhibit these processes or for vascular-related drug targeting in these disorders.
xi. Administration Protocols, Schedules, Doses, and Formulations
The molecules herein and agonists and antagonists thereto are pharmaceutically useful as a prophylactic and therapeutic agent for various disorders and diseases as set forth above.
Therapeutic compositions of the 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, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
Additional examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial 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. 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. For all administrations, 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 incoφorating a PRO polypeptide or antagonist thereof into formed articles. Such articles can be used in modulating endothelial cell growth and angiogenesis. In addition, tumor invasion and metastasis may be modulated with these articles.
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
If the detergent employed is non-ionic, it may, for example, be polysorbates (e g , POLYSORBATE™ (TWEEN™) 20, 80, etc ) or poloxamers (e g , POLOXAMER™ 188) The use of non-ionic surfactants permits the formulation to be exposed to shear surface stresses without causing denaturation of the polypeptide Further, such surfactant-containing formulations may be employed in aerosol devices such as those used in a pulmonary dosing, and needleless jet injector guns (see, e g , EP 257,956)
An isotonifier may be present to ensure isotonicity of a liquid composition of the PRO polypeptide or antagonist thereto, and includes polyhydric sugar alcohols, preferably tπhydπc or higher sugar alcohols, such as glycerin, erythπtol, 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 Examples of sustained- release matrices include polyesters, hydrogels (e g , poly(2-hydroxyethyl-methacrylate) as described by Langer et al , J Biomed Mater Res , 15 167-277 ( 1981 ) and Langer, Chem Tech , 12 98-105 (1982) or poly(vιnylalcohol)), 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-vinyl acetate (Langer et al , supra), degradable lactic acid-glyco c acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyπc acid (EP 133,988)
While polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in lmmunogenicity Rational strategies can be devised for protein stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophi zing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions
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. 77 4030-4034 (1980), EP 52,322, EP 36,676, EP 88,046, EP 143,949, EP 142,641 , Japanese patent application 83-1 18008, U S Patent Nos 4,485,045 and 4,544,545, and EP 102,324 Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy
The therapeutically effective dose of 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 be administered on an empirical basis
With the above guidelines, the effective dose generally is within the range of from about 0 001 to about 1 0 mg/kg, more preferably about 0 01-1 0 mg/kg, most preferably about 0 01-0 1 mg/kg
For non-oral use in treating human adult hypertension, it is advantageous to administer PRO polypeptide in the form of an injection at about 0 01 to 50 mg, preferably about 0 05 to 20 mg, most preferably 1 to 20 mg, per kg body weight, 1 to 3 times daily by intravenous injection For oral administration, a molecule based on the PRO polypeptide is preferably administered at about 5 mg to 1 g, preferably about 10 to 100 mg, per kg body weight, 1 to 3 times daily It should be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0 5 ng/mg protein Moreover, for human administration, 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 For example, the addition of other known growth factors, such as IGF-I, to the final composition may also affect the dosage Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomoφhometπc determinations, and 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, peπtumoral, mtralesional, or peπlesional 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
If a peptide or small molecule is employed as an antagonist or agonist, it is preferably administered orally or non-orally in the form of a liquid or solid to mammals
Examples of pharmacologically acceptable salts of molecules that form salts and are useful hereunder include alkali metal salts (e , sodium salt, potassium salt), alkaline earth metal salts (e g , calcium salt, magnesium salt), ammonium salts, organic base salts (e g , pyπdine salt, tπethylamme salt), inorganic acid salts (e g , hydrochloπde, sulfate, nitrate), and salts of organic acid (e g , acetate, oxalate, p-toluenesulfonate)
For compositions herein that are useful for bone, cartilage, tendon, or ligament regeneration, the therapeutic method includes administering the composition topically, 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
The choice of matrix material is based on biocompatibi ty, biodegradability, mechanical properties, cosmetic appearance, and interface properties The particular application of the compositions will define the appropriate formulation Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tπcalcium phosphate, hydroxyapatite, polylactic acid, polyglycohc acid, and polyanhydπdes Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen Further matrices are comprised of pure proteins or extracellular matrix components Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tπcalcium phosphate The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability One specific embodiment is a 50 50 (mole weight) copolymer of lactic acid and glyco c acid in the form of porous particles having diameters ranging from 150 to 800 microns In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the polypeptide compositions from disassociating from the matrix
One suitable family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,ethylcellulose,hydoxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, one preferred being cationic salts of carboxymethylcellulose (CMC) Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and poly(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 desoφtion of the polypeptide (or its antagonist) from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide (or its antagonist) the opportunity to assist the osteogenic activity of the progenitor cells
xu Combination Therapies
The effectiveness of the PRO230, PR0216 or PRO302 polypeptide or an agonist or antagonist thereof in preventing or treating the disorder m question may be improved by administering the active agent serially or in combination with another agent that is effective for those puφoses, either in the same composition or as separate compositions
For example, for treatment of cardiac hypertrophy, PRO polypeptide therapy can be combined with the administration of inhibitors of known cardiac myocyte hypertrophy factors, e g , inhibitors of α-adrenergic agonists such as phenylephπne, endothelin- 1 inhibitors such as BOSENTAN™ and MOXONODIN™, 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
Fortreatment of cardiac hypertrophy associated with hypertension, the PRO polypeptide can be administered in combination with β-adrenergic receptor blocking agents, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol, ACE inhibitors, e g , quinapπl, captopril, enalapπl, ramipπl, benazepπl, fosmopπl, or lisinopπl, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichloφhenamide, acetazolamide, or indapamide, and/or calcium channel blockers, e g , diltiazem, nifedipine, verapamil, or nicardipine Pharmaceutical compositions comprising the therapeutic agents identified herein by their generic names are commercially available, and are to be administered following the manufacturers' instructions for dosage, administration, adverse effects, contraindications, etc See, e g , Physicians' Desk Reference (Medical Economics Data Production Co Montvale, N J , 1997), 51th Edition
Preferred candidates for combination therapy in the treatment of hypertrophic cardiomyopathy are β- adrenergic-blocking drugs (e , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol), verapamil, difedipine, or diltiazem Treatment of hypertrophy associated with high blood pressure may require the use of antihypertensive drug therapy, using calcium channel blockers, e g , diltiazem, nifedipine, verapamil, or nicardipine, β-adrenergic blocking agents, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichloφhenamide, acetazolamide, or indapamide, and/or ACE-inhibitors, e g , quinapπl, captopril, enalapπl, ramipπl, benazepπl, fosinopπl, or hsinopπl
For other indications, 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 These agents include various growth factors such as EGF, PDGF, TGF-α or TGF-β, IGF, FGF, and CTGF
In addition, PRO polypeptides or their antagonists used to treat cancer may be combined with cytotoxic, chemotherapeutic, or growth-inhibitory agents as identified above A lso, for cancer treatment, 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
xiu Articles of Manufacture
An article of manufacture such as a kit containing PRO230, PR0216 or PRO302 polypeptide or antagonists thereof useful for the diagnosis or treatment of the disorders described above comprises at least a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes The containers may be formed from a variety of materials such as glass or plastic The container holds a composition that is effective for diagnosing or treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) The active agent in the composition is the PRO230, PR0216 or PRO302 polypeptide or an agonist or antagonist thereto The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice The article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use The article of manufacture may also comprise a second or third container with another active agent as described above E Antibodies
Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments that may inhibit the production or the gene product of the genes identified herein and/or reduce the activity of the gene products
l Polyclonal Antibodies
Methods of preparing polyclonal antibodies are known to the skilled artisan Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or 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 one skilled in the art without undue experimentation
n Monoclonal Antibodies
The antι-PRO230, antι-PR0216 or antι-PRO302 antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstem, Nature. 256 495 (1975) In a hybridoma method, a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro
The immunizing agent will typically include the PRO230, PR0216 or PRO302 polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell 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 medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells For example, if the parental cells lack the enzyme hypoxanthine guanme phosphoπbosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopteπn, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT- deficient cells
Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia Human myeloma and mouse-human heteromyelomacell lines also have been described forthe production of human monoclonal antibodies Kozbor, J Immunol , 133 3001 ( 1984), Brodeur 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 Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) Such techniques and assays are known in the art The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem , 1_07 220 (1980)
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods Goding, supra Suitable culture media for this puφose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin puπficationproceduressuch 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 Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U S Patent No 4,816,567, Morrison et al , supra) or by covalentlyjoinmg to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobu n polypeptide Such a non-immunoglobuhn polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimenc bivalent antibody
The antibodies may be monovalent antibodies Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosslinking Alternatively, the relevant cysteine residues are substituted with another ammo acid residue or are deleted so as to prevent crosslinking
In vitro methods are also suitable for preparing monovalent antibodies Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art
111 Human and Humanized Antibodies
The antι-PRO230, antι-PR0216 or antι-PRO302 antibodies may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e , 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 In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence The humanized antibody preferably also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin Jones etal , Nature, 321 522-525 (1986), Riechmann etal . Nature, 332 323-329 (1988), Presta, Curr Op Struct Biol . 2 593-596 (1992)
Methods for humanizing non-human antibodies are well known in the art Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humanization can be essentially performed following the method of Winter and co-workers (Jones et al , Nature, 321 522-525 ( 1986). Riechmann et al , Nature, 332 323-327 ( 1988), Verhoeven et al , Science, 239 1534- 1536 ( 1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such "humanized" antibodies are 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 In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
Human antibodies can also be produced using various techniques known in the art, including phage display libraries Hoogenboom and Winter, J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581 (1991) The techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1985) and Boerner 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, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, and in the following scientific publications: Marks et al, Bio/Technology. 10: 779-783 ( 1992); Lonberg etal. Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild etal. Nature Biotechnology, H: 845-851 (1996); Neuberger, Nature Biotechnology. J4: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol.. 13: 65-93 (1995).
iv. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO230, PR0216 or PRO302 polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/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 (antibody-antigen combining sites) can be fused to immunoglobulin constant-domain sequences. The fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (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. For further details of generating bispecific antibodies, see, for example, Suresh et al, Methods in Enzymology, 121 : 210 (1986).
v. Heteroconjugate Antibodies
Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune-system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection. WO 91/00360; WO 92/200373; EP 03089. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this puφose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
vi. Effector Function Engineering
It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e g , the effectiveness of the antibody m treating cancer For example, cysteine resιdue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region The homodimeπc antibody thus generated may have improved 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) Homodimeπc antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al , Cancer Research. 53 2560-2565 (1993) Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See, Stevenson et al , Anti-Cancer Drug Design. 3 219-230 ( 1989)
vii Immunoconiugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (/ e , a radioconjugate)
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abπn 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, sapaonaπa officinalis inhibitor, gelonm, mitogel n, restπctocin, phenomycin, enomycin, and the tπcothecenes A variety of radionuchdes are available for the production of radioconjugated antibodies Examples include 2l2Bι, l3 lI, ,3'In, 90Y, and l86Re
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such asN-succιnιmιdyl-3-(2-pyπdyldιthιol)propιonate (SPDP), ιmιnothιolane (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 bιs-(p-dιazonιumbenzoyl)-ethylenedιamιne), dnsocyanates (such as tolyene 2,6-dnsocyanate), and bis- active fluorine compounds (such as l ,5-dιfluoro-2,4-dιnιtrobenzene) For example, a ricin immunotoxin can be prepared as described in Vitetta et al , Science, 238 1098 (1987) Carbon- 14-labeled l-ιsothιocyanatobenzyl-3- methyldiethylene tnaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody See, W094/1 1026
In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e g avidin) that is conjugated to a cytotoxic agent (e , a radionucleotide)
viii Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al Proc Natl Acad Sci USA. 82: 3688 (1985); Hwang et al, Proc. Natl. Acad. Sci. USA, 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).
ix. Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a PRO230, PR0216 or PRO302 polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders as noted above and below in the form of pharmaceutical compositions.
If the PRO230, PR0216 or PRO302 polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, 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. For example, based upon the variable- region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the puφose intended.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in 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. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
x. Methods of Treatment using the Antibody
It is contemplated that the antibodies to a PRO230, PR0216 or PRO302 polypeptide may be used to treat various cardiovascular, endothelial, and angiogenic conditions as noted above.
The antibodies are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred.
Other therapeutic regimens may be combined with the administration of the antibodies of the instant invention as noted above. For example, if the antibodies are to treat cancer, the patient to be treated with such antibodies may also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service. Ed., M.C. Perry (Williams & Wilkins: Baltimore, MD, 1992). The chemotherapeutic agent may precede, or follow administration of the antibody, or may be given simultaneously therewith. The antibody may be combined with an anti-estrogen compound such as tamoxifen or EVISTA™ or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
If the antibodies are used for treating cancer, it may be desirable also to administer antibodies against other tumor-associated antigens, such as antibodies that bind to one or more of the ErbB2, EGFR, ErbB3, ErbB4, or VEGF receptor(s). These also include the agents set forth above. Also, the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances. Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to the patient. Sometimes, it may be beneficial also to administer one or more cytokines to the patient. In a preferred embodiment, the antibodies herein are co- administered with a growth-inhibitory agent. For example, the growth-inhibitory agent may be administered first, followed by an antibody of the present invention. However, simultaneous administration or administration of the antibody of the present invention first is also contemplated. Suitable dosages for the growth-inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth-inhibitory agent and the antibody herein.
In one embodiment, vascularization of tumors is attacked in combination therapy. The anti-PRO polypeptide antibody and another antibody (e.g., anti-VEGF) are administered to tumor-bearing patients at therapeutically effective doses as determined, for example, by observing necrosis of the tumor or its metastatic foci, if any. This therapy is continued until such time as no further beneficial effect is observed or clinical examination shows no trace of the tumor or any metastatic foci. Then TNF is administered, alone or in combination with an auxiliary agent such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin, anti-heregulin antibody, D-factor, interleukin- 1 (IL- 1 ), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), or agents that promote microvascular coagulation in tumors, such as anti-protein C antibody, anti-protein S antibody, or C4b binding protein (see, WO 91/01753, published 21 February 1991 ), or heat or radiation.
Since the auxiliary agents will vary in their effectiveness, it is desirable to compare their impact on the tumor by matrix screening in conventional fashion. The administration of anti-PRO polypeptide antibody and TNF is repeated until the desired clinical effect is achieved. Alternatively, the anti-PRO polypeptide antibody is administered together with TNF and, optionally, auxiliary agent(s). In instances where solid tumors are found in the limbs or in other locations susceptible to isolation from the general circulation, the therapeutic agents described herein are administered to the isolated tumor or organ. In other embodiments, a FGF or PDGF antagonist, such as an anti-FGF or an anti-PDGF neutralizing antibody, is administered to the patient in conjunction with the anti- PRO polypeptide antibody. Treatment with anti-PRO polypeptide antibodies preferably may be suspended during periods of wound healing or desirable neovascularization.
For the prevention or treatment of cardiovascular, endothelial, and angiogenic disorder, the appropriate dosage of an antibody herein will depend on the type of disorder to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic puφoses, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.
For example, depending on the type and severity of the disorder, about 1 μg/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily or weekly dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated or sustained until a desired suppression of disorder symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging.
xi. Articles of Manufacture with Antibodies
An article of manufacture containing a container with the antibody and a label is also provided. Such articles are described above, wherein the active agent is an antι-PRO230, antι-PR0216 or antι-PRO302 antibody
xn Diagnosis and Prognosis of Tumors using Antibodies
If the indication for which the antibodies are used is cancer, while cell-surface proteins, such as growth receptors over expressed in certain tumors, are excellent targets for drug candidates or tumor (e g , cancer) treatment, the same proteins along with PRO polypeptides find additional use in the diagnosis and prognosis of tumors For example, antibodies directed against the PRO polypeptides may be used as tumor diagnostics or prognostics
For example, antibodies, including antibody fragments, can be used qualitatively or quantitatively to detect the expression of genes including the gene encoding the PRO polypeptide The antibody preferably is equipped with a detectable, e , fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluoπmetry, 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 For this puφose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample This procedure also allows for determining the distribution of the marker gene product in the tissue examined It will be apparent to those skilled in the art that a wide variety of histological methods are readily available for in situ detection
The following Examples are offered for illustrative puφoses only, and are not intended to limit the scope of the present invention in any way
The disclosures of all patent and literature references cited in the present specification are hereby incoφorated by reference in their entirety
EXAMPLES Commercially available reagents referred to in the Examples were used according to manufacturer's instructions unless otherwise indicated The source of those cells identified in the following Examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, VA Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks Sambrook et al , supra, Ausubel et al , Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N Y , 1989), Innis et al PCR Protocols A Guide to Methods and Applications (Academic Press, Ine N Y , 1990), Harlow et al Antibodies A Laboratory Manual (Cold Spring Harbor Press Cold Spring Harbor, 1988), Gait, Oligonucleotide Synthesis (IRL Press Oxford, 1984), Freshney, Animal Cell Culture. 1987, Co gan et al , Current Protocols in Immunology, 1991 EXAMPLE 1
Isolation of cDNA Clones Encoding Human PRO230 (a tubulointerstitial nephritis antigen homolog)
The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al. Methods in Enzymology, 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA30857. An EST proprietary to Genentech was employed in the consensus assembly and is herein designated DNA20088. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above.
Based on the DNA30857 consensus sequence, oligonucleotides were synthesized: 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO230. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al. , Current Protocols in Molecular Biology, supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
PCR primers (forward and reverse) were synthesized: forward PCR primer:
5'-TTCGAGGCCTCTGAGAAGTGGCCC-3' (SEQ ID NO:3) reverse PCR primer:
5'-GGCGGTATCTCTCTGGCCTCCC-3' (SEQ ID NO:4) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30857 sequence which had the following nucleotide sequence: hybridization probe:
5'-TTCTCCACAGCAGCTGTGGCATCCGATCGTGTCTCAATCCATTCTCTGGG-3' (SEQ ID NO:5)
In order to screen several libraries for a source of a full-length clone, 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
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PRO230 polypeptide (designated herein as DNA33223-1 136 [Figure 1 , SEQ ID NO 1 ]) and the derived protein sequence for that PRO230 polypeptide
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 100-102 and a stop signal at nucleotide positions 592-594 (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 (SEQ ID NO 2) evidences the presence of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PRO230 sequence (Figure 2, SEQ ID NO 2) evidences the following a signal peptide from about amino acid 1 to about 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 about amino acid 1 17 to about amino acid 121, from about amino acid 126 to about amino acid 130, and from about amino acid 137 to about amino acid 141 , N-myπstoylation sites from about amino acid 21 to about ammo acid 27, from about amino acid 39 to about ammo acid 45, from about amino acid 44 to about amino acid 50, and from about amino acid 104 to about amino acid 1 10, and an amidation site from about amino acid 26 to about ammo acid 30 Clone DNA33223-1 136 has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no 209264
An analysis of the Dayhoff database (version 35 45 SwissProt 35), using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 2 (SEQ ID NO 2), evidenced sequence identity between the PRO230 amino acid sequence and tubulointerstitial nephritis antigen
EXAMPLE 2 Isolation of cDNA Clones Encoding Human PRQ216 (an osteomodulin/fibromodulin homolog)
The complete cDNA sequence of DNA33087 is disclosedin GenBank under accession numbers AB0001 14 1 and AB009589 1 (human osteomodulin) A related, but probably different protein, corneal keratin sulfate, is disclosed in Funderburgh et al , J Biol Chem , 27_1 31431-31436 (1996) For this particular EXAMPLE, the clone was isolated as follows
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. 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 DNA28754. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above.
Based on the DNA28754 consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0216. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1 -1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al. Current Protocols in Molecular Biology, supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
PCR primers (forward and reverse) were synthesized: forward PCR primer:
5'-TCACGATGATCCTGACAATGC-3' (SEQ ID NO:8) reverse PCR primer:
5'-AATAATGAAGGTCAAAGTGCCCTT-3' (SEQ ID NO:9) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA28754 sequence which had the following nucleotide sequence: hybridization probe:
5'-TGCTCCTTCTTGTTCTGGGCTCTCATG-3' (SEQ ID NO: 10)
In order to screen several libraries for a source of a full-length clone, 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.
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. Analysis of the full-length PR0216 sequence (Figure 4, SEQ ID NO:7) evidences the following: N-linked glycosylation sites from about amino acid 1 13 to about amino acid 1 17, from about amino acid 121 to about amino acid 125, from about amino acid 187 to about amino acid 191, from about amino acid 242 to about amino acid 246, and from about amino acid 316 to about amino acid 320; casein kinase II phosphorylation sites from about amino acid 189 to about amino acid 193, and from about amino acid 247 to about amino acid 251 ; tyrosine kinase phosphorylation sites from about amino acid 268 to about amino acid 275, and from about amino acid 300 to about amino acid 307; an N-myristoylation site from about amino acid 230 to about amino acid 236; and leucine zipper patterns from about amino acid 146 to about amino acid 168, and from about amino acid 217 to about amino acid 239. Clone DNA33087 has been deposited with ATCC on October 16, 1997 and is assigned ATCC deposit no. 209381.
This clone is the same as human osteomodulin submitted by I. Ohno to GenBank on December 26, 1996 (AB0001 14 1) and submitted by I. Ohno et al, to GenBank on December 5, 1997 (AB009589-1 ).
EXAMPLE 3 Isolation of cDNA Clones Encoding Human PRO302 (vitellogenic carboxypeptidase homolog) The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al. Methods in Enzymology, 266:460-480 (1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above. This consensus sequence is herein designated DNA35953. In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above.
Based on the DNA35953 consensus sequence, oligonucleotides were synthesized: 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO302. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al. , Current Protocols in Molecular Biology, supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
PCR primers (forward and reverse) were synthesized: forward PCR primer 1 :
5'-GTCCGCAAGGATGCCTACATGTTC-3' (SEQ ID NO: 13) forward PCR primer 2:
5'-GCAGAGGTGTCTAAGGTTG-3' (SEQ ID NO: 14) reverse PCR primer:
5'-AGCTCTAGACCAATGCCAGCTTCC-3' (SEQ ID NO: 15) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA35953 sequence which had the following nucleotide sequence: hybridization probe:
5'-GCCACCAACTCCTGCAAGAACTTCTCAGAACTGCCCCTGGTCATG-3' (SEQ ID NO: 16)
In order to screen several libraries for a source of a full-length clone, 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.
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a full-length PRO302 polypeptide (designated herein as DN A40370- 1217 [Figure 5, SEQ ID NO: 1 1 ]) and the derived protein sequence for that PRO302 polypeptide.
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 34-36 and a stop signal at nucleotide positions 1390-1392 (Figure 5, SEQ ID NO: 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 (SEQ ID NO: 12) evidences the presence of important polypeptide domains as shown in Figure 6, wherein the locations given for those important polypeptide domains are approximate as described above. Analysis of the full-length PRO302 sequence (Figure 6, SEQ ID NO: 12) evidences the following: a signal peptide from about amino acid 1 to about amino acid 25; N-linked glycosylation sites from about amino acid 64 to about amino acid 68, from about amino acid 126 to about amino acid 130, and from about amino acid 362 to about amino acid 366; a cAMP- and cGMP-dependent protein kinase phosphorylation site from about amino acid 101 to about amino acid 105; casein kinase II phosphorylation sites from about amino acid 204 to about amino acid 208, from about amino acid 220 to about amino acid 224, from about amino acid 280 to about amino acid 284, from about amino acid 284 to about amino acid 288, from about amino acid 351 to about amino acid 355, and from about amino acid 449 to about amino acid 453; and N-myristoylation sites from about amino acid 22 to about amino acid 28, from about amino acid 76 to about amino acid 82, from about amino acid 79 to about amino acid 85, from about amino acid 80 to about amino acid 86, from about amino acid 1 19 to about amino acid 125, from about amino acid 169 to about amino acid 175, from about amino acid 187 to about amino acid 193, from about amino acid 195 to about amino acid 201 , from about amino acid 331 to about amino acid 337, from about amino acid 332 to about amino acid 338, and from about amino acid 360 to about amino acid 366. Clone DNA40370- 1217 has been deposited with ATCC on November 21 , 1997 and is assigned ATCC deposit no. 209485.
An analysis of the Dayhoff database (version 35.45 SwissProt 35). using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 6 (SEQ ID NO: 12), evidenced sequence identity between the PRO302 amino acid sequence and vitellogenic carboxypeptidase.
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:
Protocol: Human venous umbilical vein endothelial cells (HU VEC, Cell Systems) (passage number less than 8 from primary) are mixed with type I rat tail collagen, final concentration 2.6 mg/ml at a density of 6 x 105 cells/ml and plated at 50 μl per well on a 96-well plate. The gel is allowed to solidify for 1 hr at 37°C, then 50 μl per well of M 1 9 culture media supplemented with 1 % FBS and a PRO230 polypeptide sample (at dilutions of 1 %, 0.1%, and 0.01%, respectively) is added along with 1 μM 6-FAM-FITC dye to stain vacuoles while they are forming. Cells are incubated at 37°C/5% CO-, for 48 hrs, fixed with 3.7% formalin at room temperature for 10 minutes, washed with PBS five times, then stained with Rh-Phalloidin at 4°C overnight followed by nuclear staining with 4 μM DAPI.
1. Apoptosis Assay
This assay will identify factors that facilitate cell survival in a 3-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.
2. Vacuoles Assay This assay will identify factors that stimulate endothelial vacuole formation and lumen formation in the presence of bFGF and VEGF (40ng/ml).
A positive result is equal to or greater than 2. 1 = vacuoles present in less than 20% of cells, 2 = vacuoles present in 20-50% of cells, 3 = vacuoles present in greater than 50% of cells. This assay is designed to identify factors that are involved in stimulating pinocytosis, ion pumping, permeability, and junction formation. 3. Tube Formation Assay
This assay is to identify factors that stimulate endothelial tube formation in a 3-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).
A positive result is equal to or greater than 2. 1 = cells are all round, 2 = cells are elongated, 3 = cells are forming tubes with some connections, 4 = cells are forming complex tubular networks. This assay would identify factors that may be involved in stimulating tracking, chemotaxis, or endothelial shape change.
The results clearly demonstrate that more complex tube formation occurs with PRO230-IgG and PRO230- poly-His samples at 1 % dilution compared with buffer controls ( 10 mM HEPES/0.14M NaCl/4% mannitol, pH 6.8) at 1% dilution.
EXAMPLE 5 Induction of c-fos in Endothelial Cells
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 04 cells/well. The day after plating, the cells were starved by removing the growth media and treating the cells with 100 μl/well test samples and controls (positive control: growth media; negative control: 10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8). The cells were incubated for 30 minutes at 37°C, in 5% C02. The samples were removed, and the first part of the bDNA kit protocol (Chiron Diagnostics, cat. #6005-037) was followed, where each capitalized reagent/buffer listed below was available from the kit.
Briefly, the amounts of the TM Lysis Buffer and Probes needed for the tests were calculated based on information provided by the manufacturer. The 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 μl of Capture Hybridization Buffer was added to each b-DNA well needed, followed by incubation for at least 30 minutes. The test plates with the cells were removed from the incubator, and the media was gently removed using the vacuum manifold. 100 μl of Lysis Hybridization Buffer with Probes were quickly pipetted into each well of the microtiter plates. The plates were then incubated at 55 °C for 15 minutes. Upon removal from the incubator, the plates were placed on the vortex mixer with the microtiter adapter head and vortexed on the #2 setting for one minute. 80 μl of the lysate was removed and added to the bDNA wells containing the Capture Hybridization Buffer, and pipetted up and down to mix. The plates were incubated at 53 °C for at least 16 hours.
On the next day, the second part of the 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/μl) in AL Hybridization Buffer. The hybridization mixture was removed from the plates and washed twice with Wash A. 50 μl of Amplifier Working Solution was added to each well and the wells were incubated at 53 °C for 30 minutes. The plates were then removed from the incubator and allowed to cool for 10 minutes. The Label Probe Working Solution was prepared by making a 1 : 100 dilution of Label Concentrate (40 pmoles/μl) in AL Hybridization Buffer. After the 10-minute cool-down period, the amplifier hybridization mixture was removed and the plates were washed twice with Wash A. 50 μl of Label Probe Working Solution was added to each well and the wells were incubated at 53 °C for 15 minutes. After cooling for 10 minutes, the Substrate was warmed to room temperature. Upon addition of 3 μl of Substrate Enhancer to each ml of Substrate needed for the assay, the plates were allowed to cool for 10 minutes, the label hybridization mixture was removed, and the plates were washed twice with Wash A and three times with Wash D. 50 μl of the Substrate Solution with Enhancer was added to each well. The plates were incubated for 30 minutes at 37°C and RLU was read in an appropriate luminometer.
The replicates were averaged and the coefficient of variation was determined. The measure of activity of the fold increase over the negative control (HEPES buffer described above) value was indicated by chemiluminescence units (RLU). Samples that show an at least two- fold value over the negative control value were considered positive.
PR0216 assayed "positive" two times:
(1 ) Negative Control = 4.18 RLU Positive control = 14.95 RLU PRO216 at 0.01 % = 10.33 RLU
(2) Negative control = 6.35 RLU Positive control = 30.46 RLU PRO216 at 0.01% = 13.37 RLU
EXAMPLE 6 Guinea Pig Vascular Leak This assay is designed to determine whether PRO302 polypeptide shows the ability to induce vascular permeability.
Hairless guinea pigs weighing 350 grams or more were anesthetized with Ketamine (75-80 mg/kg) and 5 mg/kg Xylazine intramuscularly. Test samples containing the PRO302 polypeptide or a physiological buffer without the test polypeptide are injected into skin on the back of the test animals with 100 μl per injection site intradermally. There were approximately 16-24 injection sites per animal. One ml of Evans blue dye (1% in PBS) is then injected intracardially. Skin vascular permeability responses to the compounds (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 μg/100 μl is used as a positive control, inducing a response of 15-23 mm diameter.
The results are shown in Table 4 below.
Table 4 PRO Name Hours Post-Injection mm Blueness (diameter)
PRO302 1.0 9.0
PRO302 6.0 9.0
EXAMPLE 7 In situ Hybridization
In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis, and aid in chromosome mapping.
In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision, 1: 169-176 ( 1994), using PCR-generated 33P-labeled riboprobes. Briefly, formalin-fixed, paraffin- embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37 °C, and further processed for in situ hybridization as described by Lu and Gillett, supra. A (33-P)UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55 °C overnight. The slides were dipped in Kodak NTB2™ nuclear track emulsion and exposed for 4 weeks.
— P-Riboprobe synthesis
6.0 μl (125 mCi) of 33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed-vacuum dried. To each tube containing dried 33P-UTP, the following ingredients were added:
2.0 μl 5x transcription buffer
1.0 μl DTT (100 mM)
2.0 μl NTP mix (2.5 mM: 10 μl each of 10 mM GTP, CTP & ATP + 10 μl H20)
1.0 μl UTP (50 μM) l .O μl RNAsin
1.0 μl DNA template (1 μg)
1.0 μl H,O
1.0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually) The tubes were incubated at 37°C for one hour A total of 1 0 μl RQ1 DNase was added, followed by incubation at 37 °C for 15 minutes A total of 90 μl TE (10 M Tπs pH 7 6/1 mM EDTA pH 8 0) was added, and the mixture was pipetted onto DE81 paper The remaining solution was loaded in a MICROCON-50™ ultrafiltration unit, and spun using program 10 (6 minutes) The filtration unit was inverted over a second tube and spun using program 2 (3 minutes) After the final recovery spin, a total of 100 μl TE was added, then 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR II™
The probe was run on a TBE/urea gel A total of 1-3 μl of the probe or 5 μl of RNA Mrk III was added to 3 μl of loading buffer After heating on a 95 °C heat block for three minutes, the gel was immediately placed on ice The wells of gel were flushed, and the sample was loaded and run at 180-250 volts for 45 minutes The gel was wrapped in plastic wrap (SARAN™ brand) and exposed to XAR film with an intensifying screen in a -70 °C freezer one hour to overnight
— P-Hvbndization
A Pretreatment of frozen sections
The slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes The trays were placed in a 55 CC 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) After deproteination in 0 5 μg/ml protemase K for 10 minutes at 37 °C (12 5 μl of 10 mg/ml stock in 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0 5 x SSC for 10 minutes at room temperature The sections were dehydrated in 70%, 95%, and 100% ethanol, 2 minutes each
B Pretreatment of paraffin-embedded sections
The slides were deparaffinized, placed in SQ H,0, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time The sections were deproteinated in 20 μg/ml protemase K (500 μl of 10 mg/ml in 250 ml RNase-free RNase buffer, 37 °C, 15 minutes) for human embryo tissue, or 8 x protemase K ( 100 μl in 250 ml Rnase buffer, 37 °C, 30 minutes) for formalin tissues Subsequent rinsing in 0 5 x SSC and dehydration were performed as described above
C Prehybridtzation
The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper The tissue was covered with 50 μl of hybridization buffer (3 75 g dextran sulfate + 6 ml SQ H20), vortexed, and heated in the microwave for 2 minutes with the cap loosened After cooling on ice, 18 75 ml formamide, 3 75 ml 20 x SSC, and 9 ml SQ H,0 were added, and the tissue was vortexed well and incubated at 42 °C for 1-4 hours
D Hybridization
1 0 x 106 cpm probe and 1 0 μl tRNA (50 mg/ml stock) per slide were heated at 95 °C for 3 minutes The slides were cooled on ice, and 48 μl hybridization buffer was added per slide After vortexing, 50 μl 33P mix was added to 50 μl prehybndization on the slide The slides were incubated overnight at 55 °C
E Washes
Washing was done for 2x10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0 25 M EDTA, V = L). followed by RNAseA treatment at 37°C for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer = 20 μg/ml), The slides were washed 2 x10 minutes with 2 x SSC, EDTA at room temperature The stringency wash conditions were as follows 2 hours at 55 °C, 0 1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA, Vr=4L)
F Oligonucleotides
In situ analysis was performed on two of the DNA sequences disclosed herein The oligonucleotides employed for these analyses are as follows
(1 ) DNA33223-1 136 (PRO230) 33223pl
5'-GGATTCTAATACGACTCACTATAGGGCGGCGATGTCCACTGGGGCTAC-3' (SEQ ID NO 17) 33223p2
5'- CTATGAAATTAACCCTCACTAAAGGGACGAGGAAGATGGGCGGATGGT-3' (SEQ ID NO 18)
(2) DNA33087 (PRO216) 33087pl
5'-GGATTCTAATACGACTCACTATAGGGCTGGATGGGCTAGTAAACTTGA-3' (SEQ ID NO 19) 33087p2 5'-CTATGAAATTAACCCTCACTAAAGGGACCCTTCTGCTCCTTCTTGTT-3' (SEQ ID NO 20)
G Results
In situ analysis was performed on the above two DNA sequences disclosed herein The results from these analyses are as follows ( 1) DNA33223- 1 136 (PRO230)
Sections showed an intense signal associated with arterial and venous vessels in the fetus In arteries, the signal appeared to be confined to smooth-muscle/peπcyte 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
In summary, PRO230 is a molecule that shows relatively specific vascular expression in the fetus as well as in some adult organs Expression was also observed in the fetal lens and the adult gallbladder
In a secondary screen, vascular expression was observed in fetal blocks, similar to that observed above Expression was on vascular smooth muscle, rather than endothelium Expression was also seen in smooth muscle of the developing oesophagus, so this molecule is not vascular specific Expression was examined in four lung and four breast carcinomas Substantial expression was seen in vascular smooth muscle of at least three out of four lung cancers and two out of four breast cancers In addition, in one breast carcinoma, expression was observed in pentumoral stromal cells of uncertain histogenesis (possibly myofibroblasts) No endothelial cell expression was observed in this study (2) DNA33087 (PRO216)
Sections showed strong specific expression in osteoblasts at all sites of enchondral and peπosteal new bone formation Additional sites of expression included the developing pulmonary arterial and aortic trunks Otherwise, all fetal tissues tested negative The fetal tissues examined included placenta, umbilical cord, brain, spinal cord, eye, optic nerve, trachea, lung, heart, thymus, liver, spleen, esophagus, small intestine, pancreas, adrenal, thyroid, body wall, and lower limb All of the adult tissues were negative The adult tissues examined included liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung and skin PR0216 has a probable role in control of bone matrix deposition and/or osteoblast growth All adult tissues in the multiblock were positive for beta-actin
EXAMPLE 8 Use of PRO230. PRQ216 or PRO302 as a Hybridization Probe
The following method describes use of a nucleotide sequence encoding PRO230, PR0216 or PRO302 as a hybridization probe
DNA comprising the coding sequence of full-length or mature PRO230, PR0216 or PRO302 (as shown in Figures 1 , 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 transcriptional terminator, and an argU gene
The hgation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic-resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing
Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger-scale culture The cells are then grown to a desired optical density, during which the expression promoter is turned on
After culturing the cells for several more hours, the cells can be harvested by 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
EXAMPLE 10 Expression of Nucleic Acid Encoding PRO230, PRQ216 or PRO302 in Mammalian Cells
This Example illustrates preparation of a potentially glycosylated form of PRO230, PR0216 or PRO302 by recombinant expression in mammalian cells
The vector, pRK5 (see, EP 307,247, published March 15, 1989), is employed as the expression vector Optionally, the PRO230, PR0216 or PRO302 DNA is 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)
In one embodiment, the selected host cells are 293 cells Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and or antibiotics About 10 μg DNA of pRK5-(DNA encoding PRO230, PR0216 or PRO302) is mixed with about 1 μg DNA encoding the VA RNA gene (Thimmappaya et al , Cell, 3J_ 543 ( 1982)) and dissolved in 500 μl of 1 M Tπs-HCl, 0 1 mM EDTA, 0 227 M CaCl, To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7 35), 280 mM NaCl, 1 5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25°C The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds The 293 cells are then washed with serum-free medium, fresh medium is added, and the cells are incubated for about 5 days
Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml 35S-cysteιne and 200 μCi/ml 35S-methιonιne After a 12-hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of the PRO230, PR0216 or PRO302 polypeptide The cultures containing transfected cells may undergo further incubation (in serum-free medium) and the medium is tested in selected bioassays
In an alternative technique, the gene encoding PRO230, PR0216 or PRO302 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Natl Acad Sci . 12 7575 (1981 ) 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-(DNA encoding PRO230, PR0216 or PRO302) is added The cells are first concentrated from the spinner flask by 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 μg/ml bovine insulin, and 0 1 μg/ml bovine transfernn After about four days, the conditioned media is centnfuged and filtered to remove cells and debris The sample containing the expressed gene encoding the PRO230, PR0216 or PRO302 polypeptide can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography
In another embodiment, the gene encoding PRO230, PR0216 or PRO302 can be expressed in CHO cells The pRK5-(DNA encoding PRO230, PR0216 or PRO302) nucleic acid can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35S-methιonιne After determining the presence of PRO230, PR0216 or PRO302 polypeptide, the culture medium may be replaced with serum-free medium Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested The medium containing the expressed PRO230, PR0216 or PRO302 can then be concentrated and purified by any selected method
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-Hιs-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 Finally, the CHO cells can be transfected (as described above) with the SV40-dπven vector Labeling may be performed, as described above, to verify expression The culture medium containing the expressed gene encoding the poly-Hιs-tagged-PRO230, -PR0216 or -PRO302 can then be concentrated and purified by any selected method, such as by Nι2+-chelate affinity chromatography
PRO230 and PR0216 were stably expressed in CHO cells by the above described method
EXAMPLE 1 1 Expression of Nucleic Acid Encoding PRO230, PRQ216 or PRO302 in Yeast The following method describes recombinant expression of the gene encoding PRO230, PR0216 or PRO302 in yeast
First, yeast expression vectors are constructed for intracellular production or secretion of PRO230, PR0216 or PRO302 from the ADH2/GAPDH promoter. DNA encoding PRO230, PR0216 or PRO302 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the gene encoding PRO230, PR0216 or PRO302. For secretion, DNA encoding PRO230, PR0216 or PRO302 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO230, PR0216 or PRO302 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of the gene encoding PRO230, PR0216 or PRO302.
Yeast cells, such as yeast strain AB 1 10, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% 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.
EXAMPLE 12
Expression of Nucleic Acid Encoding PRO230, PRQ216 or PRO302 in Baculovirus-infected Insect Cells
The following method describes recombinant expression in Baculovirus-infected insect cells.
The sequence coding for PRO230, PR0216 or PRO302 is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PRO230, PR0216 or PRO302 or the desired portion of the coding sequence of PRO230, PR0216 or PRO302 [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incoφorate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into 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). Briefly, 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 μm 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 25 ml of loading buffer The filtered cell extract is loaded onto the column at 0 5 ml per minute The column is washed to baseline A28o with loading buffer, at which point fraction collection is started Next, the column is washed with a secondary wash buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 6 0), which elutes non-specifically- bound protein After reaching A280 baseline again, the column is developed with a 0 to 500 mM lmidazole gradient in the secondary wash buffer One ml fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni 2+-NTA-conjugated to alkaline phosphatase (Qiagen) Fractions containing the eluted Hιs,0-tagged-PRO230, -PR0216 or -PRO302, respectively, are pooled and dialyzed against loading buffer
Alternatively, purification of the IgG-tagged (or Fc tagged)-PRO230, -PR0216 or -PRO302 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography
PRO230 and PR0216 were 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
Following PCR amplification, the respective coding sequences were subcloned into a baculovirus expression vector (pb PH IgG for IgG fusions and pb PH His c for poly-His tagged proteins), and the vector and Baculogold® baculovirus DNA (Pharmingen) were co-transfected into 105 Spodoptera 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 Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOl) of 10 Cells were incubated for 3 days at 28 °C The supernatant was harvested and the expression of the constructs in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 ml of Ni 2+-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining
The first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate 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 For the poly-His tagged constructs, 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 After loading, 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% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80 °C lmmunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows The conditioned media were pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5 The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tπs buffer, pH 9 The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity of the proteins was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal ammo acid sequencing by Edman degradation
Alternatively, a modified baculovirus procedure may be used incoφorating hιgh-5 cells In this procedure, the DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIE 1 - 1 (Novagen) The pIE 1 - 1 and pIE 1 -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus lei promoter in stably-transformed insect cells The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lei -mediated gene expression in unmfected insect cells as well as the hr5 enhancer element pIE l -1 and pIEl -2 include the translation initiation site and can be used to produce fusion proteins Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) was amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incoφorate flanking (selected) restriction enzyme sites The product was then digested with those selected restriction enzymes and subcloned into the expression vector For example, derivatives ofpIEl-1 can include the Fc region of human IgG (pb PH IgG)oran 8 hιstιdιne (pb PH His) tag downstream (3'-of) the desired sequence Preferably, the vector construct is sequenced for confirmation
Hιgh-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no C02, NO pen/strep For each 150 mm plate, 30 μg of pIE based vector containing the sequence was mixed with 1 ml Ex-Cell medium (Media Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401 -78P (note this media is light sensitive)), and in a separate tube, 100 μl of CellFectin (CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium The two solutions were combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex-Cell media was added to the 2 ml of DNA/CellFECTIN mix and this is layered on hιgh-5 cells that have been washed once with Ex-Cell media The plate is then incubated in darkness for 1 hour at room temperature The DNA/CellFECTIN mix is then aspirated, and the cells are washed once with Ex-Cell to remove excess CellFECTIN, 30 ml of fresh Ex-Cell media was added and the cells are incubated for 3 days at 28 °C. The supernatant was harvested and the expression of the sequence in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 ml of Ni 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. For the poly-His tagged constructs, the protein comprising the sequence is purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni 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. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then subsequently desalted into a storage buffer containing 10 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. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the sequence was assessed by SDS polyacrylamide gels and by N-terminal 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 incoφorating high-5 cells.
EXAMPLE 13 Preparation of Antibodies that Bind PRO230, PRQ216 or PRO302
This Example illustrates preparation of monoclonal antibodies that can specifically bind PRO230, PR0216 or PRO302.
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified PRO230, PR0216 or PRO302 fusion proteins containing PRO230, PR0216 or PRO302, and cells expressing the gene encoding PRO230, PR0216 or PRO302 on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO230, PR0216 or PRO302 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1 to 100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional immunization injections Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect antι-PRO230, antι-PR0216 or antι-PRO302 antibodies
After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO230, PR0216 or PRO302 Three to four days later, the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU 1, available from ATCC, No CRL 1597 The fusions generate hybridoma cells that can then be plated in 96-well tissue culture plates containing HAT medium to inhibit proliferation of non- fused cells, myeloma hybrids, and spleen cell hybrids
The hybridoma cells will be screened in an ELISA for reactivity against PRO230, PR0216 or PRO302 Determination of "positive" hybridoma cells secreting the desired monoclonal 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 antι-PRO230, antι-PR0216 or antι-PRO302 monoclonal antibodies Alternatively, 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
Deposit of Material
The following mateπal(s) has/have been deposited with the American Type Culture Collection, 10801 University Blvd , Manassas, VA 201 10-2209, USA (ATCC) Material ATCC Pep No Deposit Date
DNA33223-1 136 209264 September 16, 1997
DNA33087 209381 October 16, 1997
DNA40370- 1217 209485 November 21 , 1997
This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Puφose of Patent Procedure and the Regulations thereunder (Budapest Treaty) This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Ine , and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U S patent or upon laying open to the public of any U S or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U S Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC § 122 and the Commissioner's rules pursuant thereto (including 37 CFR § 1 14 with particular reference to 886 OG 638)
The assignee of the present application has agreed that if a culture of the mateπal(s) on deposit should die or be lost or destroyed when cultivated under suitable conditions, the mateπal(s) will be promptly replaced on notification with another of the same. Availability of the deposited material(s) is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct(s) deposited, since the deposited embodiment(s) is/are intended as single illustration(s) of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material(s) herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A composition comprising a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereof, in admixture with a pharmaceutically acceptable carrier.
2. The composition of Claim 1 comprising a therapeutically effective amount of said polypeptide or said agonist or antagonist thereof.
3. The composition of Claim 1, wherein the agonist is an anti-PRO230, anti-PR0216 or anti-PRO302 antibody.
4. The composition of Claim 1 , wherein the antagonist is an anti-PRO230, anti-PR0216 or anti-PRO302 antibody.
5. The composition of Claim 1 further comprising a cardiovascular, endothelial, angiogenic or angiostatic agent.
6. A method of preparing the composition of Claim 1 comprising admixing a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereof, with a pharmaceutically acceptable carrier.
7. An article of manufacture comprising:
(1 ) a composition comprising (a) a PRO230, PR0216 or PRO302 polypeptide, (b) an agonist of a PRO230, PR0216 or PRO302 polypeptide, or (c) an antagonist of a PRO230, PR0216 or PRO302 polypeptide, in admixture with a pharmaceutically acceptable carrier;
(2) a container containing said composition; and
(3) a label affixed to said container, or a package insert included in said container, referring to the use of said composition, in the treatment of a cardiovascular, endothelial, and angiogenic disorder.
8. The article of manufacture of Claim 7, wherein said agonist is an anti-PRO230, anti-PR0216 or anti- PRO302 antibody.
9. The article of manufacture of Claim 7, wherein said antagonist is an anti-PRO230, anti-PR0216 or anti- PRO302 antibody.
10. The article of manufacture of Claim 7, wherein said composition comprises a therapeutically effective amount of said polypeptide or agonist or antagonist thereof, in admixture with said pharmaceutically acceptable carrier.
1 1. A method for identifying an agonist of a PRO230, PR0216 or PRO302 polypeptide comprising:
(a) contacting cells and a test compound to be screened under conditions suitable for the induction of a cellular response normally induced by a PRO230, PR0216 or PRO302 polypeptide; and
(b) determining the induction of said cellular response to determine if the test compound is an effective agonist, wherein the induction of said cellular response is indicative of said test compound being an effective agonist.
12. The method of Claim 1 1 , wherein the cellular response normally induced by said polypeptide is stimulation of cell proliferation.
13. A method for identifying a compound that inhibits an activity of a PRO230, PR0216 or PRO302 polypeptide comprising contacting a test compound with said polypeptide under conditions and for a time sufficient to allow the test compound and polypeptide to interact and determining whether the activity of said polypeptide is inhibited.
14. A method for identifying a compound the inhibits an activity of a PRO230, PR0216 or PRO302 polypeptide comprising the steps of:
(a) contacting cells and a test compound to be screened in the presence of said polypeptide under conditions suitable for the induction of a cellular response normally induced by said polypeptide; and
(b) determining the induction of said cellular response to determine if the test compound is an effective antagonist.
15. The method of Claim 14, wherein the cellular response normally induced by said polypeptide is stimulation of cell proliferation.
16. A method for identifying a compound that inhibits the expression of a PRO230, PR0216 or PRO302 polypeptide in cells that normally expresses the polypeptide, wherein the method comprises contacting the cells with a test compound under conditions suitable for allowing expression of said polypeptide and determining whether the expression of said polypeptide is inhibited.
17. An agonist of a PRO230, PR0216 or PRO302 polypeptide.
18. An antagonist of a PRO230, PR0216 or PRO302 polypeptide.
19. A compound that inhibits the expression of a PRO230, PR0216 or PRO302 polypeptide in a mammalian cell which expresses said polypeptide.
20. The compound of Claim 19, wherein said compound is an antisense oligonucleotide.
21. An isolated antibody that binds to a PRO230, PR0216 or PRO302 polypeptide.
22. The antibody of Claim 21 which is a monoclonal antibody.
23. The antibody of Claim 21 which is an antibody fragment.
24. The antibody of Claim 21 which is a single-chain antibody.
25. A method for diagnosing a disease or susceptibility to a disease which is related to a mutation in a PRO230, PR021 or PRO302 polypeptide-encoding nucleic acid sequence comprising determining the presence or absence of said mutation in said polypeptide-encoding nucleic acid sequence, wherein the presence or absence of said mutation is indicative of the presence of said disease or susceptibility to said disease.
26. A method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises analyzing the level of expression of a gene encoding a PRO230, PR0216 or PRO302 polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in said mammal.
27. A method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises detecting the presence or absence of a PRO230, PR0216 or PRO302 polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of said polypeptide in said test sample is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in said mammal.
28. A method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal comprising (a) contacting an anti-PRO230, anti-PR0216 or anti-PRO302 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between said antibody and a PRO230, PR0216 or PRO302 polypeptide in the test sample, wherein the formation of said complex is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in the mammal.
29. A method for determining the presence of a PRO230, PR0216 or PRO302 polypeptide in a sample comprising contacting a sample suspected of containing said polypeptide with an anti-PRO230, anti-PR0216 or anti-PRO302 antibody and determining binding of said antibody to a component of said sample.
30. A cardiovascular, endothelial or angiogenic disorder diagnostic kit comprising an anti-PRO230, anti- PR0216 or antι-PRO302 antibody and a carrier in suitable packaging
31 A method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereof
32 The method according to Claim 31 , wherein the mammal is human
33 The method of Claim 32, wherein the human has suffered myocardial infarction
34 The method of Claim 32, wherein the human has cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration
35 The method of Claim 34, wherein the cardiac hypertrophy is characterized by the presence of an elevated level of PGF2α
36 The method of Claim 31 , wherein the PRO230, PR0216 or PRO302 polypeptide is administered together with a cardiovascular, endothelial or angiogenic agent
37 The method of Claim 34, wherein the PRO230, PR0216 or PRO302 polypeptide is administered following primary angioplasty
38 The method of Claim 31 , wherein the cardiovascular, endothelial or angiogenic disorder is cancer
39 The method of Claim 38, wherein the PRO230, PR0216 or PRO302 polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent or a cytotoxic agent
40 The method of Claim 31 wherein said agonist is an antι-PRO230, antι-PR0216 or antι-PRO302 antibody
41 The method of Claim 32 wherein said antagonist is an antι-PRO230, antι-PR0216 or antι-PRO302 antibody
42 A method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal a nucleic acid molecule that encodes a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereof
43. The method of Claim 42 wherein said agonist is an anti-PRO230, anti-PR0216 or anti-PRO302 antibody.
44. The method of Claim 42 wherein said antagonist is an anti-PRO230, anti-PR0216 or anti-PRO302 antibody.
45. The method of Claim 42, wherein the mammal is human.
46. The method of Claim 42, wherein the nucleic acid molecule is administered via ex vivo gene therapy.
47. A recombinant retroviral particle comprising a retroviral vector consisting essentially of (1 ) a promoter, (2) nucleic acid encoding a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereof, and (3) a signal sequence for cellular secretion of the polypeptide, wherein the retroviral vector is in association with retroviral structural proteins.
48. 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 ( 1 ) promoter, (2) nucleic acid encoding a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereof, and (3) 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.
49. A method for inhibiting endothelial cell growth in a mammal comprising administering to the mammal a PR0216 polypeptide or agonist thereof, wherein endothelial cell growth in said mammal is inhibited.
50. A method for stimulating endothelial cell growth in a mammal comprising administering to the mammal a PRO230 polypeptide or agonist thereof, wherein endothelial cell growth in said mammal is stimulated.
51. A method of inhibiting endothelial cell growth in a mammal comprising administering to the mammal an antagonist of a PRO230 polypeptide, wherein endothelial cell growth in said mammal is inhibited.
52. A method of stimulating endothelial cell growth in a mammal comprising administering to the mammal an antagonist of a PR0216 polypeptide, wherein endothelial cell growth in said mammal is stimulated.
53. A method for inhibiting angiogenesis induced by a PRO302 polypeptide in a mammal comprising administering a therapeutically effective amount of an anti-PRO302 antibody to the mammal, wherein said angiogenesis is inhibited.
54 A method for stimulating angiogenesis induced by a PRO320 polypeptide in a mammal comprising administering a therapeutically effective amount of said polypeptide to the mammal, wherein said angiogenesis is stimulated
55 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an ammo acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7), and Figure 6 (SEQ ID NO 12)
56 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in Figure 1 (SEQ ID NO 1), Figure 3 (SEQ ID NO 6), and Figure 5 (SEQ ID NO 1 1)
57 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the full-length coding sequence of the nucleotide sequence shown in Figure 1 (SEQ ID NO 1 ), Figure 3 (SEQ ID NO 6), and Figure 5 (SEQ ID NO 1 1 )
58 Isolated nucleic acid having at least 80% nucleic acid sequence identity to the full-length coding sequence of the DNA deposited under ATCC accession number 209264, 209381 , or 209485
59 A vector comprising the nucleic acid of any one of Claims 55 to 58
60 The vector of Claim 59 operably linked to control sequences recognized by a host cell transformed with the vector
61 A host cell comprising the vector of Claim 59
62 The host cell of Claim 61 , wherein said cell is a CHO cell
63 The host cell of Claim 61 , wherein said cell is an E colt
64 The host cell of Claim 61 , wherein said cell is a yeast cell
65 The host cell of Claim 61 , wherein said cell is a Baculovirus infected insect cell
66 A process for producing a PRO230, PR0216 or PRO302 polypeptide comprising culturing the host cell of Claim 61 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture
67. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), and Figure 6 (SEQ ID NO: 12).
68. An isolated polypeptide scoring at least 80% positives when compared to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), and Figure 6 (SEQ ID NO: 12).
69. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence encoded by the full-length coding sequence of the DNA deposited under ATCC accession number 209264, 209381 , or 209485.
70. A chimeric molecule comprising a polypeptide according to any one of Claims 67 to 69 fused to a heterologous amino acid sequence.
71. The chimeric molecule of Claim 70, wherein said heterologous amino acid sequence is an epitope tag sequence.
72. The chimeric molecule of Claim 70, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.
73. An antibody which specifically binds to a polypeptide according to any one of Claims 67 to 69.
74. The antibody of Claim 73, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.
75. Isolated nucleic acid having at least 80% nucleic acid sequence identity to:
(a) a nucleotide sequence encoding the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO: 12), lacking its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO: 12), with its associated signal peptide; or
(c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO: 12), lacking its associated signal peptide.
76. An isolated polypeptide having at least 80% amino acid sequence identity to:
(a) the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO: 12), lacking its associated signal peptide; (b) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO: 12), with its associated signal peptide; or
(c) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO: 12),lacking its associated signal peptide.
PCT/US1999/028214 1994-09-08 1999-11-29 Promotion or inhibition of angiogenesis and cardiovascularization WO2001019987A1 (en)

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
AU23907/00A AU2390700A (en) 1999-07-20 1999-12-20 Polypeptidic compositions and methods for the treatment of tumors
PCT/US1999/030999 WO2001005836A1 (en) 1999-07-20 1999-12-20 Polypeptidic compositions and methods for the treatment of tumors
PCT/US2000/004414 WO2001004311A1 (en) 1999-07-07 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU28839/00A AU2883900A (en) 1999-07-07 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002362427A CA2362427A1 (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
PCT/US2000/005841 WO2000053758A2 (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
AU35144/00A AU3514400A (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
EP00913764A EP1220905A2 (en) 1999-03-08 2000-03-02 Composition and methods for the treatment of immune related diseases
KR1020017011406A KR20010103046A (en) 1999-03-08 2000-03-02 Compositions and Methods for the Treatment of Immune Related Diseases
JP2000603379A JP2004516227A (en) 1999-03-08 2000-03-02 Compositions and methods for treating immune-related diseases
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 (en) 1999-07-28 2000-07-28 Method of preventing the injury or death of retinal cells and treating ocular diseases
US09/902,713 US20030082541A1 (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
US09/902,853 US20020192659A1 (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
US09/902,615 US20030092002A1 (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
US09/902,634 US20030082540A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,572 US20030108983A1 (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
US09/902,736 US20030049676A1 (en) 1997-09-17 2001-07-10 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
US09/903,823 US20030104381A1 (en) 1997-09-17 2001-07-11 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
US09/903,520 US20030054401A1 (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
US09/904,119 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
US09/903,943 US20030054349A1 (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
US09/903,806 US20030130489A1 (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
US09/905,291 US20020160374A1 (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
US09/904,938 US20030211569A1 (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
US09/904,766 US20030152999A1 (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
US09/905,088 US20030073077A1 (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
US09/905,125 US6664376B2 (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
US09/904,920 US6806352B2 (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
US09/905,075 US7169906B2 (en) 1997-09-17 2001-07-13 PRO211 polypeptides
US09/904,532 US7151160B2 (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
US09/904,462 US6878807B2 (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
US09/904,485 US20030064367A1 (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
US09/905,449 US6965011B2 (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
US09/904,956 US20030049622A1 (en) 1995-12-01 2001-07-14 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
US09/906,646 US6852848B2 (en) 1997-09-17 2001-07-16 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
US09/906,777 US20030148371A1 (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
US09/906,838 US7070979B2 (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
US09/906,815 US7094567B2 (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
US09/907,979 US20030082542A1 (en) 1994-09-08 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
US09/907,728 US20030190611A1 (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
US09/907,794 US6635468B2 (en) 1997-09-17 2001-07-17 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
US09/907,652 US20030104469A1 (en) 1997-09-17 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
US09/907,824 US20020197671A1 (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
US09/909,320 US7074592B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides nucleic acid encoding
US09/909,064 US6818449B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/908,576 US20040005553A1 (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
US10/121,041 US20030077776A1 (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
US10/121,040 US20030082759A1 (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
US10/121,059 US20030190721A1 (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
US10/121,051 US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,045 US20030073210A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,054 US20030199054A1 (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
US10/121,053 US20030199053A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,056 US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,052 US20030199052A1 (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
US10/121,062 US20030077779A1 (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
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,048 US20030199051A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,058 US20030190720A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,057 US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,055 US20030190718A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,156 US20030194792A1 (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
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,291 US20030199058A1 (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
US10/123,261 US20030068796A1 (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
US10/123,292 US20030073211A1 (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
US10/123,262 US20030049816A1 (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
US10/123,236 US20030068795A1 (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
US10/123,322 US20030199059A1 (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
US10/123,157 US20030190725A1 (en) 1997-03-31 2002-04-15 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
US10/123,905 US20030087344A1 (en) 1997-06-18 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
US10/123,912 US20030100087A1 (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
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,906 US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/124,822 US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,819 US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,817 US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,813 US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/124,823 US20030199062A1 (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
US10/124,824 US20030077659A1 (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
US10/124,821 US20030199023A1 (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
US10/124,818 US20030082763A1 (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
US10/125,927 US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/125,931 US20030199063A1 (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
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/127,831 US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,689 US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/137,865 US20030032155A1 (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
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/140,024 US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,474 US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,470 US20030022331A1 (en) 1997-03-31 2002-05-06 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
US10/140,865 US20030207420A1 (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
US10/140,809 US20030207418A1 (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
US10/140,860 US7307151B2 (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
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,928 US20030068798A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,760 US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/143,113 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
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
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
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/146,792 US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,730 US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 US20030082765A1 (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
US10/158,791 US20030207429A1 (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
US10/160,498 US20030073216A1 (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 (en) 1998-09-14 1999-09-13 Promotion or inhibition of angiogenesis and cardiovascularization
PCT/US1999/021090 WO2000015796A2 (en) 1998-09-16 1999-09-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US99/21090 1999-09-15

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
PCT/US1999/021547 Continuation-In-Part WO2000015797A2 (en) 1994-09-08 1999-09-15 Compositions and methods for the treatment of immune related diseases
PCT/US1999/021090 Continuation-In-Part WO2000015796A2 (en) 1994-09-08 1999-09-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US1999/023089 Continuation-In-Part WO2000021996A2 (en) 1994-09-08 1999-10-05 Methods and compositions for inhibiting neoplastic cell growth

Related Child Applications (3)

Application Number Title Priority Date Filing Date
PCT/US1999/028313 Continuation-In-Part WO2000032221A2 (en) 1994-09-08 1999-11-30 Promotion or inhibition of angiogenesis and cardiovascularization
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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WO1995029242A1 (en) * 1994-04-26 1995-11-02 The Children's Medical Center Corporation Angiostatin and method of use for inhibition of angiogenesis
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Publication number Priority date Publication date Assignee Title
WO2003041652A2 (en) * 2001-11-13 2003-05-22 Millennium Pharmaceuticals, Inc. Method of using 18080, a human serine carboxypeptidase family member
WO2003041652A3 (en) * 2001-11-13 2003-08-14 Millennium Pharm Inc Method of using 18080, a human serine carboxypeptidase family member

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