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EP1159419A1 - Promotion or inhibition of angiogenesis and cardiovascularization - Google Patents

Promotion or inhibition of angiogenesis and cardiovascularization

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Publication number
EP1159419A1
EP1159419A1 EP00912015A EP00912015A EP1159419A1 EP 1159419 A1 EP1159419 A1 EP 1159419A1 EP 00912015 A EP00912015 A EP 00912015A EP 00912015 A EP00912015 A EP 00912015A EP 1159419 A1 EP1159419 A1 EP 1159419A1
Authority
EP
European Patent Office
Prior art keywords
seq
antι
polypeptide
pro
pro840
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00912015A
Other languages
German (de)
French (fr)
Inventor
Avi J. Ashkenazi
Kevin P. Baker
Napoleone Ferrara
Hanspeter Gerber
Mary E. Gerritsen
Audrey Goddard
Austin L. Gurney
Kenneth J. Hillan
Scot A. Marsters
Nicholas F. Paoni
Robert M. Pitti
Colin K. Watanabe
P. Mickey Williams
William I. Wood
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
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/US1999/005028 external-priority patent/WO1999046281A2/en
Priority claimed from PCT/US1999/012252 external-priority patent/WO1999063088A2/en
Priority claimed from PCT/US1999/020111 external-priority patent/WO2000012708A2/en
Priority claimed from PCT/US1999/021090 external-priority patent/WO2000015796A2/en
Priority claimed from PCT/US1999/028409 external-priority patent/WO2000032778A2/en
Priority claimed from PCT/US1999/028313 external-priority patent/WO2000032221A2/en
Priority claimed from PCT/US1999/028565 external-priority patent/WO2000037638A2/en
Priority claimed from PCT/US2000/000219 external-priority patent/WO2000053753A2/en
Priority claimed from PCT/US2000/004342 external-priority patent/WO2000078961A1/en
Priority claimed from PCT/US2000/004341 external-priority patent/WO2000053756A2/en
Priority claimed from PCT/US2000/004414 external-priority patent/WO2001004311A1/en
Application filed by Genentech Inc filed Critical Genentech Inc
Publication of EP1159419A1 publication Critical patent/EP1159419A1/en
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • the present invention relates to compositions and methods useful for promoting or inhibiting angiogenesis and or cardiovascularization 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 116 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 f ibrosis (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.
  • 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.
  • 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).
  • APN atrial natriuretic peptide
  • 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
  • CT-1 cardiotrophin- 1
  • catecholamines catecholamines
  • adrenocorticosteroids angiotensin
  • prostaglandins angiotensin
  • 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
  • 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, angiotensin converting enzyme (ACE) inhibitors such as qumap ⁇ l, captop ⁇ l, enalap ⁇ l, ramip ⁇ l, benazep ⁇ l, fosinop ⁇ l, and lisinop ⁇ l, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, and indapamide, and calcium channel blockers, e g , diltiazem, nifedipine,
  • 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
  • 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 a I .
  • 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 .
  • TNK t-PA a Tl 03N, Nl 17Q, KHRR(296-299)AAAA t-PA variant, Keyt et al , Proc Natl Acad Sci USA.91 3670 3674 ( 1994)
  • TNK t-PA a Tl 03N, Nl 17Q, KHRR(296-299)AAAA t-PA variant, Keyt et al , Proc Natl Acad Sci USA.91 3670 3674 ( 1994)
  • 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
  • FGF basic and acidic fibroblast growth factois
  • PD-ECGF platelet-derived endothelial cell growth factor
  • VEGF vascular endothelial growth factor
  • hVEGF human VEGF
  • hVEGF-related proteins The 121 amino acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 1 16 and 159 in h VEGF
  • the 189-amino 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 amino acid protein differs from h VEGF by virtue of an insertion of 41 amino acids at residue 1 16 in h VEGF Houck et al , Mol Endoc ⁇ n
  • angiogenesis which im ohes the formation of new blood vessels from preexisting endothelium is implicated in the pathogenesis of a vanetv of disorders
  • vanetv of disorders 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 reaction of transplanted corneal tissue and other tissues rheumatoid arthritis and psoriasis Folkman etfl/ , J Biol Chem .267 10931 -10934 (1992).
  • angiogenesis appears to be crucial for the transition from hyperplasia to neoplasm, and for providing nourishment to the growing solid tumor Fo kman etal .
  • 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 ef a/ . Lancet. 340 1 120-1124 (1992), Macchia ⁇ m et al . Lancet.
  • VEGF vascular endothelial cell proliferation
  • Ferrara et al Endocr Rev . j_8 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
  • VEGF has been shown to be a key mediator of neovascula ⁇ zation associated with tumors and intraocular disorders Ferrara et al , Endocr Rev .
  • VEGF mRNA is overexpressed by the majority of human tumors examined Berkman et al , J Chn Invest . 91 153-1 9 (1993), Brown et al Human Pathol 26 86-91 (1995). Brown etal , Cancer Res .53 4727-4735 (1993). Mattern gfa/ . 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 , __ Engl J Med .
  • TGF- ⁇ transforming growth factor beta
  • IGFBPs insulin-like growth factor binding proteins
  • IGF insulin-like growth factor
  • the present i ention concerns compositions and methods for promoting oi inhibiting angiogenesis and/or cardiovascularization in mammals
  • the present invention is based on the identification of proteins that test positive in various cardiovascular assays that test promotion or inhibition of certain biological activities Accordingly, the proteins are believed to be useful drugs for the diagnosis and/or treatment (including prevention) of disorders where such effects are desired, such as the promotion or inhibition of angiogenesis, inhibition or stimulation of vascular endothelial cell growth, stimulation of growth or proliferation of vascular endothelial cells, inhibition of tumor growth, inhibition of angiogenesis-dependent tissue growth, stimulation of angiogenesis-dependent tissue growth, inhibition of cardiac hypertrophy and stimulation of cardiac hypertrophy, e g , for the treatment of congestive heart failure
  • 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 angiogemc 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 foi 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 comprising an anti-PRO antibody in admixture with a pharmaceutically acceptable carrier
  • the composition comprises a therapeutically effective amount ofthe 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 Pieserved liquid pharmaceutical formulations might contain multiple doses of the anti-PRO antibody, and might, theiefore, be suitable for repeated use
  • the antibody is a monoclonal antibody, an antibody fragment,
  • the present invention provides an article of manufacture comprising (a) a composition of matter comprising a PRO polypeptide or agonist or antagonist thereof,
  • composition may comprise a therapeutically effective amount of the PRO polypeptide or the 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 act ⁇ ⁇ t> 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
  • this method comprises the steps of
  • 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
  • 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
  • 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
  • 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 desc ⁇ bed above
  • the invention provides an isolated antibody that binds a PRO polypeptide
  • the antibody is a monoclonal antibody, which preferably has non-human complementa ⁇ ty-determining-region (CDR) residues and human framework-region (FR) residues
  • CDR non-human complementa ⁇ ty-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 oi absence of a PRO polypeptide in a test sample of tissue cells obtained from said mammal,
  • 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 angiogenic disorder is cardiac hypertrophy and said PRO polypeptide is administered together with a cardiovascular, endothelial or angiogenic agent
  • a cardiovascular, endothelial or angiogenic agent for this purpose is selected from the group consisting of an antihypertensive drug, an ACE inhibitor, an endothelm receptor antagonist and a thrombolytic agent If a thrombolytic agent is administered, preferably the PRO polypeptide is administeied following administration of such agent More preferably, the thrombolytic agent is recombinant human tissue plasminogen activator
  • the cardiovascular, endothelial or angiogenic disordei is cardiac hypertrophy and the PRO polypeptide is administered following primary angioplasty for the treatment of acute preferably wherein the mammal is further exposed to angioplasty or a cardiovascular, endothelial, or angiogenic agent
  • the cardiovascular, endothelial oi 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-i elated 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 (
  • 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
  • the mammal is human and the endothelial cell growth is associated with a tumor or a retinal disorder
  • the invention provides a method foi 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 antibod)
  • the mammal is human
  • 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
  • the invention provides a method for inhibiting endothelial cell growth in a mammal comprising administering to the mammal a PR0333, PR0364, PR0877 PR0879, PR0882 or PR0885 polypeptide or agonist thereof, wherein endothelial cell growth in said mammal is inhibited
  • the invention provides a method for stimulating endothelial cell growth in a mammal comprising administering to the mammal a PRO 179, PR0321 , PRO840, PR0844, PR0846, PR0878 oi PR0879 polypeptide or agonist thereof, wherein endothelial cell growth in said mammal is stimulated
  • the invention provides a method for inhibiting endothelial cell giowth in a mammal comprising administering to the mammal an antagonist of a PRO 179, PR0321 , PRO840, PR0844 PR0846 PR0878 or PR0879 polypeptide, wherein endothelial cell growth in said mammal is inhibited
  • the invention provides a method for stimulating endothelial cell growth in a mammal comprising administering to the mammal an antagonist of a PR0333, PR0364, PR0877 PR0879,
  • PR0882 or PR0885 polypeptide wherein endothelial cell growth in said mammal is stimulated
  • the invention provides a method for inducing cardiac hypertrophy in a mammal comprising administering to the mammal a PRO205, PR0882 or PR0887 polypeptide or agonist thereof, wherein cardiac hypertrophy in said mammal is induced
  • the invention provides a method for reducing cardiac hypertrophy in a mammal comprising administering to the mammal a PR0238, PR0878 or PRO 1760 polypeptide or agonist thereof, wherein cardiac hypertrophy in said mammal is reduced
  • the invention provides a method for inducing cardiac hypertrophy in a mammal comprising administering to the mammal an antagonist of a PR0238, PR0878 or PROl 760 polypeptide, wherein cardiac hypertrophy in said mammal is induced
  • the invention provides a method for reducing cardiac hypertrophy in a mammal comprising administering to the mammal an antagonist of a PRO205, PR0882 or PR0887 polypeptide, wherein cardiac hypertrophy in said mammal is reduced
  • the invention provides a method for inhibiting angiogenesis induced by a PRO 179, PR0321 , PRO840, PR0844, PR0846, PR0878 or PR0879 polypeptide comprising administering a therapeutically effective amount of an anti-PRO 179, ant ⁇ -PR0321 , ant ⁇ -PRO840, ant ⁇ -PR0844, ant ⁇ -PR0846, ant ⁇ -PR0878 or anti- PR0879 antibody to the mammal, wherein said angiogenesis is inhibited
  • the invention provides a method for stimulating angiogenesis induced by a
  • PROl 79, PR0321 , PRO840, PR0844, PR0846, PR0878 or PR0879 polypeptide comprising administering a therapeutically effective amount of said polypeptide to the mammal, wherein said angiogenesis is stimulated
  • 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% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternativeh at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternativel ⁇ at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternativeh at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96%
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively
  • Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain deleted or transmembrane domain-inacti vated, or is complementary to such encoding nucleotide sequence wherein the tiansmembrane doma ⁇ n(s) of such polypeptide are disclosed herein Therefore soluble extracellular domains of the herein described PRO polypeptides are contemplated
  • nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 1 10 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least
  • 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% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity alternativeh at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity alternatively at least about 91 % amino acid sequence identity, alternate eh at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about
  • the invention concerns an isolated PRO polypeptide comprising an am o acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% ammo acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% ammo acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% am o acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% am o
  • the invention concerns an isolated PRO polypeptide comprising an amino acid sequence scoring at least about 80% positives, alternatively at least about 81 % positives, alternatively at least about 82% positives, alternatively at least about 83% positives, alternatively at least about 84% positives, alternatively at least about 85% positives, alternatively at least about 86% positives alternatively at least about 87% positives alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 907r positives, alternatively at least about 91 % positives, alternatively at least about 92% positives, alternatively at least about 93% positives alternatively at least about 94% positives, alternatively at least about 95%- positiv es alternatively at least about 96% positives, alternatively at least about 97%- positives, alternatively at least about 98% positives and alternatively 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 amino acid
  • the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded bv a nucleotide sequence that encodes such an amino acid sequence as hereinbefoie described Processes for producing the same are also herein described, wherein those processes comprise cultu ⁇ ng a host cell comprising a vector w hich comprises the approp ⁇ ate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
  • 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 cultu ⁇ ng 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
  • 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 cultu ⁇ ng host cells under conditions suitable foi expression of the desired polypeptide and recovering the desired polypeptide from the cell culture
  • the invention provides chime ⁇ c molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence
  • chime ⁇ c 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 or below described polypeptides
  • the antibodv is a monoclonal antibody, humanized antibody antibody fragment or single-chain antibody
  • the invention provides o gonucleotide 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 1 ) of a native sequence PRO 179 cDN A wherein SEQ ID NO 1
  • ID NO 1 is a clone designated herein as "DNA16451 -1388"
  • 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 3) of a native sequence PR0238 cDNA, wherein SEQ ID NO 3 is a clone designated herein as "DNA35600-1 162"
  • Figuie 4 shows the amino acid sequence (SEQ ID NO 4) derived from the coding sequence of SEQ ID NO 3 shown in Figure 3
  • Figure 5 shows a nucleotide sequence (SEQ ID NO 5) of a native sequence PR0364 cDNA, wherein SEQ
  • ID NO 5 is a clone designated herein as "DNA47365-1206"
  • Figure 6 shows the amino acid sequence (SEQ ID NO 6) derived from the coding sequence of SEQ ID NO 5 shown in Figure 5
  • Figure 7 shows a nucleotide sequence (SEQ ID NO 7) of a native sequence PR0844 cDNA, wherein SEQ ID NO 7 is a clone designated herein as "DNA59838-1462"
  • Figure 8 shows the amino acid sequence (SEQ ID NO 8) derived from the coding sequence of SEQ ID NO 7 shown in Figure 7
  • Figure 9 shows a nucleotide sequence (SEQ ID NO 9) of a native sequence PR0846 cDNA, wherein SEQ ID NO 9 is a clone designated herein as "DNA44196- 1353"
  • Figure 10 shows the amino acid sequence (SEQ ID NO 10) derived from the coding sequence of SEQ ID NO
  • Figure 1 1 shows a nucleotide sequence (SEQ ID NO 1 1 ) of a native sequence PRO 1760 cDNA, wherein SEQ ID NO 11 is a clone designated herein as "DNA76532-1702"
  • Figure 12 shows the amino acid sequence (SEQ ID NO 12) derived from the coding sequence of SEQ ID NO 1 1 shown in Figure 1 1
  • Figure 13 shows a nucleotide sequence (SEQ ID NO 13) of a native sequence PRO205 cDNA, wherein SEQ ID NO 13 is a clone designated herein as "DNA30868"
  • Figure 14 shows the amino acid sequence (SEQ ID NO 14) derived from the coding sequence of SEQ ID NO 13 shown in Figure 13
  • Figure 15 shows a nucleotide sequence (SEQ ID NO 15) of a native sequence PR0321 cDNA, wherein SEQ
  • ID NO 15 is a clone designated herein as "DNA34433"
  • Figure 16 shows the ammo acid sequence (SEQ ID NO 16) derived from the coding sequence of SEQ ID NO 15 shown in Figure 15
  • Figure 17 shows a nucleotide sequence (SEQ ID NO 17) of a native sequence PR0333 cDNA, wherein SEQ ID NO 17 is a clone designated herein as "DNA41374'
  • Figure 18 shows the amino acid sequence (SEQ ID NO 18) derived from the coding sequence of SEQ ID NO 17 shown in Figure 17
  • Figure 19 shows a nucleotide sequence (SEQ ID NO 19) of a native sequence PRO840 cDNA, wherein SEQ ID NO 19 is a clone designated herein as "DNA53987”
  • Figure 20 shows the amino acid sequence (SEQ ID NO 20) derived from the coding sequence of SEQ ID NO 20
  • Figure 21 shows a nucleotide sequence (SEQ ID NO 21 ) of a nati ve sequence PR0877 cDN A, wherein SEQ ID NO 21 is a clone designated herein as ' DNA58120"
  • Figure 22 shows the amino acid sequence (SEQ ID NO 22) derived from the coding sequence of SEQ ID NO 21 shown in Figure 21
  • Figure 23 shows a nucleotide sequence (SEQ ID NO 23) of a native sequence PR0878 cDNA, wherein SEQ ID NO 23 is a clone designated herein as "DNA58121"
  • Figure 24 shows the amino acid sequence (SEQ ID NO 24) de ⁇ ved from the coding sequence of SEQ ID NO 24
  • Figure 25 shows a nucleotide sequence (SEQ ID NO 25) of a native sequence PR0879 cDNA, wherein SEQ ID NO 25 is a clone designated herein as "DNA58122"
  • Figure 26 shows the amino acid sequence (SEQ ID NO 26) derived from the coding sequence of SEQ ID NO 25 shown in Figure 25
  • Figure 27 shows a nucleotide sequence (SEQ ID NO 27) of a native sequence PR0882 cDNA, wherein SEQ ID NO 27 is a clone designated herein as "DNA58125"
  • Figure 28 shows the amino acid sequence (SEQ ID NO 28) derived from the coding sequence of SEQ ID NO 27 shown in Figure 27
  • Figure 29 shows a nucleotide sequence (SEQ ID NO 29) of a native sequence PR0885 cDNA, wherein SEQ
  • ID NO 29 is a clone designated herein as "DNA58128"
  • Figure 30 shows the amino acid sequence (SEQ ID NO 30) derived from the coding sequence of SEQ ID NO 29 shown in Figure 29
  • Figure 31 shows a nucleotide sequence (SEQ ID NO 31 ) of a native sequence PR0887 cDNA, wherein SEQ ID NO 31 is a clone designated herein as "DNA58130"
  • Figure 32 shows the amino acid sequence (SEQ ID NO 32) derived from the coding sequence of SEQ ID NO 31 shown in Figure 31
  • cardiothelial and angiogenic disordei cardiovascular, endothelial and angiogenic dysfunction' cardiovascular, endothelial or angiogenic disoider' and cardiovascular, endothelial oi angiogenic dysfunction are used interchangeably and refer in part to systemic disorders that affect vessels, such as diabetes melhtus, as well as diseases of the vessels themselves such as of the arteries, capillaries, veins, and/oi lymphatics This would include indications that stimulate angiogenesis and/oi cardiov ascula ⁇ zation, and those that inhibit angiogenesis and or cardiovascularization
  • disorders include for example, arterial disease, such as atherosclerosis, hypertension inflammatory vascu tides, Reynaud s disease and Reynaud s phenomenon, aneurysms and arterial restenosis, venous and lymphatic disorders such as thrombophlebitis hmphangitis, and lymphedema and other vascular disorders such as peripheral vascular
  • hemangioma capillary and cavernous
  • glomus tumors telangiectasia
  • bacillaiy angiomatosis hemangioendothehoma
  • 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
  • Hypertrophy 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
  • cardiac hypertrophy is defined as an increase in mass ofthe 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 appears to play a critical role in determining the nature of the response
  • the early stage of cardiac hypertrophy is usually characterized morphologically by increases in the size of myofib ⁇ ls 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
  • Heart failure refers to an abnormality of cardiac function wheie the heart does not pump blood at the rate needed for the requirements of metabolizing tissues
  • the heart failure can be caused by a numbei of factors, including lschemic, congenital, rheumatic, or ldiopathic forms
  • CHF Consgestive heart failure
  • CHF congestive heart failure
  • cardiac output the volume of blood pumped by the heart over time
  • CHF structural and hemodynamic damages occur While these damages have a variety of manifestations, one characteristic symptom is v ent ⁇ culai hypertrophy
  • CHF is a common end result of a number of va ⁇ ous cardiac disorders
  • Myocardial infarction generally results from atherosclerosis of the coronary arteries, often with superimposed coronary thrombosis It may be divided into two major types transmural infarcts, in which myocardial necrosis involves the full thickness ofthe ventrii
  • 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 diastohc performance Fouad e/ ⁇ / J Am Coll Cardiol , 4 1 00- 1506 (1984). Smith et al .
  • hypotrophic cardiomyopathy Another complex cardiac disease associated with cardiac hypertrophy is "hypertrophic cardiomyopathy" This condition is characterized by a great diversity of morphologic, functional, and clinical features (Maron et al , N Engl J Med , 316 780-789 (1987). Sp ⁇ to era/ , N Engl J Med .320 749-755 ( 1989), Louie and Edwards, Pro g . Cardiovasc Pis .36 275 308 (1994). Wigle etal .
  • the causative factors of hypertrophic cardiomyopathy are also diverse and little understood
  • mutations in genes encoding sarcome ⁇ c proteins are associated with hypertrophic cardiomyopathy
  • ⁇ -myosin heavy chain mutations may account for approximately 30 to 40 percent of cases of familial hypertrophic cardiomyopathy Watkins etal . N Engl j Med .326 1108-1 114 (1992). Schwartz etal. Circulation 91 532-540
  • Supravalvular "aortic stenosis” is an inherited vascular disordei 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 lntracardiac pressure resulting in myocardial hypertrophy and eventually heart failure and death The pathogenesis of this disorder is not fully understood, but hypertrophy and possibly hyperplasia of medial smooth muscle are prominent featuies of this disorder It has been reported that molecular variants of the elastin gene are involved in the development and pathogenesis of aortic stenosis.
  • Valvular regurgitation occurs as a result of heart diseases resulting in disorders of the cardiac valves
  • Va ⁇ ous 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 ⁇ ovent ⁇ cular orifices and operation of the heart
  • Defects such as the na ⁇ owing of the valve stenosis or the defectiv e closing of the valve result in an accumulation of blood in the heart cavity or regurgitation of blood past the valve If unco ⁇ ected, prolonged valvular stenosis or insufficiency may result in cardiac hypertrophy and associated damage to the heart muscle, which may eventually necessitate valve replacement
  • 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' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and
  • cytotoxic agent 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 , 1 I I, 125 1, 9U Y, and 186 Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer
  • examples of chemotherapeutic agents include alkylating agents, fohc acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics, py ⁇ midine analogs, 5-fluorourac ⁇ l, cisplatin, purine nucleosides, amines, amino acids, t ⁇ azol nucleosides, or corticosteroids
  • Specific examples include Ad ⁇ amycin, Doxorubicin, 5-Fluorourac ⁇ l, Cytosme arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate,
  • Cisplatin Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C Mitoxantrone, V creistine, Vinorelbme, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopte ⁇ n Dactinomycin, Mitomyc s, 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 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 vitio 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 a ⁇ est Classical M-phase blockers include the vincas (vinc ⁇ stine and vinblastine), taxol and topo
  • tumor necrosis factor an antibody capable of inhibiting or neutiahzmg the angiogenic activity of acidic or basic FGF or hepatocyte growth factor (HGF), an antibody capable of inhibiting or neutiahzmg the coagulant activities of tissue factor, protein C, or protein S (see WO 91/01753, published 21 Februai) 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 ofthe agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial effect, such as an anti-hypertrophic effect, for an extended period of time
  • mammal forpurposes 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
  • specific examples of these include ang ⁇ otens ⁇ n-II receptor antagonists, endothelm receptor antagonists such as, for example.
  • BOSENTANTM and MOXONODINTM interferon-gamma (IFN- ⁇ ), des-aspartate-angiotensin I, thrombolytic agents, e g , streptokinase, urokinase, t-PA, and a t-PA variant specifically designed to have longei half-life and very high fibrin specificity, TNK t-PA (a Tl 03N, Nl 17Q, KHRR(296-299)AAAA t-PA variant. Keyt et al , Proc Nati Acad Sci USA.
  • 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 , quinap ⁇ l, captop ⁇ l, enalap ⁇ l, ramip ⁇ l, benazep ⁇ l, fosinop ⁇ l, and lisinop ⁇ l, diuretics, e g chlorothiazide, hydrochlorothiazide hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide acetazolamide, and indap
  • ACE angiotensin
  • PDGF epidermal growth factor
  • CTGF corthelial growth factor
  • FGF epidermal growth factor
  • TGF- ⁇ and TGF- ⁇ "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
  • cytotherapeutic agents such as cytotoxic agents, chemotherapeutic agents, growth-inhibitory agents, apoptotic agents, and other agents to treat cancer, such as anti- HER-2, ant ⁇ -CD20, and other bioactive and organic chemical agents
  • a "therapeutically effective amount" of an active agent such as a PRO polypeptide or agonist or antagonist thereto or an anti-PRO antibody refers to an amount effective in the treatment of a cardiovascular, endothelial or angiogenic disorder in a mammal and can be determined empirically
  • an "effective amount" of an active agent such as a PRO polypeptide or agonist or antagonist thereto or an anti-PRO antibody refers to an amount effective for carrying out a stated purpose, wherein such amounts may be determined empirically for the desired effect
  • PRO polypeptide and "PRO” as used herein and when immediately followed by a numerical designation refer to va ⁇ ous 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 co ⁇ esponding PRO polypeptide derived from nature Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means
  • native sequence PRO polypeptide specifically encompasses naturally-occurring truncated or secrete
  • 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 methion e residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionme residues located either upstream or downstream from the am o acid position 1 in the figures mav be employed as the starting amino acid residue for the PRO polypeptides
  • the PRO polypeptide ' extracellular domain refers to a form of the PRO polypeptide which is essentially free ofthe transmembrane and cytoplasmic domains
  • ECD ECD will hav e less than 1 % of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0 5% of such domains
  • 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 v ary 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 nucle
  • PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more am o acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence
  • a PRO polypeptide variant will have at least about 80% ammo acid sequence identity alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% ammo acid sequence identity,
  • ely at least about 86% amino acid sequence identity alternatively at least about 87% amino acid sequence identity alternatively at least about
  • 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
  • sequences can be m upper- or lower-case an may contain ambiguity
  • 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 lmax, /* lengths of stripped file names */ static ⁇ J[2], /* jmp index for a path */ static nc[2], /* number at start of current line */ static m[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 starfP LINE], /* set by stars() *//
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ] + + , ps[ ⁇ ] + + ,
  • *py + + *px; else if ( ⁇ slower(*px))
  • *py++ toupper(*px); if ( ⁇ ndex("ATGCU",*(py-l))) natgc + + ; ⁇ ⁇
  • Percent (%) amino acid sequence identity with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent -sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are withm 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
  • 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 of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison ammo acid sequence of interest (t 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,
  • nucleotides in length alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more
  • Percent (%) nucleic acid sequence identity with respect to the PRO polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST 2, ALIGN, ALIGN 2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measu ⁇ ng 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 ⁇ dent ⁇ t ⁇ values are obtained as described below bv using the sequence comparison computer program ALIGN 2, wherein the complete source code for the ALIGN 2 program is
  • % 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))
  • % 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 (/ e , the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynu
  • PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) , Figure 10 (SEQ ID NO 10), Figure 12 (SEQ ID NO 12), F ⁇ gu ⁇ e l4 (SEQ ID NO 14), Figure 16 (SEQ ID NO 16), Figure 18 (SEQ ID NO 18), Figure 20 (SEQ ID NO 20), Figure 22 (SEQ ID NO 22), Figure 24 (SEQ ID NO 24), Figure 26 (SEQ ID NO 26), Figure 28 (SEQ ID NO 28), Figure 30 (SEQ ID NO 30), and Figure 32 (SEQ ID NO 32), respectively
  • PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide
  • 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 prefe ⁇ ed substitution (as defined in Table 3 below) of the amino acid residue of interest
  • the % value of positives of a given ammo 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
  • Isolated when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones and other proteinaceous or non-protemaceous solutes
  • the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal am o 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 ofthe PRO natural environment will not be present Ordinarily, howe ⁇ er isolated polypeptide will be prepared by at
  • 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 PRO polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence
  • a ⁇ bosome 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
  • enhancers do not have to be contiguous Linking is accomplished by hgation at convenient restriction sites If such sites do not exist, the synthetic ohgonucleotide adaptors or linkers are used in accordance with conventional practice
  • 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 chlo ⁇ de/0 0015 M sodium c ⁇ trate/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 album ⁇ n/0 1 % F ⁇ coll/0 1 % polyv ⁇ nylpyrrohdone/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, s
  • Modely-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 mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50 °C
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like
  • epitope-tagged when used herein refers to a chime ⁇ c polypeptide comprising a PRO polypeptide fused to a "tag polypeptide"
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity 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 PRO variants refers to form(s) of PRO proteins that retain the biologic and/or lmmunologic activities of a native or naturally-occurring PRO 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 melhtus 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 mitogemc 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 mitoge c, angiogenic, oi other biological activity of PRO polypeptides and thus are useful for the treatment of diseases or disorders characterized by undesirable excessive neovascula ⁇ zation, including by way of example tumors and especially solid malignant tumors, rheumato
  • 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 Abs
  • immunoglobulins Igs are glycoproteins having the same structural characteristics
  • 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 heterotetrame ⁇ c 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 intracham disulfide bridges Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains Each light chain has a variable domain at one end (V L ) and a constant domain at its other end, the constant domain of the light chain is aligned with the first constant domain ot 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
  • 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 othei chain, contribute to the formation of the antigen-bindmg site of antibodies See, Kabat et al , NIH Publ No 91 3242, Vol I, pages 647-669 ( 1991 )
  • Antibody fragments' comprise a portion of an intact antibody preferably the antigen-binding or v ariable region of the intact antibody
  • antibody fragments include Fab, Fab', F(ab')-,, 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 (CHI) of the heavy chain Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CHI domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine res ⁇ due(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 ot immunoglobulins are well known
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population ot substantially homogeneous antibodies, i 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 ot 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
  • 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 co ⁇ esponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to co ⁇ esponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies 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.
  • 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.
  • 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 ofthe 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.
  • Fc immunoglobulin constant region
  • the humanized antibody includes a PRIMATIZEDTM antibody wherein the antigen-binding region ofthe antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
  • Single-chain Fv or “sFv” antibody fragments comprise the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv 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 Hollinger et al, Proc. Nati. Acad. Sci. USA. 90: 6444-6448 (1993).
  • an "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes
  • 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 other composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody
  • the label may be detectable by itself (e g , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable Radionuchdes that can serve as detectable labels include, for example, 1-131, 1-123, 1-125, Y-90, Re-188, At-211 , 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 sihcones
  • the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275,149
  • a "liposome” is a small vesicle composed of various types of hpids, phosphohpids and/or surfactant that is useful for delivery of a drug (such as the PRO polypeptide or antibodies thereto disclosed herein) to a mammal
  • a drug such as the PRO polypeptide or antibodies thereto disclosed 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 amino acid sequence comprising at least the binding site of a receptor or a hgand
  • 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
  • Tyr (Y) trp, phe, thr, ser phe Val (V) lie, leu, met, phe, ala, norleucine leu
  • Substantial modifications in function or immunological identity ofthe PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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
  • 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 consen ative 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 alamne scanning, and PCR mutagenesis Site-directed mutagenesis [Carter et al Nucl Acids Res 13 4331 (1986), Zoller etal , Nucl Acids Res , 10 6487 ( 1987)], cassette mutagenesis [Wells et al , Gene, 34 31 (1985)] restriction selection mutagenesis [Wells et al Philos Trans R Soc London SerA, 317 41 ( 1986)1 or other known techniques can be performed on the cloned DNA to produce the PROl 79, PR0238, PR0364 PR0844, PR0846, PROl 760, PRO205, PR0321 PR0333 PRO840, PR0877, PR0878, PR0879 PR0882 PR0885 or PR0887 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 ammo acids
  • ammo acids include alanme, glycine, serine, and cysteine
  • Alanine is typically a prefe ⁇ ed 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 lsote ⁇ c amino acid can be used
  • PRQ844, PRQ846. PRO1760.
  • PRQ333 PRO840. PRQ877. PRQ878. PRQ879. PRQ882. PRQ885 and PRQ887
  • Covalent modifications of PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 and PR0887 are included within the scope of this invention
  • One type of covalent modification includes reacting targeted amino acid residues of a PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide with an organic de ⁇ vatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 De ⁇ vat
  • Another type of covalent modification of the PROl 79, PR0238, PR0364 PR0844 PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR088 oi PR0887 polypeptide included withm the scope of this invention comprises altering the native glycosylation pattern ot the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PROl 79, PR0238, PR0364, PR0844. PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840.
  • PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 (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 PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887.
  • 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may be accomplished by altering the amino acid sequence.
  • the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 (for O-linked glycosylation sites).
  • PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205. PR0321. PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PRO 179, PR0238, PR0364. PR0844. PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al, Arch. Biochem. Biophys.. 259:52 ( 1987) and by Edge et al, Anal.
  • 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. Enzvmol.. 138:350 ( 1987).
  • PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 comprises linking the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333.
  • PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 ofthe present invention may also be modified in a way to form a chimeric molecule comprising PR0179, PR0238, PR0364, PR0844, PR0846. PRO1760, PRO205, PR0321. PR0333, PRO840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887 fused to another, heterologous polypeptide or amino acid sequence.
  • such a chimeric molecule comprises a fusion of the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887
  • Tag polypeptides include the Flag peptide [Hopp et al , BioTechnology. 6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science, 255 192-194 (1992)], an ⁇ -tubuhn epitope peptide [Skinner et al , J Biol Chem .
  • the chimeric molecule may comprise a fusion of the PROl 79, PR0238,
  • the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge CHI
  • PRQ333 PRO840 PRQ877 PRQ878 PRQ879. PRQ882. PRQ885 and PRQ887
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0179, PR0238, PR0364, PR0844 PR0846, PRO1760 PRO205, PRO321 , PRO333, PRO840, PRO877 PR0878, PR0879, PR0882 PR0885 or PR0887
  • cDNAs encoding PROl 79, PR0238, PR0 64 PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879 PR0882, PR0885 or PR0887 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below It is noted that proteins pioduced in separate expression rounds may be given different PRO numbei s but the UNQ number is unique tor any given DNA and the encoded protein and will not be changed.
  • PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptides by culturing cells transformed or transfected with a vector containing nucleic acid encoding PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptides.
  • PROl 79 PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879. PR0882, PR0885 or PR0887.
  • the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321, PR0333, PRO840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide sequence, or portions thereof may be produced by direct peptide synthesis using solid-phase techniques. See, e.g.
  • PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333.
  • PRQ885 or PRQ887 i. Isolation of DNA Encoding PROl 79. PRQ238. PRQ364. PRQ844. PRQ846. PROl 760. PRQ205. PRQ321. PRQ333. PRO840. PRQ877. PRQ878. PRQ879. PRQ882. PRQ885 or PRQ887
  • PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA encoding PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887 and to express it at a detectable level. Accordingly, DNAs encoding human PROl 79, human PR0238.
  • human PR0364, human PR0844, human PR0846, human PRO 1760, human PRO205, human PR0321. human PR0333. human PRO840, human PR0877, human PR0878, human PR0879, human PR0882, human PR0885 or human PR0887 can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in the Examples.
  • PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879. PR0882, PR0885 or PR0887 polypeptide may also be obtained from a genomic library or by ohgonucleotide synthesis
  • Probes such as antibodies to the PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 etal , supra
  • An alternative means to isolate the gene encoding PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 is to use PCR methodology Sambrook et al , supra, Dieffenbach et al , PCR Primer A Laboratory Manual (New York Cold Spring Harbor Laboratory Press
  • 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 tor the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse transcribed into cDNA
  • Host cells are transfected or transformed with expression or cloning vectors desc ⁇ bed herein tor PROl 79, PR0238 PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877 PR0878, PR0879 PR0882, PR0885 or PR0887 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 w ithout 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 Butlei , ed (IRL Press, 1991 ) and Sambrook et al , supia
  • transfection is performed using standaid techniques appropriate to such cells
  • the calcium treatment employing calcium chloride, as described in Sambrook et al , supta, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers
  • Infection with A ⁇ t obacterium tumefaciens is used for transformation of certain plant cells, as desc ⁇ bed by Shaw et al, Gene, 23: 315 (1983) and WO 89/05859 published 29 June 1989.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram- positive organisms, for example, Enterobacteriaceae such as E. coli.
  • eubacteria such as Gram-negative or Gram- positive organisms
  • Enterobacteriaceae such as E. coli.
  • E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325); and K5 772 (ATCC 53,635).
  • Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter.
  • Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations.
  • 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 kari ; E.
  • co// W31 10 strain 37D6 which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kari; E. coli W31 10 strain 40B4. which is strain 37D6 with a non- kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990.
  • in vitro methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887.
  • Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature.290: 140 [ 1981 ]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No.
  • K. lactis MW98-8C, CBS683, CBS4574; Louvencourtef ⁇ - .J. Bacteriol.J37 [1983]
  • K. fragilis ATCC 12.424
  • K. bulgaricus ATCC 16,045)
  • K. wickeramii ATCC 24,178
  • K. waltii ATCC 56,500
  • K. drosopl larum ATCC 36,906; Van den Berg et al, Bio/Technology. 8: 135 (1990)
  • K . thermotolerans K.
  • Schwanniomyces such as Schwaniuoi ces occidentals (EP 394,538 published 31 October 1990), and filamentous fungi such as, e g Neurospora, Penic ⁇ lium, Tolypocladium (WO 91/00357 published 10 January 1991 ), and Aspergillus hosts such as A mdulans (Ballance et al , Biochem Biophys Res Commun , 1 12 284-289 [1983]. Tilburn et al . Gene. 26 205-221 ri983].
  • 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, anteopsi , 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 .
  • the nucleic acid (e g , cDNA or genomic DNA) encoding PRO 179, PR0238 PR0364, PR0844, PR0846 PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 may be inserted into a rephcable vector for cloning (amplification ot the DNA) or for expression
  • a rephcable vector for cloning (amplification ot the DNA) or for expression
  • the vector may tor example, be in the form ot a plasmid cosmid.
  • nucleic acid sequence may be inserted into the vector by a va ⁇ ety of procedures
  • DNA is inserted into an appropriate restriction endonuclease s ⁇ te(s) using techniques known in the art
  • Vector components generally include, but are not limited to, one or more ot a signal sequence it the sequence is to be secreted, an origin of replication, one or more marker genes, an enhancei element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more ot these components employs standard hgation techniques that are known to the skilled artisan
  • PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760 PRO205 PR0321 , PR0333 PRO840 PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 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 piotein or polypeptide
  • the signal sequence may be a component ofthe vector, oi it may be a part of the DNA encoding PRO 179, PR0238 PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840 PR0877.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicilhnase lpp or heat-stable enterotoxin II leaders
  • yeast secretion the signal sequence may be, e g , the yeast invertase leader, alpha factor leadei (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 , ampicilhn, neomycm, methotrexate, or tetracychne, (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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887, 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 Nati 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 e/ ⁇ / , Gene, 7 141 (1979).
  • the trp ⁇ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No 44076 or PEP4-1 Jones, Genetics.
  • Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840 PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 to direct mRNA synthesis
  • Promoters recognized by a variety of potential host cells are well known Promoters suitable tor 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 (trp) promoter system (Goeddel, Nucleic Acids Res .
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S D ) sequence operably linked to the DNA encoding PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333 PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887
  • S D Shine-Dalgarno
  • suitable promoting sequences for use with yeast hosts include the promoters tor 3- phosphoglycerate kinase (Hitzeman et al , J Biol Chem .
  • glycolytic enzymes Hess et al , J Adv Enz y me Re .7 149 ( 1968), Holland, Biochemistry, V_ 4900 (1978)
  • enolase glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase 3-phosphoglycerate mutase, pyruvate kinase, t ⁇ osephosphate 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
  • nucleic acid transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, and Simian Virus 40 (SV40), by heterologous mammalian promoters, e g , the actin promoter or an immunoglobulin promoter, and by heat-shock promoters, provided such promoters are compatible with the host cell systems
  • viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a
  • 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)
  • 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
  • Gene amplification and/or expression may be measured in a sample directly, for example by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc Nati Acad Sci USA, 77 5201 -5205 (1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-prote 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
  • PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321.
  • a suitable detergent solution e g , TRITON-XTM 100
  • PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell-lys g agents It may be desired to purify the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205 PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide from recombinant cell proteins oi polypeptides
  • the following procedures are exemplary of suitable purification procedures by tractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, foi example, Sephadex G-
  • PRQ885 or PRQ887 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 endothelm antagonist activity include a rat heart ventricle binding assay where the polypeptide is tested for its ability to inhibit lodimzed endothel ⁇ n-1 binding in a receptor assay, an endothelm receptor binding assay testing for intact cell binding of radiolabeled endothelm- 1 using rabbit renal artery vascular smooth muscle cells, an inositol phosphate accumulation assay where functional activity is determined in Rat-1 cells by measuring intra-cellular levels of second messengers, an arachidonic acid release assay that measures the ability of added compounds to reduce endothehn-stimulated arachidonic acid release in cultured vascular smooth muscles, in vitro (isolated vessel) studies using endothehum 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 endothehum)
  • Assays for wound-healing activity include, for example, those described in Winter, Epidermal Wound Healing Maibach, HI and Rovee, DT, eds (Year Book Medical Publishers, Inc , Chicago), pp 71-1 12, as modified by the article of Eaglstein and Mertz, J Invest Dermatol , l ⁇ 382-384 (1978)
  • An assay to screen for a test molecule relating to a PRO polypeptide that binds an endothelm B t (ETB,) receptor polypeptide and modulates signal transduction activity involves providing a host cell transformed with a DNA encoding endothelm B, receptor polypeptide, exposing the cells to the test candidate, and measuring endothelm B, receptoi signal transduction activity, as described, e g , in U S Pat No 5,773,223
  • cardiac hypertrophy 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 bv Piper et al "Adult ventricular rat heart muscle cells" in Cell Culture Techniques in Heart and Vessel Research, H M Piper ed (Berlin Sp ⁇ nger-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, ⁇ (PGF ⁇ J have been shown to induce a spreading response in these adult cells
  • PGF 1 ⁇ or PGF 2c analogs (e g fluprostenol) and phenyleph ⁇ ne by various potential inhibitors ot cardiac hypertrophy is then tested
  • an in vno assay is a test for inhibiting cardiac hypertiophy 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 ⁇ )
  • rats with pathologic cardiac hypertrophy induced by myocardial infarction have chronically elevated levels of extractable PGF, ⁇ in their myocardium Lai et al , Am J Physiol (Heart Circ Physiol ), 271 H2197 H2208 (1996)
  • factors that can inhibit the effects of tlupiostenol 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 eight of heart, ventricles, and left ventricle (normalized by body weight) relative to
  • 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 g , subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e g colon cancer cells implanted in colonic tissue See, e g , PCT publication No WO 97/335
  • the cells introduced into such animals can be derived from known tumor/cancei cell lines, such as any of the above listed tumor cell lines, and, for example, the B 104-1 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene), / ⁇ 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 Karmah et al , Br J Cancer. 48 689-696 (1983)
  • Tumor cells can be introduced into animals such as nude mice by a variety of procedures
  • the subcutaneous (s c ) space in mice is very suitable for tumor implantation
  • Tumors can be transplanted s c as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions
  • 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 rae ⁇ -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 etal , Cancer Research. 54 4726-4728 (1994) and Too et al , Cancer Research. 55 681-684 (1995) This model is based on the so-called “METAMOUSETM” sold by AntiCancer, Inc , (San Diego, California)
  • Tumors that arise in animals can be removed and cultured in vitw 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 aie 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 Palladino et al , J Immunol .
  • 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 10x 10'' to 10xl 0 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
  • the Lewis lung (3LL) carcinoma of mice which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model Efficacy this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small-cell carcinoma of the lung (SCCL)
  • SCCL small-cell carcinoma of the lung
  • 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 tumoi cells survive For further information about this tumor model see, Zacharski, Haemostasis.
  • recombinant (transgemc) animal models can be engineered by introducing the coding portion of the PRO gene identified herein into the genome of animals of interest, using standard techniques for producing transgemc animals
  • Animals that can serve as a target for transgemc 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 micro jection (U S Patent No 4,873,191), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al , Proc Nati Acad Sci USA.
  • transge c 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 etal , Proc Nati Acad Sci USA, 89 6232-636 (1992)
  • the expression ofthe transgene in transgemc 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 PRO polypeptide identified here
  • 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 PRO 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
  • gene amplification and/or gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription ot mRNA (Thomas, Proc Nati Acad Sci USA.77 5201 5205 ( 1980)), dot blotting (DNA analysis) or in situ hybi idization, using an appropriately labeled probe, based on the sequences provided herein
  • Alternativeh 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 lmmunohistochemical staining of 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 PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO DNA and encoding a specific antibody epitope
  • General techniques for generating antibodies, and special protocols for in situ hybridization are provided hereinbelow
  • m Antibody Binding Studies The results of the cardiovascular, endothelial, and angiogenic study can be further verified by antibody binding studies, in which the ability of anti-PRO antibodies to inhibit the effect ofthe PRO polypeptides on endothelial cells or other cells used in the cardiovascular, endothelial, and angiogenic assays is tested Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and lmmunoprecipitation 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-immunoglobuhn 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 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 , tumoi cells can be tested by using cells or cells lines that have been identified as being stimulated or inhibited by the PRO polypeptide herein
  • Such cells include, for example, those set forth in the Examples below
  • suitable tumor cells include, for example, stable tumor cells lines such as the B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and /as-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 B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and /as-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
  • the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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
  • nucleic acid is injected directly into the patient, usually at the sites where the PR0179, PR0238, PR0364, PR0844, PR0846.
  • PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide is required i e , the site of synthesis of the PRO 179, PR0238, PR0364, PR0844, PR0846.
  • PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide if known, and the site (e g , wound) where biological activity of PR0179, PR0238, PR0364, PR0844 PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840 PR0877, PR0878, PR0879, PR0882 PR0885 orPR0887 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 within porous membranes that are implanted into the patient (see, e g , U S Pat Nos 4,892.538 and 5,283,187)
  • 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 prefe ⁇ ed in vivo nucleic acid transfer techniques include transtection with viral or non-viral vectors (such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) and hpid-based systems (useful hpids for hpid-mediated transfer ot the gene are, for example, DOTMA, DOPE, and DC-Choi, see, e g , Tonk son 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
  • This invention is also related to the use of the gene encoding the PRO 179 PR0238 PR0364, PR0844, PR0846 PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877 PR0878 PR0879, PR0882, PR0885 or PR0887 polypeptide as a diagnostic Detection of a mutated form of the PRO 179 PR0238 PR0364, PR0844 PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 polypeptide will allow a diagnosis of a cardiovascular, endothelial and angiogenic disease or a susceptibility to a cardiovascular, endothelial, and angiogenic disease, such as a tumoi , since mutations in the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 PR0333 PRO840,
  • Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S I protection or the chemical cleavage method, for example, Cotton et al , Proc Nati 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 ot restriction enzymes, e g restriction fragment length polymorphisms (RFLP) and Southern blotting of genomic DNA
  • methods such as hybridization, RNase protection, chemical cleavage direct DNA sequencing, or the use ot restriction enzymes, e g restriction fragment length polymorphisms (RFLP) and Southern blotting of genomic DNA
  • vn 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 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 neovascularization associated with tumor formation, since an altered level of this PRO polypeptide may be indicative of such disorders.
  • a competition assay may be employed wherein antibodies specific to the PRO polypeptide are attached to a solid support and the labeled PRO polypeptide and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of the PRO polypeptide in the sample.
  • 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.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • 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 co ⁇ esponding 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.
  • 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.
  • FISH requires use of the clones from which the gene encoding the PROl 79, PR0238, PR0364, PR0844, PR0846. PRO 1760, PRO205, PR0321 , PR0333, PRO840. PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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).
  • 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 PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide (agonists) or prevent the effect of the PROl 79, PR0238, PR0364 PR0844, PR0846, PRO 1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide (antagonists)
  • Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide encoded by the genes
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art All assays for antagonists are common in that they call for contacting the drug candidate with a PRO 179,
  • the interaction is binding and the complex formed can be isolated oi detected in the reaction mixture
  • PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO 1760 PRO205.
  • an immobilized antibody e g , a monoclonal antibody, specific for the PRO 179 PR0238 PR0364, PR0844 PR0846, PRO1760, PRO205, PR0321 , PR0333 PRO840, PR0877, PR0878.
  • PR0879 PR0882, PR0885 or PR0887 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 suiface 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 PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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
  • 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 etal , Proc Nati Acad Sci USA.
  • 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
  • yeast expression system described in the foregoing publications (generally refe ⁇ ed 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-/ ⁇ cZ reporter gene under control of a GAL4-act ⁇ vated 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
  • PR0879, PR0882, PR0885 or PR0887 polypeptide identified herein and other mtra- 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 react ⁇ on(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 ot 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 H-thymidine and harvested onto glass fiber filters (phD, Cambridge Technology, Watertown, MA) Mean ' H- thymidine incorporation (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA) Significant 3 (H) thymidine incorporation indicates stimulation of endothelial cell proliferation
  • 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)thym ⁇ d ⁇ ne incorporation 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 Cohgan et al Current Protocols in Immun , 1 (2) Chapter 5 ( 1991 )
  • expression cloning is employed wherein polyadenylated RNA is prepared from a cell
  • the labeled PRO polypeptide can be photoaffinity- hnked 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 ohgonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor
  • 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, t ⁇ ple-hehx molecules, etc , that inhibit the expression and/or activity of the target gene product
  • potential antagonists include an ohgonucleotide 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 ldiotypic 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 t ⁇ ple-hehx 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 ohgonucleotide of from about 10 to 40 base pairs in length
  • a DNA ohgonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see, Lee etal , Nucl Acids Res .
  • RNA ohgonucleotide 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, oligodeoxy ⁇ bonucleotides 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 activ ity of the PRO polypeptide
  • small molecules include, but are not limited to, small peptides or peptide-hke 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 turthei 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 t ⁇ ple-hehx formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides
  • the base composition of these oligonucleotides is designed such that it promotes t ⁇ ple-hehx formation via Hoogsteen base-pairing rules, which generally require sizeable stretches ot purines or py ⁇ midines on one strand of a duplex
  • base-pairing rules which generally require sizeable stretches ot purines or py ⁇ midines on one strand of a duplex
  • the PRO polypeptides, or agonists or antagonists thereto, that have activity in the cardiovascular, angiogenic, and endothelial assays described herein, and/or whose gene product has been found to be localized to the cardiovascular system, are likely to have therapeutic uses in a variety of cardiovascular, endothelial, and angiogenic disorders including systemic disorders that affect vessels, such as diabetes melhtus
  • Their therapeutic utility could include diseases of the arteries, capillaries, veins, and/or lymphatics
  • 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
  • the increase in angiogenesis mediated by the PRO polypeptide or antagonist would be beneficial to lschemic 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
  • vascular tumors such as haemangioma, tumor angiogenesis, neovascularization in the retina choroid or cornea associated with diabetic retinopathy or premature infant ret opathy or macular degeneration and proliferative vitreoretinopathy, rheumatoid arthritis Crohn s disease, atherosclerosis, ovarian hyperstimulation, psoriasis endomet ⁇ osis associated with neovascularization, restenosis subsequent to balloon angioplasty, scar tissue overproduction, for example, that seen in a keloid that forms after surgery
  • angiogenesis is desired such as peripheral vascular disease, hypertension, inflammatory vascuhtides, 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 mtimal 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 vascuhtides include giant cell arte ⁇ tis, Takayasu's arte ⁇ tis, polyarte ⁇ tis nodosa (including the microangiopathic form), Kawasaki's disease, microscopic polyangiitis, Wegener's granulomatosis, and a va ⁇ ety 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
  • thromboophlebitis 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 ot the lymphatic system that are congenital, often cystic, malformations of the lymphatics that usually occui in newborns Cystic tumors tend to grow into the adjacent tissue Cystic tumors usually occur in the cervical and axillary legion 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 G ⁇ ener et al , Lymphology. 4 140-144 (1971 )
  • tumor angiogenesis involves vascula ⁇ zation 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, a ⁇ henoblastomas, cervical carcinomas, endomet ⁇ al carcinoma, endomet ⁇ al hyperplasia, endomet ⁇ osis, fibrosarcomas, cho ⁇ ocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastom
  • AMD Age-related macular degeneration
  • 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 no ⁇ o 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 oi regeneration of other tissues, such as organs (including, for example, pancreas, liver intestine, kidney, skin, oi endothehum), muscle (smooth, skeletal or cardiac), and vascular (including vascular endothehum) tissue or foi promoting the growth of cells comprising such tissues
  • organs including, for example, pancreas, liver intestine, kidney, skin, oi endothehum
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothehum
  • 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 precursoi tissues oi cells, or for inhibiting the growth of tissues described above
  • a PRO polypeptide or antagonist thereto may also be used in the treatment of pe ⁇ odontal 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/hgament-hke 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/hgament-hke 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 othei 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
  • the PRO polypeptide or its antagonist may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, ; e , for the treatment of central and peripheral nervous system disease and neuropathies as well as mechanical and traumatic disorders, that involve degeneration, death, 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 injuiies, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndiome
  • diseases of the peripheral nervous system such as peripheral nerve injuiies, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndiome
  • Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as
  • Endothelial cell dysfunction may be important in both the initiation of, and in regulation of the sequelae of events that occur following ischemia-repertusion injury
  • Rheumatoid arthritis is a further indication Blood vessel growth and targeting ot inflammatory cells through the vasculature is an important component in the pathogenesis of rheumatoid and seio-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 ot 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 at ⁇ al fibrillation, which develops in a substantial portion of patients diagnosed with hypertrophic cardiomyopathy
  • Non-neoplastic conditions include psoriasis, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, pe ⁇ cardial 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 earners, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate citrate, and other organic acids, antioxidants including ascorbic acid and methionme, preservatives (such as octadecy ldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol
  • 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 protamme sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts colloidal silica, magnesium t ⁇ sihcate, poly vinyl pyrrohdone, cellulose-based substances, and polyethylene glvcol
  • Carriers for topical or gel-based forms of antagonist include polysaccha ⁇ des such as sodium caiboxymethylcellulose 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
  • Another formulation comprises incorporating 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 systemtcally 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
  • the detergent employed is non-ionic, it may, for example, be polysorbates (e g , POLYSORBATETM
  • 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 lsotomcity of a liquid composition of the PRO polypeptide or antagonist thereto, and includes polyhyd ⁇ c sugar alcohols, preferably t ⁇ hyd ⁇ c or higher sugar alcohols, such as glycerin, eryth ⁇ tol, arabitol, xyhtol sorbitol, and manmtol These sugar alcohols can be used alone or in combination Alternatively, sodium chloride or other appropriate inorganic salts may be used to render the sol utions lsotonic
  • the buffer may, for example be an acetate, citrate succinate, or phosphate butter depending on the pH desired
  • the pH of one type of liquid formulation of this invention is buffeted in the range of about 4 to 8, preferably about physiological pH
  • the preservatives phenol, benzvl 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 (1 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 .
  • 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
  • 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 C, resulting in a loss of biological activity and possible changes in lmmunogemcity 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, lyophihzing 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 pei se DE 3,218, 121 , Epstein et al Proc Nati Acad Sci USA.
  • the liposomes are ot the small (about 200-800 Angstroms) unilamellar type in which the hpid 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, vaiy depending on such factors as the pathological condition to be treated (including pievention).
  • the method ot administration the type of compound being used for treatment, any co-therapy involved the patient s age, weight, general medical condition, medical history, etc , and its determination is well with the skill of a practicing physician Accordingly, it will be necessary for the therapist to titer the dosage and modify the route ot administration as required to obtain the maximal therapeutic effect
  • the PRO polypeptide has a na ⁇ ow host range, tor 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,
  • the effective dose generally is withm 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, pyrogemcity, 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 in 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, histomorphometric determinations, and tetracychne labeling
  • the route of PRO polypeptide or antagonist or agonist administration is in accord with known methods e g , by injection or infusion by intravenous, intramuscular, intracerebral, intraperitoneal, intracerobrospinal, subcutaneous, intraocular, intraarticular, intrasynovial, mtrathecal, 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, intralesional, or pe ⁇ lesional routes, to exert local as well as systemic therapeutic effects
  • the intraperitoneal 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
  • compositions herein that are useful for bone, cartilage, tendon, or ligament regeneration include administering the composition topically systemically, or locally as an implant or dev ice 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 ot bone, cartilage or tissue
  • sequestering agents include cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydoxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, one preferred being catiomc salts of carboxymethylcellulose (CMC)
  • CMC carboxymethylcellulose
  • Other prefe ⁇ ed sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and poly(v ⁇ nyl alcohol)
  • the amount of sequestering agent useful herein is 0 5-20 wt%, preferably 1 -10 wt%, based on total formulation weight, which represents the amount necessary to prevent desorption of the polypeptide (or its antagonist) from the polymer mat ⁇ x and to provide appropriate handling ofthe composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix,
  • PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or an agonist or antagonist thereof in preventing or treating the disorder in question may be improved by administering the active agent serially or in combination with another agent that is effective for those purposes, either in the same composition or as separate compositions For example, for treatment of cardiac hypertrophy .
  • PRO polypeptide therapy can be combined with the administration of inhibitors of known cardiac myocyte hypertrophy factors, e g , inhibitors of ⁇ -adrenergic agonists such as phenyleph ⁇ ne, endothelm- 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
  • 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, captop ⁇ l, enalap ⁇ l, ramip ⁇ l, benazep ⁇ l, fosmop ⁇ l, or lisinop ⁇ l, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, or mdapamide, and/or calcium channel
  • Preferred candidates for combination therapy m the treatment of hypertrophic cardiomyopathy are ⁇ - adrenergic-blocking drugs (e g , propranolol, timolol, tertalolol, carteolol, nadolol betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol), verapamil, difedipine, or diltiazem
  • Treatment of hypertrophy associated with high blood pressure may require the use of antihypertensive drug therapy, using calcium channel blockers, e g , diltiazem, nifedipine, verapamil, or nicardipine, ⁇ -adrenergic blocking agents, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide,
  • PRO polypeptides or their antagonists used to treat cancer mav be combined with cytotoxic, chemotherapeutic, or growth-inhibitory agents as identified above Al so, 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 hypei or hypotension, renal impairment, etc
  • the dose will additionally depend on such factors as the type ot 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 PRO 179, PR0238, PR0364 PR0844, PR0846, PRO 1760,
  • PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882 PR0885 or PR0887 polypeptide or agonists 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 v ials 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 PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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.
  • the article of manufacture may also comprise a second or third container with another active agent as described above.
  • 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.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or a fusion protein thereof.
  • immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants 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.
  • Monoclonal Antibodies The anti-PROl 79, anti-PR0238, anti-PR0364, anti-PR0844. anti-PR0846. anti-PRO 1760, anti-PRO205. anti-PR0321, anti-PR0333. anti-PRO840, anti-PR0877, anti-PR0878. anti-PR0879, anti-PR0882, anti-PR0885 or anti-PR0887 antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 ( 1975).
  • 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.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the PROl 79, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or a fusion protein thereof
  • 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 Goding, Monoclonal Antibodies Principles and Practice (New York Academic Press, 1986), pp 59-103
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin Usually, rat or mouse myeloma cell lines are employed The hybridoma cells
  • Prefe ⁇ ed 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, tor instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies Kozbor, J Immunol , 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications (Marcel Dekker. Inc New York, 1987) pp 51-63
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 clones may be subcloned by limiting dilution procedures and grown by standard methods Goding, supra Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures 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 ohgonucleotide 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 prefe ⁇ ed 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 4,81
  • antibodies may further comprise humanized antibodies or human antibodies
  • Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobulins immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab 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
  • 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' v a ⁇ able 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 .
  • humanized antibodies are chimeric antibodies (U S Patent No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the co ⁇ esponding 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 etal . Monoclonal Antibodies and Cancer Therapy. Alan R L ⁇ ss, p 77 (1985) and Boerner etal , J Immunol .
  • human antibodies can be made by introducing human immunoglobulin loci into transgemc 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 rea ⁇ angement, 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 ⁇ n thefollow ⁇ ng sc ⁇ ent ⁇ f ⁇ c publ ⁇ cat ⁇ ons Marks etal . Bio/Technology.
  • 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 PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide
  • the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit
  • Methods for making bispecific antibodies are known in the art Traditionally the recombinant production of bispecific antibodies is based on the co expression of two immunoglobulin heavy-chain/hght-cham pairs, wheie the two heavy chains have different specificities Milstein and Cuello.
  • Heteroconjugate antibodies are composed ot 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
  • the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosshnking agents
  • immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond
  • suitable reagents for this purpose include lminothiolate and methyl-4- mercaptobuty ⁇ midate 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 , 126 1 191 - 1 195 ( 1992) and Shopes, J Immunol , M8 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 Drug
  • 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 activ e fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeiuginosa), ricin A chain, ab ⁇ n A chain, modeccin A chain, alpha-sarcm, Aleut ites fordu proteins, dianthin proteins, Plntolaca ameucana proteins (PAPI PAPII, and PAP-S), momordica charantia inhibitor, curcm, crotin sapaona ⁇ a officinahs inhibitoi, gelo n mitogelhn, rest ⁇ ctocin, phenomycin, enomycin, and the t ⁇ cothecenes A v a ⁇ ety of radionuchdes are available for the production of radioconjugated antibodies Examples include - 1 Bi "'I ⁇
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succ ⁇ mm ⁇ dyl-3-(2-py ⁇ dyld ⁇ th ⁇ ol) propionate (SPDP) lminothiolane (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), dusocyanates (such as tolyene 2,6 diisocyanate), and bis- active fluorine compounds (such as 1.5 d ⁇ fluoro-2,4-d ⁇ n ⁇ trobenzene)
  • the antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e g , avidin) that is conjugated to a cytotoxic agent (e g , a radionucleotide)
  • a "receptor” such as streptavidin
  • a ligand e g , avidin
  • cytotoxic agent e g , a radionucleotide
  • the antibodies disclosed herein may also be formulated as immunohposomes
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein etal , Proc Nati Acad Sci USA. 82 3688 (1985), Hwang et al , Proc Nati Acad Sci USA. 77 4030 (1980), and U S Pat Nos 4,485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5,013,556
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a hpid composition comprising phosphatidylcholine, cholesterol, and PEG-de ⁇ vatized phosphatidylethanolamme (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 .
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome See, Gabizon et al , J National Cancer Inst . 81 (19) 1484 (1989)
  • PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbetore, can be administered for the treatment of various disorders as noted above and below in the form of pharmaceutical compositions If the PROl 79, PR0238, PR0364, PR0844. PR0846 PROl 760, PRO205, PR0321 , PR0333 PRO840,
  • PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred
  • hpofections 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/oi produced by recombinant DNA technology See, e g , Marasco et al , Proc Nati Acad Sci USA 90 7889-7893 ( 1993)
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each othei Alternatively , or in addition, the composition may comprise an agent that enhances its function, such as. for example a cytotoxic agent, cytokme, chemotherapeutic agent, or growth-inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interf acial 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
  • 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 include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g , films, or microcapsules
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2 hydroxyethyl-methacrylate), or poly(v ⁇ nylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and ⁇ ethyl-L glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid glycohc acid copolymer and leuprohde acetate), and poly-D-(-)-3-hydroxybuty ⁇ c acid While polymers such as ethylene-vinyl a
  • the antibodies to a PR0179, PR0238, PR0364, PR0844 PR0846, PROl 760 PRO205, PR0321 , PR0333, PRO840, PR0877 PR0878, PR0879, PR0882, PR0885 or PR0887 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, lntracerobrosp al, subcutaneous, intra-articular, intrasynovial, mtrathecal oral topical, or inhalation routes Intravenous administration of the antibody is preferred
  • chemotherapeutic agents may be administered to the patient Preparation and dosing schedules tor such chemotherapeutic agents may be used according to manufacturers instructions or as determined empirically by the skilled practitionei 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 onap ⁇ stone (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, 01 VEGF receptor(s) These also include the agents set forth above Also, the antibody is suitably administered serially or combination with radiological treatments, whether involving l ⁇ adiation 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 coadministered 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 (sy
  • anti-hereguhn antibody D-factor, interleukin- 1 (IL-1), ⁇ nterleuk ⁇ n-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
  • IL-1 interleukin- 1
  • IL-2 ⁇ nterleuk ⁇ n-2
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • 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
  • auxiliary agents will vary in their effectiveness, it is desirable to compare their impact on the tumor by matrix screening in conventional fashion.
  • 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)
  • 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
  • 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 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 purposes previous therapy, the patient s clinical history and response to the antibody, and the discretion ot 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 foi administration to the patient whethei 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 anti-PROl 79, ant ⁇ -PR0238, ant ⁇ -PR0364, ant ⁇ -PR0844, anti- PR0846, ant ⁇ -PRO1760, ant ⁇ -PRO205, ant ⁇ -PR0321 , ant ⁇ -PR0333, ant ⁇ -PRO840, ant ⁇ -PR0877, ant ⁇ -PR0878, ant ⁇ -PR0879, ant ⁇ -PR0882, ant ⁇ -PR0885 or ant ⁇ -PR0887 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 g , 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 immunofluorescence 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 present invention uses standard procedures of recombinant DNA technology, such as those described heremabove 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)Jnn ⁇ s / ⁇ / , PCR Protocols A Guide to Methods and Applications (Academic Press, Inc N Y , 1990), Harlow et al , Antibodies A Laboratory Manual (Cold Spring Harbor Press Cold Spring Harbor, 1988). Gait. Ohgonucleotide Synthesis (IRL Press Oxford, 1984), Freshnev. Animal Cell Culture, 1987, Cohgan etal , Current Protocols in Immunology, 1991
  • EXAMPLE 1 Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNA Encoding Therefor
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases
  • the EST databases included public databases (e g , GenBank), and proprietary databases (e g , LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA)
  • the search was performed using the computer program BLAST or BLAST-2 [Altschul etal , Methods in Enzymology.
  • oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length
  • the probe sequences are typically 40-55 bp in length
  • 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
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site see, Holmes et al , Science 253 1278 1280 (1991 )) in the unique Xhol and Notl sites
  • 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
  • a secondary cDNA library was generated in order to preferentially represent the 5' ends of the primary cDNA clones
  • Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY 0 using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above)
  • the double stranded cDNA was sized to 500- 1000 bp, linkered with blunt to Notl adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector pSST- AMY 0 is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites
  • cDNAs cloned into this vector that are
  • DNA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml) The bacteria and vector mixture was then electroporated as recommended by the manufacturer Subsequently. SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37 °C for 30 minutes The transformants were then plated onto 20 standard 150 mm LB plates containing ampicilhn and incubated for 16 hours (37°C) Positive colonies were scraped oft the plates and the DNA was isolated from the bacterial pellet using standard protocols, e g , CsCl-gradient The purified DNA was then earned on to the yeast protocols below
  • the yeast methods were divided into three categories (1 ) Transformation of yeast with the plasmid/cDNA combined vector, (2) Detection and isolation of yeast clones secreting amylase, and (3) PCR amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis
  • yeast strain used was HD56-5A (ATCC-90785) This strain has the following genotype MAT alpha, ura3-52 leu2-3, leu2-l 12, h ⁇ s3-l 1 , h ⁇ s3-15, MAL + , SUC + , GAL +
  • yeast mutants can be employed that have deficient post-translational pathways Such mutants may have translocation deficient alleles in vec71 , vet72, sec62.
  • antagonists including antisense nucleotides and/oi ligands which interfere with the normal operation of these genes, other proteins implicated in this post translation pathw ay (e g , SEC ⁇ lp, SEC72p, SEC62p, SEC63p, TDJlp or SSA lp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressmg yeast Transformation was performed based on the protocol outlined by Gietz et al , Nucl Acid Res , 20 1425 (1992) Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and grown overnight at 30 °C The YEPD broth was prepared as described in Kaiser et al , Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p.
  • the cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM T ⁇ s-HCl, 1 mM EDTA pH 7 5, 100 mM L ⁇ 2 OOCCH 3 ), and resuspended into LiAc/TE (2 5 ml)
  • Transformation took place by mixing the prepared cells ( 100 ⁇ 1) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 ⁇ g, vol ⁇ 10 ⁇ l) in microfuge tubes The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 ⁇ l, 40% polyethylene glycol-4000, 10 mM T ⁇ s-HCl, 1 mM EDTA, 100 mM L ⁇ 2 OOCCH 3 , pH 7 5) was added This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes The cells were then heat shocked at 42°C for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 ⁇ l, 10 mM T ⁇ s-HCl, 1 mM EDTA pH 7 5) followed by recent ⁇ fugation The cells were then diluted into TE (1 ml) and a
  • the selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al , Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p 208- 210 (1994) Transformants were grown at 30 °C for 2-3 days The detection of colonies secreting amylase was performed by including red starch in the selective growth media Starch was coupled to the red dye (Reactive Red- 120, Sigma) as per the procedure described by Biely et al , Anal Biochem .
  • SCD-Ura synthetic complete dextrose agar lacking uracil
  • the coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0 15% (w/v), and was buffered with potassium phosphate to a pH of 7 0 (50-100 mM final concentration)
  • the positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies
  • Well isolated single colonies positive for amylase secretion were detected by direct incorporation ot red starch into buffered SCD-Ura agar Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly
  • PCR was then performed as follows a Denature 92°C, 5 minutes b 3 cycles of Denature 92°C, 30 seconds
  • the underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY 0 when no insert was present
  • the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers
  • the signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides su ⁇ ounding the first and optionally the second methionme codon(s)
  • ATG at the 5 -end of the sequence or sequence fragment under consideration
  • the nucleotides following the first ATG must code for at least 35 unambiguous ammo acids without any stop codons It the first ATG has the required amino acids, the second is not examined If neither meets the requirement the candidate sequence is not scored
  • the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation paiameters) known to be associated with secretion signals Use of this algorithm resulted in the identification of numerous polypeptide-encoding nucleic acid sequences
  • ohgonucleotide probes were then generated from the sequence of the DNA10028 molecule and used to screen a human fetal liver (LIB6) library prepared as described in paragraph 1 of Example 2 above
  • the cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the
  • PRO 179 polypeptide shows significant similarity to the angiopoietin family of proteins, thereby indicating that PRO 179 may be a novel angiopoietin family member More specifically, an analysis ofthe Dayhoff database (version 35 45 SwissPiot 35) evidenced significant homology between the PRO 179 amino acid sequence and the following Dayhoff sequences
  • DNA30908 Based on the DNA30908 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 PR0238 PCR primers (forward and reverse) were synthesized based upon the DNA30908 sequence Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA30908 sequence In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supia, with the PCR primer pair A positive library was then used to isolate clones
  • the ohgonucleotide sequences used in the above procedure were the following forward PCR primer 1
  • RNA for construction of the cDNA libraries was isolated from human fetal liver tissue
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with 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 ot 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 tor PR0238 [herein designated as DNA35600-1 162] ( Figure 3, SEQ ID NO 3) and the de ⁇ ved protein sequence for PR0238
  • 1 162 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 134-
  • the predicted polypeptide precursor is 310 amino acids long ( Figure 4, SEQ ID NO 4), and has an estimated molecular weight ot about 33.524 daltons and a pi of about 9 55
  • PR0238 may be a novel reductase
  • Ohgonucleotide probes based upon the DNA44825 and " ⁇ consen01>" consensus sequences were then synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) tor use as probes to isolate a clone ofthe full-length coding sequence for PR0364
  • Forward and rev erse PCR primers generally range from 20-30 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
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Cu ⁇ ent 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 ohgonucleotide and one of the primer pairs
  • hybridization probes were constructed from the consensus DNA44825 sequence which had the following nucleotide sequences hybridization probe (44825 pi ) 5'-GAGGAGTGCTGTTCCGAGTGGGACTGCATGTGTGTCCAGC-3 * (SEQ ID NO 45) hybridization probe (44825 GITR p) 5'-AGCCTGGGTCAGCGCCCCACCGGGGGTCCCGGGTGCGGCC-3' (SEQ ID NO 46)
  • DNA from the libraries was screened by PCR amplification with the PCR primer pairs identified above A positive library was then used to isolate clones encoding the PR0364 gene using the probe oligonucleotides and one of the PCR primers
  • RNA for construction of the cDNA libraries was isolated from human bone ma ⁇ ow tissue
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed with oligo dT containing a Notl site, 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
  • PR0364 amino acid sequence indicates that portions of it possess homology to members of the tumor necrosis factor receptor family, thereby indicating that PR0364 may be a novel member of the tumor necrosis factor receptor family
  • the intracellular domain of PR0364 contains a motif (in the region of amino acids 207-214) similar to the minimal domain within CD30 receptor shown to be required for TRAF2 binding and which is also present within TNFR2
  • cysteine- ⁇ ch domains characteristic of the TNFR family (see, Naismith and Sprang, Trends Biochem Sci , 23 74-79 (1998)), of which the third CRD has 3 rather than the more typical 4 or 6 cysteines of the TNFR family
  • the PR0364 amino acid sequence has 8 cysteines in the CRD1 relative to 5 cysteines in CRD1 of mouse GITR, and the presence of one potential N-hnked glycosylation site in the ECD as compared to 4 potential
  • PR0364 represents the human counterpart or ortholog to the mouse GITR protein reported by Nocentini et al
  • PR0844 may be a novel proteinase inhibitor More specifically, an analysis of the Dayhoff database (version 35 45 SwissProt 35) evidenced significant homology between the PR0844 amino acid sequence and at least the following Dayhoff sequences ALK1_HUMAN, P_P82403, P_P82402, ELAF_HUMAN and PJP60950
  • Example 1 above This consensus sequence is herein designated DNA39949 and " ⁇ consenl 322> Based on the DNA39949 consensus sequence and the " ⁇ consenl322>" sequnece, 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 PR0846 PCR primers (forward and reverse) were synthesized based upon the DNA39949 and " ⁇ consen 1322>" consensus sequences Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA30908 sequence
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Cureent Protocols in Molecular Biology, supia, with the PCR primer pair A positive library was then used to isolate clones encoding the PR0846 gene using the probe ohgonucleotide and one of the PCR primers
  • the ohgonucleotide sequences used in the above procedure were the following forward PCR primer (39949 f 1 )
  • a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA39949 sequence which had the following nucleotide sequence hybridization probe (39949 pi )
  • RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB227)
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • the cDNA was primed ith Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoiesis 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 etal . Science, 253 1278-1280 ( 1991 )) in the unique Xhol and Notl sites
  • Clone DNA76532-1702 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 60-62 and ending at the stop codon at nucleotide positions 624-626 ( Figure 1 1 )
  • the predicted polypeptide precursor is 188 ammo acids long ( Figure 12, SEQ ID NO 12)
  • the full-length PRO1760 protein shown Figure 12 has an estimated molecular weight of about 21 ,042 daltons and a pi of about 5 36
  • Analysis of the full-length PRO 1760 sequence shown in Figui e 12 evidences the presence of a variety of important polypeptide domains as shown in Figure 12, wherein the locations given for those important polypeptide domains are approximate as described above
  • Analysis ofthe full-length PRO 1760 sequence evidences the presence of the follo ing features a signal peptide from about amino acid 1 to about amino acid 20, N-glycosylation sites from about ammo acid 121 to about amino acid 125 and from about amino acid 171 to about amino acid 175, a
  • This assay is designed to measure the ability of PRO polypeptides to stimulate hypertrophy of neonatal heart PRO polypeptides testing positive in this assay are expected to be useful for the therapeutic treatment of various cardiac insufficiency disorders
  • Cardiac myocytes from 1-day old Harlan Sprague Dawley rats were obtained Cells (180 ⁇ l at 7 5 x lOVml, serum ⁇ 0 1 %, freshly isolated) are added on day 1 to 96-well plates previously coated with DMEM/F12 + 4% FCS Test samples containing the test PRO polypeptide or growth medium only (negative control) (20 ⁇ l/well) are added directly to the wells on day 1 PGF (20 ⁇ l/well) is then added on day 2 at a final concentration of 10 6 M The cells are then stained on day 4 and visually scored on day 5, wherein cells showing no increase in size (as compared to negative controls) are scored 0 0, cells showing a small to moderate increase in size (a
  • VEGF Vascular Endothelial Growth Factor
  • Assay 9 The ability of various PRO polypeptides to inhibit VEGF stimulated proliferation of endothelial cells was tested. Polypeptides testing positive in this assay are useful for inhibiting endothelial cell growth in mammals where such an effect would be beneficial, e.g., for inhibiting tumor growth.
  • ACE bovine adrenal cortical capillary endothelial cells
  • DMEM 10% calf serum, 2 mM glutamine, and IX penicillin/streptomycin/fungizone.
  • Control wells included the following: (1 ) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGF plus
  • test samples 5 ng/ml LIF.
  • poly-his tagged PRO polypeptides in 100 microliter volumes
  • the cell cultures were incubated for 6-7 days at
  • the activity of PRO polypeptides was calculated as the percent inhibition of VEGF (3 ng/ml) stimulated proliferation (as determined by measuring acid phosphatase activity at OD 405 nm) relative to the cells without stimulation.
  • TGF-beta was employed as an activity reference at 1 ng/ml, since TGF-beta blocks 70-90% of VEGF- stimulated ACE cell proliferation.
  • the results, as shown in TABLE 5 below, are indicative of the utility ofthe PRO polypeptides in cancer therapy and specifically in inhibiting tumor angiogenesis.
  • the numerical values (relative inhibition) shown in TABLE 5 are determined by calculating the percent inhibition of VEGF stimulated proliferation by the PRO polypeptides relative to cells without stimulation and then dividing that percentage into the percent inhibition obtained by TGF- ⁇ at 1 ng/ml which is known to block 70-90% of VEGF stimulated cell proliferation. The results are considered positive if the PRO ploypeptide exhibits 30% or greater inhibition of
  • VEGF stimulation of endothelial cell growth (relative inhibition 30% or greater).
  • Assay 34 Induction of c-fos in Endothelial Cells (Assay 34) This assay is designed to determine whether PRO polypeptides show the ability to induce c-fos in endothelial cells PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of conditions or disorders where angiogenesis would be beneficial including, for example wound healing, and the like (as would agonists of these PRO polypeptides) Antagonists of the PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of cancerous tumors
  • Human venous umbilical vein endothelial cells (HUVEC Cell Systems) in growth media (50% Ham s F12 w/o GHT low glucose, and 50% DMEM without glycine with NaHC03, 1 % glutamme, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF) were plated on 96-well microtiter plates at a cell density of 1 x 10 cells/well The day after plating, 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 1 O mM HEPES J 40 mM NaCl 4% (w/v) mannitol pH 6 8) The cells were incubated for 30 minutes at 37 °C in 5%- CO, The samples were removed and the first part of the bDNA kit protocol (Chiron Diagnostics, cat #6005 037) was followed where each capitalized reagent/buffer listed below was available from the kit Briefly, the amounts
  • 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.
  • 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.
  • PRO polypeptides testing positive in this assay are expected to be useful for the therapeutic treatment of various cardiac insufficiency disorders
  • DMEM/F12 plus 4% FCS 200 ⁇ l/well
  • Assay media included DMEM/F12 (with 2 44 gm bicarbonate), 10 ⁇ g/ml transfer ⁇ n, 1 ⁇ g/ml insulin, 1 ⁇ g/ml aprotinin, 2 mmol/L glutamine, 100 U/ml penicillin G, 100 ⁇ g/ml streptomycin Protein buffer containing mannitol (4%) gave a positive signal (score 3 5) at 1/10 (0 4%) and 1/100 (0 04%), but not at 1/1000 (0 004%) Therefore the test sample buffer containing mannitol is not run
  • PR0878 polypeptide provided a score of less than 0 in the above assay
  • EXAMPLE 15 Induction of Endothelial Cell Apoptosis (Assay 73)
  • the ability of PRO polypeptides to induce apoptosis in endothelial cells was tested in human v enous umbilical vein endothelial cells (HUVEC, Cell Systems)
  • a positiv e test in the assay is indicative of the usefulness of the polypeptide in therapeutically treating tumors as well as vascular disordei s where inducing apoptosis of endothelial cells would be beneficial
  • PRO polypeptides to induce apoptosis in endothelial cells was tested in human venous umbilical vein endothelial cells (HUVEC, Cell Systems), using a 96-w ell format in 0% serum media supplemented with 100 ng/ml VEGF (As HUVEC cells are easily dislodged from the plating surface, all pipetting in the wells must be done as gently as practicable )
  • the medium was aspirated and the cells washed once with PBS 5 ml of 1 x trypsin was added to the cells in a T-175 flask and the cells were allowed to stand until thev were released from the plate (about 5-10 minutes)
  • Trypsimzation was stopped by adding 5 ml of growth media The cells were spun at 1000 rpm for 5 minutes at 4 °C The media was aspirated and the cells were resuspended in 10 ml of 10% serum complemented medium (Cell
  • test PRO polypeptide samples were added in triplicate at dilutions of 1 %, 0 33% and 0 1 1 % Wells without cells were used as a blank and wells with cells only were used as a negative control As a positive control 1 3 serial dilutions of 50 ⁇ l of a 3x stock of staurospo ⁇ ne were used.
  • Annexm-V a member of the calcium and phosphohpid binding proteins, to detect apoptosis
  • Annexin V - Biotin stock solution 100 ⁇ g/ml were diluted in 4 6 ml 2 x Ca 2+ binding buffer and 2 5% BSA (1 25 dilution) 50 ⁇ ls of the diluted Annexin V - Biotm solution were added to each well (except controls) to a final concentration of 1 0 ⁇ g/ml
  • the samples were incubated for 10-15 minutes with Annexin-Biotin prior to direct addition of 35 S-Streptav ⁇ d ⁇ n 35 S-Streptav ⁇ d ⁇ n was diluted in 2x Ca 2+ Binding buffer, 2 5% BSA and was added to all wells at a final concentration of 3 x 10 4 cpm/well
  • the plates were then sealed, centrifuged at 1000 rpm for 15 minutes and placed on orbital shaker for 2 hours The analysis was performed on 1450 M ⁇ crobeta T ⁇ lux (Wallac) The results are shown in TABLE 7 below where percent above background represents the percentage amount of counts per minute
  • This assay is designed to determine whether PRO polypeptides of the present invention show the ability to inhibit neonatal heart hypertrophy induced by LIF and endothel ⁇ n-1 (ET-1 )
  • a test compound that provides a positiv e response in the present assay would be useful for the therapeutic treatment of cardiac insufficiency diseases or disorders characterized or associated with an undesired hypertrophy of the cardiac muscle
  • Cardiac myocytes from 1 -day old Harlan Sprague Dawley rats ( 180 ⁇ l at 7 5 ⁇ 10 4 /ml serum ⁇ 0 1 freshly isolated) are introduced on day 1 to 96-well plates previously coated with DMEM/F12 + 4%FCS Test PRO polypeptide samples or growth medium alone (negative control) are then added directly to the wells on day 2 in 20 ⁇ l volume LIF + ET-1 are then added to the wells on day 3 The cells are stained attei an additional 2 days in culture and are then scored visually the next day A positive in the assay occurs when the PRO polypeptide treated myocytes are visually smaller on the average or less numerous than the untreated myocytes PR0238 and PRO 1760 polypeptides tested positive in this assay
  • EXAMPLE 17 Stimulation of Endothelial Tube Formation - Sprout formation This assay is designed to determine whether PRO polypeptides show the ability to promote endothelial vacuole and lumen formation in the absence of exogenous growth factors
  • PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of disorders where endothelial vacuole and/or lumen formation would be beneficial including, for example, where the stimulation of pinocytosis, ion pumping, vascular permeability and/or junctional formation would be beneficial
  • HUVEC cells passage ⁇ 8 from primary
  • type I rat tail collagen final concentration 2 6 mg/ml
  • EXAMPLE 18 Induction of Endothelial Cell Apoptosis (ELISA) (Assay 109)
  • PRO polypeptides to induce apoptosis in endothelial cells was tested in human venous umbilical vein endothelial cells (HUVEC, Cell Systems) using a 96-well format, in 0% serum media supplemented with 100 ng/ml VEGF, 0 1 % BSA, lX penn/strep A positive result in this assay indicates the usefulness of the polypeptide for therapeutically treating any of a variety of conditions associated with undesired endothelial cell growth including, for example, the inhibition of tumor growth
  • the 96-well plates used were manufactured by Falcon (No 3072) Coating of 96 well plates were prepared by allowing gelati zation to occur foi >30 minutes with 100 ⁇ l of 0 2% gelatin in PBS solution The gelatin mix was aspirated thoroughly before plating HUVEC cells at a final concentration of 2 x 10 4 cells/ml in 10% serum containing medium - 100 ⁇ l volume per well The cells were grown for 24 hours before adding test samples
  • 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
  • the tubes were incubated at 37°C for one hour A total of I 0 ⁇ l RQ1 DNase was added, followed by incubation at 37 °C for 15 minutes A total of 90 ⁇ l TE (10 mM Tris pH 7 6/1 mM EDTA pH 8 0) was added and the mixture was pipetted onto DE81 paper The remaining solution was loaded in a MICROCON 50 ultrafiltration unit, and spun using program 10 (6 minutes) The filtration unit was inverted over a second tube and spun using program 2 (3 minutes) After the final recovery spin, a total of 100 ⁇ 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 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 dehydrated in 70%, 95%, and 100% ethanol, 2 minutes each
  • the slides were deparaffinized, placed in SQ H 2 0, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time
  • the sections were deproteinated in 20 ⁇ g/ml proteinase K (500 ⁇ l of 10 mg/ml in 250 ml
  • RNase-free RNase buffer 37 °C, 15 minutes
  • 8 x proteinase K 100 ⁇ l in 250 ml Rnase buffer 37 °C, 30 minutes

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Abstract

Compositions and methods are disclosed for stimulting 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 cardiovascularization 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 116 percent.
CHF is a syndrome characterized by left ventricular dysfunction, reduced exercise tolerance, impaired quality of life, and markedly shortened life expectancy. The sine qua non of heart failure is an inability 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 f ibrosis (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 etal, FASEB J.. 5: 3037-3046 (1991 ); Chien etal., 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. 12: 208-217 (1991); Inoue et al, Proc. Natl. Acad. Sci. USA. 86: 2863-2867 (1989); Yanagisawa and Masaki, Trends Pharm. Sci.. K): 374-378 (1989); U.S. Patent No. 5,573,762 (issued November 12, 1996). Further exemplary factors that have been identified as potential mediators 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, endothelm (including endothehn-1 , 2, and -3 and big endothelm), 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 et al , Br Heart J . 44 488-98 ( 1980), Harrison et al , Circulation, 29 84-98 (1964) Verapamil has been descπbed 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 etal . Circulation.65 499-507 (1982), Betocchi efα/ . 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 Wigleefα/ . 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 qumapπl, captopπl, enalapπl, ramipπl, benazepπl, fosinopπl, and lisinopπl, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, 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 ventπculai muscle mass, but the Doppler indices of diasto c 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 etal ,
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 pressuie o\erload cardiac hypertrophy, in experimental rats
Agents that increase cardiac contractility directly (lontropic agents) weie 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, JJ2 209-217 ( 1997) Reddy et al , Curr Opin Cardiol . 12 233-241 (1997) Beta-adrenergic receptor blockers have recenth 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 sun lval 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 cardιotropιn-1 or antagonists thereof, or growth hormone and/or insulin-like giowth factor I in the treatment of CHF Another treatment modality is heart transplantation, but this is limited by the a\ aιlabιht\ of donor hearts Endothelm is a vasoconstπcting peptide comprising 21 amino acids, isolated from swine arterial endothelial culture supernatant and structurally determined Yanagisawa et al , Nature, 332 41 1 -415 (1988) Endothelm was later found to exhibit various actions, and endothelm antibodies as endothelm antagonists have proven effective in the treatment of myocardial infarction, renal failure, and other diseases Since endothelm is present in live bodies and exhibits vasoconstπcting 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 Endothelm 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 endothelm B receptor for identifying endothelm receptor antagonists is described in U S Pat No 5,773,223 Current therapy for heart failure is primarily directed to using angiotensin-con verting enzyme (ACE) inhibitors, such as captopπl, and diuretics These drugs improve hemodynamic profile and exercise tolerance and reduce the incidence of morbidity and mortality in patients with CHF Kramer et al , Circulation, 67(4) 807-816 (1983), Captopπl Multicenter Research Group, J A C C . 2(4) 755-763 (1983), The CONSENSUS Trial Study Group. N Engl J Med . 316(23) 1429-1435 (1987). The SOLVD Investigators. N Engl J Med .325(5) 293-302 (1991 ) Further, they are useful in treating hypertension, left ventricular dysfunction, atherosclerotic vascular disease, and diabetic nephropathy Brown and Vaughan, supi a However, despite proven efficacy, response to ACE inhibitors has been limited For example, while prolonging survival in the setting of heart failure, ACE inhibitors appear to slow the progression towards end-stage heart failure, and substantial numbers of patients on ACE inhibitors have functional class III heart failure Moreover, improvement of functional capacity and exercise time is only small and mortality, although reduced, continues to be high The CONSENSUS Trial Study Group, N Engl J Med .316(23) 1429-1453 ( 1987), The SOLVD Investigators, N Engl J Med . 325(5) 293-302 (1991 ), Cohn et al , N Engl J Med , 325(5) 303-310 ( 1991 ), The Captopπl-Digoxin Multicenter Research Group, JAMA.259(4) 539-544 (1988) Hence, ACE inhibitors consistently appear unable to relieve symptoms in more than 60% of heart failure patients and reduce mortality of heart failure only by approximately 15-20% For further adverse effects, see Brown and Vaughan, supra
An alternative to ACE inhibitors is represented by specific ATI receptor antagonists Clinical studies are planned to compare the efficacy of these two modalities in the treatment of cardiovascular and renal disease However, animal model data suggests that the ACE/Ang II pathway, while clearly ol ved 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 1 A, 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 hypertroph) About 750,000 patients suffer from acute myocardial infaiction (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 a I . Am J Cardiol . 61 723-728 (1988), Neuhaus ef o/ J Am Coll Cardiol . 12 581 -587 (1988), Neuhaus et al , J Am Coll Cardiol , J4 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 Tl 03N, Nl 17Q, KHRR(296-299)AAAA t-PA variant, Keyt et al , Proc Natl Acad Sci USA.91 3670 3674 ( 1994)) is particularly suitable for bolus administration However, despite all these advances, the long-term prognosis of patient survival depends greatly on the post-infarction monitoring and treatment 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 factois (FGF) (Burgess and Maciag, Annual Rev Biochem .58 575 (1989)), platelet-derived endothelial cell growth factor (PD-ECGF) (Ishikawa etal , Nature, 338 557 (1989)), and vascular endothelial growth factor (VEGF) Leung et al . Science. 246 1306 (1989), Ferrara and
Henzel. Biochem Biophys Res Commun . 161 851 (1989), Tischer et al . Biochem Biophys 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 amino acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 1 16 and 159 in h VEGF The 189-amino 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 amino acid protein differs from h VEGF by virtue of an insertion of 41 amino acids at residue 1 16 in h VEGF Houck et al , Mol Endocπn . 5 1806
(1991 ) Ferrara et al . J Cell Biochem . 47 21 1 (1991 ) Ferrara et al . Endocrine Reviews, 13 18 (1992), Keck et al , Science, 246 1309 (1989). Connolly et l , J Biol Chem 264 20017 (1989), EP 370,989 published May 30, 1990
It is now well established that angiogenesis, which im ohes the formation of new blood vessels from preexisting endothelium is implicated in the pathogenesis of a vanetv 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 reaction of transplanted corneal tissue and other tissues rheumatoid arthritis and psoriasis Folkman etfl/ , J Biol Chem .267 10931 -10934 (1992). Klagsbrun efa/ . Annu Re\ Physiol , 53 217 239 (1991 ), and Garner A , "Vascular diseases "Jn Pathobiology of Ocular Disease A Dynamic Approach Garner A , Klintworth GK eds , 2nd Edition (Marcel Dekker, NY, 1994), pp 1625 1710
In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasm, and for providing nourishment to the growing solid tumor Fo kman etal . 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 ef a/ . Lancet. 340 1 120-1124 (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, angiogenin, IL-8, etc Folkman et al , J B C . supra, and Klagsbrun et al , supis. The negative regulators so far identified include thrombospondin (Good et al , Proc Natl Acad Sci USA . 87 6624- 6628 (1990)), the 16-kιlodalton N-terminal fragment of prolactin (Clapp et al , Endocrinology, 133 1292-1299 (1993)), angiostat (O'Reilly etal , Cell. 79 315-328 (1994)), and endostatin O'Reilly et al . Cell. 88 277-285 (1996)
Work done over the last several years has established the key role of VEGF, not only in stimulating vascular endothelial cell proliferation, but also in inducing vascular permeability and angiogenesis Ferrara et al Endocr Rev . j_8 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 al , Endocr Rev . supra The VEGF mRNA is overexpressed by the majority of human tumors examined Berkman et al , J Chn Invest . 91 153-1 9 (1993), Brown et al Human Pathol 26 86-91 (1995). Brown etal , Cancer Res .53 4727-4735 (1993). Mattern gfa/ . 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 , __ Engl J Med . 331 1480-1487 (1994) Furthermore, recent studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by AMD Lopez et al , Invest Ophthalmol Vis Sci , 37 855-868 (1996) Anti-VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in nude mice
(Kim et al , Nature, 362 841 -844 (1993). Warren et l , J C n 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 lschemic retinal disorders Adamis et al , Arch Ophthalmol . 1 14 66-71 (1996) Therefore anti VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of solid tumors and various intraocular neo\ ascular disorders Such antibodies are descπbed, 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 transcπptionally 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 1178-1 182 ( 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- ke biological and immunological activities, and competes with PDGF for a particular cell surface receptor, fisp 12 (Ryseck et al , Cell Growth Differ . 2 235-233 (1991)), human vascular IBP- ke growth factor (VIGF) (WO 96/17931 ), and nov, normally arrested in adult kidney cells, which was found to be overexpressed in myeloblastosιs-assocιated-vιrus-type-1- mduced nephroblastomas Joloit et al , Mol Cell Biol . 12 10-21 ( 1992) The expression of these immediate-early genes acts as "third messengers" in the cascade of events triggered by growth factors It is also thought that they are needed to integrate and coordinate complex biological processes, such as differentiation and wound healing in which cell proliferation is a common event
As additional mitogens, insulin-like growth factor binding proteins (IGFBPs) have been shown, in complex with insulin-like growth factor (IGF), to stimulate increased binding of IGF to fibroblast and smooth muscle cell surface receptors Clemmons et al , J Chn Invest . 77 1548 ( 1986) Inhibitory effects of IGFBP on various IGF actions in vitro include stimulation of glucose transport by adipocytes, sulfate incorporation by chondrocytes, and thymidine incorporation in fibroblast Zapf et al , J Chn 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 se\eral treatment modalities for vaπous cardiovascular and oncologic disorders, there is still a need for additional therapeutic approaches
Summary of the Invention
A Embodiments
Accordingly, the present i ention concerns compositions and methods for promoting oi inhibiting angiogenesis and/or cardiovascularization in mammals The present invention is based on the identification of proteins that test positive in various cardiovascular assays that test promotion or inhibition of certain biological activities Accordingly, the proteins are believed to be useful drugs for the diagnosis and/or treatment (including prevention) of disorders where such effects are desired, such as the promotion or inhibition of angiogenesis, inhibition or stimulation of vascular endothelial cell growth, stimulation of growth or proliferation of vascular endothelial cells, inhibition of tumor growth, inhibition of angiogenesis-dependent tissue growth, stimulation of angiogenesis-dependent tissue growth, inhibition of cardiac hypertrophy and stimulation of cardiac hypertrophy, e g , for the treatment of congestive heart failure
In one embodiment, the present invention provides a composition comprising a 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 angiogemc 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 foi 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 ofthe 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 Pieserved liquid pharmaceutical formulations might contain multiple doses of the anti-PRO antibody, and might, theiefore, 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 actι\ ιt> 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 effecm e 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 undei 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 descπbed 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 complementaπty-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 oi 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 cardiovasculai endothelial oi 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 antιbod\ 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 cyto etry, fluoπmetry, or other techniques known in the art The test sample is usually obtained from an individual suspected to have a cardiovascular, endothelial or angiogenic disorder
In another embodiment, the invention provides a method for determining the presence 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 PGF2[! 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 angiogenic disorder is cardiac hypertrophy and said PRO polypeptide is administered together with a cardiovascular, endothelial or angiogenic agent The preferred cardiovascular, endothelial or angiogenic agent for this purpose is selected from the group consisting of an antihypertensive drug, an ACE inhibitor, an endothelm receptor antagonist and a thrombolytic agent If a thrombolytic agent is administered, preferably the PRO polypeptide is administeied 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 disordei is cardiac hypertrophy and the PRO polypeptide is administered following primary angioplasty for the treatment of acute preferably wherein the mammal is further exposed to angioplasty or a cardiovascular, endothelial, or angiogenic agent
In another preferred embodiment, the cardiovascular, endothelial oi 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-i elated 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 e\ 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 oi (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 foi 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 antibod) 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 In yet another embodiment, the invention provides a method for inhibiting endothelial cell growth in a mammal comprising administering to the mammal a PR0333, PR0364, PR0877 PR0879, PR0882 or PR0885 polypeptide or agonist thereof, wherein endothelial cell growth in said mammal is inhibited
In yet another embodiment, the invention provides a method for stimulating endothelial cell growth in a mammal comprising administering to the mammal a PRO 179, PR0321 , PRO840, PR0844, PR0846, PR0878 oi PR0879 polypeptide or agonist thereof, wherein endothelial cell growth in said mammal is stimulated
In yet another embodiment, the invention provides a method for inhibiting endothelial cell giowth in a mammal comprising administering to the mammal an antagonist of a PRO 179, PR0321 , PRO840, PR0844 PR0846 PR0878 or PR0879 polypeptide, wherein endothelial cell growth in said mammal is inhibited
In yet another embodiment, the invention provides a method for stimulating endothelial cell growth in a mammal comprising administering to the mammal an antagonist of a PR0333, PR0364, PR0877 PR0879,
PR0882 or PR0885 polypeptide, wherein endothelial cell growth in said mammal is stimulated
In yet another embodiment, the invention provides a method for inducing cardiac hypertrophy in a mammal comprising administering to the mammal a PRO205, PR0882 or PR0887 polypeptide or agonist thereof, wherein cardiac hypertrophy in said mammal is induced
In yet another embodiment, the invention provides a method for reducing cardiac hypertrophy in a mammal comprising administering to the mammal a PR0238, PR0878 or PRO 1760 polypeptide or agonist thereof, wherein cardiac hypertrophy in said mammal is reduced In yet another embodiment, the invention provides a method for inducing cardiac hypertrophy in a mammal comprising administering to the mammal an antagonist of a PR0238, PR0878 or PROl 760 polypeptide, wherein cardiac hypertrophy in said mammal is induced
In yet another embodiment, the invention provides a method for reducing cardiac hypertrophy in a mammal comprising administering to the mammal an antagonist of a PRO205, PR0882 or PR0887 polypeptide, wherein cardiac hypertrophy in said mammal is reduced
In yet another embodiment, the invention provides a method for inhibiting angiogenesis induced by a PRO 179, PR0321 , PRO840, PR0844, PR0846, PR0878 or PR0879 polypeptide comprising administering a therapeutically effective amount of an anti-PRO 179, antι-PR0321 , antι-PRO840, antι-PR0844, antι-PR0846, antι-PR0878 or anti- PR0879 antibody to the mammal, wherein said angiogenesis is inhibited In yet another embodiment, the invention provides a method for stimulating angiogenesis induced by a
PROl 79, PR0321 , PRO840, PR0844, PR0846, PR0878 or PR0879 polypeptide comprising administering a therapeutically effective amount of said polypeptide to the mammal, wherein said angiogenesis is stimulated
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% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternativeh at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternativel} at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternativeh at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment 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% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PRO polypeptide cDNA as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement 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% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein, oi (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 either transmembrane domain deleted or transmembrane domain-inacti vated, or is complementary to such encoding nucleotide sequence wherein the tiansmembrane domaιn(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 oi 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 foi an anti-PRO antibody or as antisense ohgonucleotide probes Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 1 10 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence 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 nucleotide molecule 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% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity alternativeh at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity alternatively at least about 91 % amino acid sequence identity, alternate eh at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein
In a further aspect, the invention concerns an isolated PRO polypeptide comprising an am o acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% ammo acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% ammo acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% am o acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% am o acid sequence identity and alternatively at least about 99% amino acid 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, alternatively at least about 81 % positives, alternatively at least about 82% positives, alternatively at least about 83% positives, alternatively at least about 84% positives, alternatively at least about 85% positives, alternatively at least about 86% positives alternatively at least about 87% positives alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 907r positives, alternatively at least about 91 % positives, alternatively at least about 92% positives, alternatively at least about 93% positives alternatively at least about 94% positives, alternatively at least about 95%- positiv es alternatively at least about 96% positives, alternatively at least about 97%- positives, alternatively at least about 98% positives and alternatively 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 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 specific aspect, the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded bv a nucleotide sequence that encodes such an amino acid sequence as hereinbefoie described Processes for producing the same are also herein described, wherein those processes comprise cultuπng a host cell comprising a vector w hich comprises the appropπate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
Another aspect 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 cultuπng 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 descπbed, 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 ofthe 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 cultuπng host cells under conditions suitable foi expression of the desired polypeptide and recovering the desired polypeptide from the cell culture
In other embodiments, the invention provides chimeπc molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence Example of such chimeπc 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 or below described polypeptides Optionally, the antibodv, is a monoclonal antibody, humanized antibody antibody fragment or single-chain antibody In yet other embodiments, the invention provides o gonucleotide 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 1 ) of a native sequence PRO 179 cDN A wherein SEQ
ID NO 1 is a clone designated herein as "DNA16451 -1388"
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 3) of a native sequence PR0238 cDNA, wherein SEQ ID NO 3 is a clone designated herein as "DNA35600-1 162"
Figuie 4 shows the amino acid sequence (SEQ ID NO 4) derived from the coding sequence of SEQ ID NO 3 shown in Figure 3 Figure 5 shows a nucleotide sequence (SEQ ID NO 5) of a native sequence PR0364 cDNA, wherein SEQ
ID NO 5 is a clone designated herein as "DNA47365-1206"
Figure 6 shows the amino acid sequence (SEQ ID NO 6) derived from the coding sequence of SEQ ID NO 5 shown in Figure 5
Figure 7 shows a nucleotide sequence (SEQ ID NO 7) of a native sequence PR0844 cDNA, wherein SEQ ID NO 7 is a clone designated herein as "DNA59838-1462"
Figure 8 shows the amino acid sequence (SEQ ID NO 8) derived from the coding sequence of SEQ ID NO 7 shown in Figure 7
Figure 9 shows a nucleotide sequence (SEQ ID NO 9) of a native sequence PR0846 cDNA, wherein SEQ ID NO 9 is a clone designated herein as "DNA44196- 1353" Figure 10 shows the amino acid sequence (SEQ ID NO 10) derived from the coding sequence of SEQ ID
NO 9 shown in Figure 9
Figure 1 1 shows a nucleotide sequence (SEQ ID NO 1 1 ) of a native sequence PRO 1760 cDNA, wherein SEQ ID NO 11 is a clone designated herein as "DNA76532-1702"
Figure 12 shows the amino acid sequence (SEQ ID NO 12) derived from the coding sequence of SEQ ID NO 1 1 shown in Figure 1 1
Figure 13 shows a nucleotide sequence (SEQ ID NO 13) of a native sequence PRO205 cDNA, wherein SEQ ID NO 13 is a clone designated herein as "DNA30868"
Figure 14 shows the amino acid sequence (SEQ ID NO 14) derived from the coding sequence of SEQ ID NO 13 shown in Figure 13 Figure 15 shows a nucleotide sequence (SEQ ID NO 15) of a native sequence PR0321 cDNA, wherein SEQ
ID NO 15 is a clone designated herein as "DNA34433"
Figure 16 shows the ammo acid sequence (SEQ ID NO 16) derived from the coding sequence of SEQ ID NO 15 shown in Figure 15
Figure 17 shows a nucleotide sequence (SEQ ID NO 17) of a native sequence PR0333 cDNA, wherein SEQ ID NO 17 is a clone designated herein as "DNA41374'
Figure 18 shows the amino acid sequence (SEQ ID NO 18) derived from the coding sequence of SEQ ID NO 17 shown in Figure 17
Figure 19 shows a nucleotide sequence (SEQ ID NO 19) of a native sequence PRO840 cDNA, wherein SEQ ID NO 19 is a clone designated herein as "DNA53987" Figure 20 shows the amino acid sequence (SEQ ID NO 20) derived from the coding sequence of SEQ ID
NO 19 shown in Figure 19
Figure 21 shows a nucleotide sequence (SEQ ID NO 21 ) of a nati ve sequence PR0877 cDN A, wherein SEQ ID NO 21 is a clone designated herein as ' DNA58120" Figure 22 shows the amino acid sequence (SEQ ID NO 22) derived from the coding sequence of SEQ ID NO 21 shown in Figure 21
Figure 23 shows a nucleotide sequence (SEQ ID NO 23) of a native sequence PR0878 cDNA, wherein SEQ ID NO 23 is a clone designated herein as "DNA58121 " Figure 24 shows the amino acid sequence (SEQ ID NO 24) deπved from the coding sequence of SEQ ID
NO 23 shown in Figure 23
Figure 25 shows a nucleotide sequence (SEQ ID NO 25) of a native sequence PR0879 cDNA, wherein SEQ ID NO 25 is a clone designated herein as "DNA58122"
Figure 26 shows the amino acid sequence (SEQ ID NO 26) derived from the coding sequence of SEQ ID NO 25 shown in Figure 25
Figure 27 shows a nucleotide sequence (SEQ ID NO 27) of a native sequence PR0882 cDNA, wherein SEQ ID NO 27 is a clone designated herein as "DNA58125"
Figure 28 shows the amino acid sequence (SEQ ID NO 28) derived from the coding sequence of SEQ ID NO 27 shown in Figure 27 Figure 29 shows a nucleotide sequence (SEQ ID NO 29) of a native sequence PR0885 cDNA, wherein SEQ
ID NO 29 is a clone designated herein as "DNA58128"
Figure 30 shows the amino acid sequence (SEQ ID NO 30) derived from the coding sequence of SEQ ID NO 29 shown in Figure 29
Figure 31 shows a nucleotide sequence (SEQ ID NO 31 ) of a native sequence PR0887 cDNA, wherein SEQ ID NO 31 is a clone designated herein as "DNA58130"
Figure 32 shows the amino acid sequence (SEQ ID NO 32) derived from the coding sequence of SEQ ID NO 31 shown in Figure 31
Detailed Description of the Invention I Definitions
The phrases "cardiovasculai endothelial and angiogenic disordei ' cardiovascular, endothelial and angiogenic dysfunction' cardiovascular, endothelial or angiogenic disoider' and cardiovascular, endothelial oi angiogenic dysfunction are used interchangeably and refer in part to systemic disorders that affect vessels, such as diabetes melhtus, as well as diseases of the vessels themselves such as of the arteries, capillaries, veins, and/oi lymphatics This would include indications that stimulate angiogenesis and/oi cardiov asculaπzation, and those that inhibit angiogenesis and or cardiovascularization Such disorders include for example, arterial disease, such as atherosclerosis, hypertension inflammatory vascu tides, Reynaud s disease and Reynaud s phenomenon, aneurysms and arterial restenosis, venous and lymphatic disorders such as thrombophlebitis hmphangitis, and lymphedema and other vascular disorders such as peripheral vascular disease, cancer such as vascular tumors, e -? , hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillaiy angiomatosis, hemangioendothehoma, 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
(hyperplasia), or both Certain organs, such as the heart, lose the ability to divide shortly after birth Accordingly, "cardiac hypertrophy" is defined as an increase in mass ofthe 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 morphologically by increases in the size of myofibπls and mitochondria, as well as by enlargement of mitochondria and nuclei At this stage, while muscle cells are larger than normal, cellular organization is largely preserved At a more advanced stage 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 longstanding hypertrophy show more obvious disruptions in cellular organization, including markedly enlarged nuclei with highly lobulated membranes, which displace adjacent myofibπls and cause breakdown of normal Z-band registration The phrase "cardiac hypertrophy" is used to include all stages of the progression of this condition, characterized by vaπous 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 wheie the heart does not pump blood at the rate needed for the requirements of metabolizing tissues The heart failure can be caused by a numbei of factors, including lschemic, congenital, rheumatic, or ldiopathic forms "Congestive heart failure" (CHF) is a progressive pathologic state wheie 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 v entπculai hypertrophy CHF is a common end result of a number of vaπous cardiac disorders "Myocardial infarction" generally results from atherosclerosis of the coronary arteries, often with superimposed coronary thrombosis It may be divided into two major types transmural infarcts, in which myocardial necrosis involves the full thickness ofthe ventrii-ular 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 Mvocardial lntaiction is known to cause both a change in hemodynamic effects and an alteration in structure in the damaged and healthy zones of the heait Thus, for example, myocardial infarction reduces the maximum cardiac output and the stroke volume of the heait 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 diastohc performance Fouad e/α/ J Am Coll Cardiol , 4 1 00- 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 morphologic, functional, and clinical features (Maron et al , N Engl J Med , 316 780-789 (1987). Spιπto era/ , N Engl J Med .320 749-755 ( 1989), Louie and Edwards, Prog. Cardiovasc Pis .36 275 308 (1994). Wigle etal . 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 etal . N Engl j Med .326 1108-1 114 (1992). Schwartz etal. Circulation 91 532-540
(1995), Marian and Roberts, Circulation. 92 1336-1347 (1995), Thierfelder et al , CeH, 77 701-712 (1994), Watkins et al , Nat Gen . ϋ 434-437 ( 1995) Besides β-myosin heavy chain, other locations of genetic mutations include cardiac troponin T, alpha topomyosm, cardiac myosin binding protein C, essential myosin light chain, and regulatory myosin light chain See. Malik and Watkins. Curr Opin Cardiol . 12 295-302 (1997) Supravalvular "aortic stenosis" is an inherited vascular disordei 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 lntracardiac pressure resulting in myocardial hypertrophy and eventually heart failure and death The pathogenesis of this disorder is not fully understood, but hypertrophy and possibly hyperplasia of medial smooth muscle are prominent featuies 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 Jul> 22, 1997
"Valvular regurgitation" occurs as a result of heart diseases resulting in disorders of the cardiac valves
Vaπous 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 atπoventπcular orifices and operation of the heart Defects such as the naπowing of the valve stenosis or the defectiv e closing of the valve result in an accumulation of blood in the heart cavity or regurgitation of blood past the valve If uncoπected, 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 othei caidiovascular endothelial and angiogenic disorders w hich 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 , 1 II, 1251, 9UY, and 186Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer Examples of chemotherapeutic agents include alkylating agents, fohc acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics, pyπmidine analogs, 5-fluorouracιl, cisplatin, purine nucleosides, amines, amino acids, tπazol nucleosides, or corticosteroids Specific examples include Adπamycin, Doxorubicin, 5-Fluorouracιl, Cytosme arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate,
Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C Mitoxantrone, V creistine, Vinorelbme, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopteπn Dactinomycin, Mitomyc s, 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 vitio 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 aπest Classical M-phase blockers include the vincas (vincπstine and vinblastine), taxol and topo
II inhibitors such as doxorubicin, daunorubicin, etoposide, and bleomycin Those agents that arrest G l also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen predmsone, dacarbazine, mechlorethamine, cisplatin, methotrexate. 5-fluoιouracιl, and ara-C Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds , Chaptei 1. entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami etal (WB Saunders Philadelphia, ! 995), especially p 13 Additional examples include tumor necrosis factor (TNF) an antibody capable of inhibiting or neutiahzmg the angiogenic activity of acidic or basic FGF or hepatocyte growth factor (HGF), an antibody capable of inhibiting or neutiahzmg the coagulant activities of tissue factor, protein C, or protein S (see WO 91/01753, published 21 Februai) 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 ofthe agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial effect, such as an anti-hypertrophic effect, for an extended period of time
"Mammal" forpurposes 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, endothelm receptor antagonists such as, for example. BOSENTAN™ and MOXONODIN™, interferon-gamma (IFN-γ), des-aspartate-angiotensin I, thrombolytic agents, e g , streptokinase, urokinase, t-PA, and a t-PA variant specifically designed to have longei half-life and very high fibrin specificity, TNK t-PA (a Tl 03N, Nl 17Q, KHRR(296-299)AAAA t-PA variant. Keyt et al , Proc Nati Acad Sci USA. 91 3670-3674 (1994)), inotropic or hypertensive agents such as digoxigenin and β-adrenergic receptor blocking agents, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol atenolol, metoprolol, and carvedilol, angiotensin converting enzyme (ACE) inhibitors, e g , quinapπl, captopπl, enalapπl, ramipπl, benazepπl, fosinopπl, and lisinopπl, diuretics, e g chlorothiazide, hydrochlorothiazide hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide acetazolamide, and indapamide, and calcium channel blockers, e g , diltiazem, nifedipine, verapamil, nicardipine One preferred category of this type is a therapeutic agent used for the treatment of cardiac hypertrophy or of a physiological condition instrumental in the development of cardiac hypertrophy, such as elevated blood pressui e aortic stenosis, or myocai dial infarction "Angiogenic agents" and "endothelial agents" are active agents that promote angiogenesis and/or endothelial cell growth, or if applicable, vasculogenesis This would include factois that accelerate wound healing, such as growth hormone, insulm-like growth factor-I (IGF-I), VEGF, VIGF. PDGF. epidermal growth factor (EGF), CTGF and members of its family. FGF, and TGF-α and TGF-β "Angiostatic agents" are active agents that inhibit angiogenesis or vasculogenesis or otherwise inhibit or prevent growth of cancer cells Examples include antibodies or other antagonists to angiogenic agents as defined above, such as antibodies to VEGF They additionally include cytotherapeutic agents such as cytotoxic agents, chemotherapeutic agents, growth-inhibitory agents, apoptotic agents, and other agents to treat cancer, such as anti- HER-2, antι-CD20, and other bioactive and organic chemical agents
In a pharmacological sense, in the context of the present invention, a "therapeutically effective amount" of an active agent such as a PRO polypeptide or agonist or antagonist thereto or an anti-PRO antibody, refers to an amount effective in the treatment of a cardiovascular, endothelial or angiogenic disorder in a mammal and can be determined empirically As used herein, an "effective amount" of an active agent such as a PRO polypeptide or agonist or antagonist thereto or an anti-PRO antibody, refers to an amount effective for carrying out a stated purpose, wherein such amounts may be determined empirically for the desired effect
The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to vaπous 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 coπesponding 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-occumng variant forms {e g , alternatively spliced forms) and naturally-occurring allelic variants ofthe polypeptide In v aπous embodiments of the inv ention, the nativ e 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 methion e residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionme residues located either upstream or downstream from the am o acid position 1 in the figures mav 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 ofthe transmembrane and cytoplasmic domains Ordinarily, a PRO polypeptide ECD will hav e 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 v ary 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 comtemplated by the present invention The approximate location ofthe "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-termmal 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 ammo 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 ' PRO polypeptide variant" means an active PRO polypeptide as defined above or below having at least about
80% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more am o acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence Ordinarily, a PRO polypeptide variant will have at least about 80% ammo acid sequence identity alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% ammo acid sequence identity, alternativ ely at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity , alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with oi without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein Oidmaπly, PRO variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 ammo acids in length, alternatively at least about 30 ammo acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or more. As shown below, Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program. This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program.
In addition, Tables 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 -8 /* value of a match with a stop */ int _day[26][26] = {
/* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/
/* A*/ 2, 0,-2 0,0,-4, 1,-1,-1,0,-1,-2, -1, 0,__M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0}, l*_*l 0, 3,-4 3, 2,-5, 0, 1,-2, 0, 0,-3,- 2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1},
/*C */ -2,-4,15 -5,-5,-4,-3,-3,-2, 0,-5,-6 ,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5},
/*D*/ 0, 3,-5 4, 3,-6, 1, 1,-2,0,0,-4,- 3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2},
/*E*/ 0, 2,-5 3,4,-5,0, 1,-2,0,0,-3,- 2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3}, /* p */ -4,-5,-4 -6,-5,9,-5,-2, 1,0,-5,2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5},
/*G*/ 1,0,-3 1,0,-5, 5,-2,-3, 0,-2,-4,- •3, 0,_M,-l,-l,-3, 1, 0, 0,-1,-7, 0,-5, 0}, /*H*/ -1, 1,-3. 1, 1,-2,-2, 6,-2,0,0,-2,- ■2, 2,_M, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0, 2},
1*1*1 -1,-2,-2 -2,-2, 1,-3,-2, 5,0,-2,2, 2,-2,_M,-2,-2,-2,-l, 0, 0, 4,-5, 0,-1,-2},
I* 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},
/*κ*/ -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}, 1*0*1 _M,_M M,_M, M, M, M, M, M, M,_M, M, M, M, 0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M},
/* p */ 1,-1,-3 -1,-1,-5,-1, 0,-2, 0,-l,-3,-2,-l, M, 6, 0,0, 1,0,0,-1,-6,0,-5,0},
/*Q*/ 0, 1,-5 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 1,-1,-1,0,-2,-5,0,-4, 3},
/*R*/ -2, 0,-4 -1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0, 1, 6, 0,-1,0,-2, 2,0,-4,0},
/*S*/ 1,0,0 0,0,-3, 1,-1,-1,0,0,-3,-2, 1,_M, 1, -1,0,2, 1,0,-1,-2, 0.-3,0}, /* Ύ *I 1, 0,-2 0,0,-3,0,-1,0, 0,0,-1,-1, 0,_M, 0, -1,-1, 1, 3,0, 0,-5,0,-3,0}, l*\]*l 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}, I* v */ 0,-2,-2 -2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2,_M,-l, ,-2,-2,-1,0,0,4,-6,0,-2,-2}, /* W*/ -6,-5,-8 -7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4,_M,-6 5,2,-2,-5, 0,-6,17, 0.0,-6}, /*X*/ 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},
/* Y */ -3,-3, 0 -4,-4, 7,-5, 0,-1, 0,-4,-l,-2,-2,_M,-5 ,-4,-4,-3,-3.0,-2,0,0,10,-4},
!*_*! 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 < stdιo.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 for mismatched bases */
#define DINSO 8 /* penalty for a gap */
#defme DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap */
#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP] /*• size of jmp (neg for dely) */ unsigned short xfMAXJMP] /* base no of jmp in seq x */ }, /* limits seq to 2A16 -1 */ struct diag { int sec /* 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 xfJMPS], /* loc of jmp (last ele before gap) */
}, char *ofιle, /* output file name */ char *namex[2], /* seq names: getseqs() * char *prog, /* prog name for err msgs */ char *seqx[2], /* seqs. getseqsQ */ int dmax, /* best diag. nw() */ int dmaxO, /* final diag */ int dna, /* set if dna maιn() */ int endgaps, /* set if penalizing end gaps */ int gapx, gapy, /* total gaps m 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_calioc(),
Page 1 of nw.h /* Needleman-Wunsch alignment program
*
* usage progs filel file2
* where filel and file2 are two dna or two protein sequences
* The sequences can be m 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 "ahgn.out"
*
* The program may create a tmp file in /tmp to hold info about traceback
* Original version developed under BSD 43 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'-Α')), 4, 8, 16, 32, 64, 128, 256, OxFFFFFFF, 1< < 10, 1< < 11, 1< < 12, 1< < 13, 1< < 14, 1< < 15, 1< < 16, 1< < 17, 1< < 18, 1< < 19, 1< <20, 1< <21, 1< <22, 1<<23, 1<<24, 1< <25|(1< <(Ε,-Α'))|(1< <('Q'-Α')) }. int ac, char *av[],
{ prog = av[0], if (ac ' = 3) { fpπntf(stderr, "usage %s filel file2\n", prog), fpπntf(stderr, "where filel and file2 are two dna or two protein sequences \n' ), fpπntf(stderr, "The sequences can be m upper- or lower-case\n"), fpπntf(stderr,"Any lines beginning with ', or ' < are ιgnored\n"), fpπntf(stderr, "Output is in the file V align out\"\n"), exιt(l),
} namex[0] = av[l], namex[l] = av[2], seqx[0] = getseq(namex[0], &len0), seqx[l] = getseq(namex[l], &lenl), xb = (dna)'' _dbval _pbval, endgaps = 0, I* 1 to penalize endgaps *l ofile = 'align out", /* output file */ nw(), /* fill in the matrix get the possible jmps */ readjmpsO, /* get the actual jmps */ pπnt(), /* 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
nw() nw
{ 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 msO, insi , /* insertion penalties */ register id, /* diagonal index */ register ij, /* 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 coIO", lenl + 1 , sizeof(int)), coll = (int *)g_calloc("to get coll ", lenl + 1, sizeof(int)); insO = (dna)? DINSO : PINSO, insi = (dna)? DINS 1 : PINS1 , smax = -10000, if (endgaps) { for (col0[0] = dely[0] = -insO, yy = 1, yy < = lenl , yy + +) { col0[yy] = delyfy ] = col0[yy-l] - insi , ndely [yy] = yy,
} col0[0] = 0, /* Waterman Bull Math Biol 84 */
} else for (yy = 1 , yy < = lenl , y\ -1- +) dely[yy] = -msO,
/* fill m 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 = -(ιns0 + ιnsl), else col l [0] = delx = col0[0] - msl , ndelx = xx,
} else { ndelx = 0, }
Page 2 of nw.c ...n seqx[l], yy = 1 , yy < = lenl ; py+ + , yy+ +) { mis + = (xbm[*px-Α']&xbm[*py-Α']y? DMAT DMIS, else mis + = _day[*px-'A'][*py-'A'],
/* update penalty for del in x seq,
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps | | ndely[yy] < MAXGAP) { if (col0[yy] - insO > = dely[yy]) { dely[yy] = col0[yy] - (lnsO + msl), ndelyfyy] = 1 , } else { delyfyy] -= insi , ndely[yy] + + , }
} else { if (col0[yy] - (msO+insl) > = delyfyy]) { dely[yy] = col0[yy] - (msO + msl), ndelyfyy] = 1 ,
} else ndelyfyy] + + ,
/* update penalty for del in y seq, * favor new del over ongong del */ if (endgaps | | ndelx < MAXGAP) { if (collfyy-1] - insO > = delx) { delx = coll[yy-l] - (msO + msl), ndelx = 1 , ndelx + + , }
} else { if (coll fyy 1] - (msO + insl) > = delx) { delx = collfyy 1] - (msO + 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 > = delyfyy]) else if (delx > = delyfyy]) { collfyy] = delx, IJ = dxfid] ljmp, if (dxfid] jp n[0] && ('dna 1 1 (ndelx > = MAXJMP && xx > dxfid] jp x[ιj] +MX) 1 1 mis > dxfid] score+DINSO)) { dxfid] ιjmρ+ + , if (+ +ιj > = MAXJMP) { wπtejmps(ιd), ij = dxfid] ljmp = 0, dxfid] offset = offset, offset + = sizeof (struct jmp) + sιzeof(offset), } } dxfid] jp nfij] = ndelx, dxfid] jp xfij] = xx, dxfid] score = delx,
} else { coll fyy] = delyfyy], ij = dxfid] ljmp, if (dxfid] jp n[0] && ('dna | | (ndelyfyy] > = MAXJMP
&& xx > dxfid] jp x[ιj] + MX) | | mis > dx[ιd] score+DINSO)) { dxfid] ιjmρ+ + , if (+ +ιj > = MAXJMP) { wπtejmps(ιd), ij = dxfid] ljmp = 0 dxfid] offset = offset, offset + = sizeof (struct jmp) + sιzeof(offset),
} } dxfid] jp nfij] = -ndelyfyy], dxfid] jp xfij] = xx, dxfid] score = delyfyy],
} if (xx = = lenO && yy < lenl) { /* last col */ if (endgaps) coll fyy] -= msO+ιnsl *(lenl v) if (col 1 [y> ] > smax) { s ax = coll fyy], dmax = id, } } } if (endgaps && xx < lenO) coll [yy-l] = ιnsO + msl *(lenO xx), if (col l fyy- 1] > smax) { smax = coll fyy 1], dmax = id,
} tmp = colO, colO = col l , col l = tmp,
}
(void) free((char *)ndely), (void) free((char *)col0), (■ oid) free((char *)coll),
Page 4 of nw c /* *
* print() - only routine visible outside this module *
* static
* getmatO - trace back best path, count matches pπnt()
* pr_ahgn() - print alignment of described in array p[] prmt()
* dumpblock() - dump a block of lines with numbers, stars pr_ahgn()
* nums() — put out a number line. dumpblock()
* putline() — put out a line (name, [num], seq, fnum]) dumpblockO
* stars() - -put a line of stars: dumpblockO
* stπpnameO - strip any path and prefix from a seqname */
#include "nw h"
#define SPC 3
#define P LINE 256 /* maximum output line */
#define P SPC 3 /* space between name or num and seq */ extern day [26] [26], int olen, /* set output line length */
FILE *fx, /* output file */ pπnto print
{ int lx, ly, firstgap, lastgap, /* overlap */ if ((fx = foρen(ofile, "w")) = = 0) { fpπntf(stderr, " %s can't write %s\n", prog, ofile), cleanup(l),
} fpπntf(fx, " < first sequence %s (length = %d)\n", namexfO], lenO), fpπntf(fx, " < second sequence %s (length = %d)\n", namexfl], lenl), olen = 60, lx = lenO, ly = lenl , firstgap = lastgap = 0, if (dmax < lenl 1) { /* leading gap in x */ pp[0] spc = firstgap = lenl - dmax - 1 , ly -= pp[0] spc,
} else if (dmax > lenl - 1) { /* leading gap in y */ ppfl] spc = firstgap = dmax - (lenl - 1), lx - = ppfl] spc,
} if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = lenO dmaxO - 1 , lx -= lastgap,
} else if (dmaxO > lenO - 1) { /* trailing gap in */ lastgap = dmaxO - (lenO - 1), ly = lastgap,
} getmat(lx, 1> , firstgap, lastgap), pr_ahgn(),
Page 1 of nwpπnt.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 */
{ mt nm , ι0, ii , sizO, sizl , char outx[32], double pet, register nO, nl , register char *p0, *pl ,
/* get total matches, score */ pO = seqxfO] + pp[l] spc, pi = seqxfl] + pp[0] spc, nO = pp[l] spc + 1 , nl = pp[0] spc + 1 , nm = 0, while ( *p0 && *pl ) {
if (xbm[*pO- A ]&xbm[*pl 'A ]) nm+ + ,
}
/* 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 < \y)-> lx ly, pet = 100 *(double)nm/(double)lλ fpπntf(fx \n ), ipπntt(tx, < %d match s in an ov erlap of %d % 2f percent sιmιlaπty\n nm, (nm = = l)9 ' es lx, pet),
Page 2 of nwpπnt.c fpπntf(fx, " < gaps in first sequence %d", gapx), .getmat if (gapx) {
(void) spπntf(outx, " (%d %s%s)", ngapx, (dna)9 "base" "residue" , (ngapx = = l)9 "s' ), fpπntf(fx, " %s' , outx), fpπntf(fx, ", gaps in second sequence %d", gapy), if (gapy) {
(void) spπntf(outx. " (%d %s%s)", ngapy, (dna)*? "base" "residue", (ngapy = = l) 's '), fpπntf(fx, " %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 + %ά 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 = = l)9 "" "s , lastgap, (dna)9 "base' "residue", (lastgap = = l)9 " " "s' ), else fpπntf(fx, " < endgaps not penahzed\n"),
static nm, /* matches in core — for checking */ static lmax, /* lengths of stripped file names */ static ■J[2], /* jmp index for a path */ static nc[2], /* number at start of current line */ static m[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 starfP LINE], /* set by stars() */
/* * print alignment of described in struct path ppf]
*/ static
Pr. .ahgnO pr align
{ int nn, /* char count */ int more, register 1 for (1 = 0, lmax = 0 I < 2, ! + +) { nn = stπpname(namex fi] ) , if (nn > lmax) lmax = nn
sizfi] = >)['] = o, psfi] = seqxfi] ,
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 (ppfi] spc) { /* leading space */ *po[ι] + + = ' ', pp[ι] spc-,
} else if (sιz[ι]) { /* in a gap */
*po[ι] + + = '-', sizfi]-,
} 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 seq9 */ if(m[ι] == ppfi] xfij [ι]]){
* we need to merge all gaps
* at this location sizfi] = ppfi] n[ιj[ι] + +]. while (mfi] == pp[ι] x[ιj[ι]l) sizfi] -+ = ppfi] n[ιj[ι] + +]
} nι[ι] + + ,
}
} if (++nn = = olen | | 'more && I τn){ dumpblockO, for (l = 0, l < 2, ι++) po[ι] = OUtfl], nn = 0,
}
}
}
/*
* dump a block of lines, including numbers, stars Pr_ all ιgn()
*/ static dumpblockO dumpblock
{ register I, for (ι = 0, l < 2, ι++) *po[ι]- = '\0',
Page 4 of nwpπnt.c ...dumpblock
(void) putc('\n', fx), for(ι = 0, l < 2, ι++) { if O-outfi] && (*out[ι] '= ' ' || *(po[ι]) '= ' ')){ if(. ==0) nums(ι), if (i == 0&&*out[l]) stars(), puthne(ι), if 0 = = 0 && *out[l]) fpπntf(fx, star), if(ι == 1) nums(ι),
}
}
* put out a nu imber line dumpblockO */ static nums(ιx) nums int lx, /* index m out[] holding seq line */
{ char nlmefP LINE], register •-J. register char *pn, *px, *py, for (pn = nl e, I = 0, l < lmax+P_SPC, ι+ + , pn++)
*pn for (I = ncfix], py = outfix], *py, py++, pn++) { if(* py = = ' ' II *py == '-') *pn = else { if (ι%10 == 0 | | (i == 1 &&nc[x] ' = D){ j = (K 0)9 -. ., for (px = pn, j, j /= 10, px-)
*px =j%10 + '0', if (l < 0)
*px = '-',
} else
*pn ι + + ,
}
}
*pn = '\0 , nc[ιx] = l, for (pn = nhne, ''-pn, pn++)
(void) putc(*pn, fx), (void) putc('\n', fx),
}
/*
* put out a line (name, fnum], seq, [num]) dumpblockO static puthne(ιx) putline
Page 5 of nwprint.c .putline int l, register char px, for (px = namex[ιx], 1 = 0; *px && *px '= ':'; px++, ι++)
(void) putc(*px, fx); for(, l < lmax + P SPC; ι++)
(void) putcO ', fx),
/* these count from 1 :
* nι[] is current element (from 1)
* ncf] is number at start of current line */ for (px = outfix]; *px; px++) (void) putc(*px&0x7F, fx); (void) putc('\n', fx);
/*
* put a line of stars (seqs always in outfO], out[l]): dumpblockO
*/ static stars() stars
{ int I; register char *p0, *pl, ex, *px; if (!*out[0] I I (*out[0] == ' '&& *(po[0]) == ' ') 11 !*out[l] I I (*out[l] == ' '&& *(po[l]) == ' ')) return; px = star, for (l = lmax+P SPC; I; ι~) *px+ + = ' '; for (pO = out[0],pl = outfl]; *p0&&*pl; p0+ + ,pl + +){ if (ιsalpha(*pO) && ιsalpha(*pl)) { if (xbm[*pO-'A']&xbm[*pl-'A']) { ex = '* , nm+ + .
} else if (!dna&&_day[*pO-'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_ahgn()
*/ static stπpname(pn) Stπpname char *pn; /* file name (may be path) */
{ register char *px, *py py = 0; for (px = pn; *px; px+ +) if (*px = -= '/') 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() — calloc() with error checkin
* readjmpsO ~ get the good jmps, from tmp file if necessary
* wntejmpsO — 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
*/ cieanup(ι) cleanup int l.
{ if (fj)
(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(file. len) getseq char *file; /* file name */ int *len, /* seq len */
{ char hne[1024], *pseq; register char *px, *py, int natgc, tlen,
FILE *fp, if ((fp = fopen(file, "r")) = = 0) { fpπntf(stderr," %s: can't read %s\n" , prog, file); exιt(l).
} tlen = natgc = 0; while (fgets(lme, 1024, fp)) { if (*hne = = ' ;' | | *hne = = ' < ' | | *hne = = ' > ') 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[2J = pseq[3] = '\0' ,
Page 1 of nwsubr.c ...getseq py = pseq + 4; *len = tlen, rewmd(fp); while (fgets(hne, 1024, fp)) { if (*lιne == ';* | | *hne == '<' | | *lme == '>') 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_calloc char *msg; /* program, calling routine */ int nx, sz; /* number and size of elements */ char *px, *calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) { fpπntf(stderr, "%s: g_calloc() failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz), exιt(l), } } return(px),
* get final jmps from dxf] or tmp file, set ppf], reset dmax: maιn()
*/ readjmps
{ int fd = -1; register 1,J, XX, if(j){
(void) fclose(fj); if ((fd = openOna e, 0_RDONLY, 0)) < 0) { fpπntf(stderr, "%s can't open() %s\n", prog, jname), cleanup(l), } } for (I = lO = ii = 0, dmaxO = dmax, xx = lenO; , ι+ +) { while (1) { for (j = dx [dmax]. ljmp, j > = 0 && dx[dmax].jp xf]] > = xx; j— )
Page 2 of nwsubr.c ...readjmps if 0 < 0 && dxld ax]. offset && fj) {
(void) lseek(fd, dx [dmax]. off set, 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 (l > = JMPS) { fpπntf(stderr, "%s: too many gaps in ahgnment\n", prog); cleanup(l); } if 0 > = 0) { siz = dx[dmax].jp.nfj]; xx = dx[dmax].jp.x[)]; if (siz < 0) { /* gap in second seq */
/* id = xx - yy + lenl - 1 */ pp[l].x[ιl] = xx - dmax + lenl - 1; gapy+ + ; ngapy -= siz; /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP 11 endgaps)? -siz : MAXGAP; ιl + + ;
} else if (siz > 0) { /* gap in first seq */ 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 < iO; j+ + , ι0~) {
. = ppfOJ.n l; pp[0].n[]] = pp[0].n[ι0]; pp[0].n[ι0] = i, i = pp[0].xD]; pp[0].xfj] = pp[0].x[ι0], pp[0].x[ι0] = i, } for = 0, ιl-,j < ιl;j+ + , ιl-) { i = pp[l].nDl; pp[l].nD] = pp[l].π[ιl]; pp[l].n[ιl] = i;
' = PPll].x[)]; pp[l]-xϋ] = PP[l].x[ιl], pp[l].x[ιl] - i, } if (fd > = 0)
(void) close(fd); if (fj) {
(void) unhnk(jname); fj =0; offset = 0; }
Page 3 of nwsubr.c /* * write a filled jmp struct offset o the prev one (if any): nw()
*/ writejmps(ix) writejmps int ix;
{ char *mktemp(); if (!fj) { if (mktemp(jname) < 0) { fprintf(stderr, " %s: can't mktemp() %s\n", prog, jna e); cleanup(l);
} if ((fj = fopen(jname, "w")) = = 0) { fprintf(stderr, " %s: can't write %s\n", prog, jname); exit(l); } }
(void) fwrite((char *)&dx[ix] .jp, sizeof(struct jmp), 1 , fj); (void) fwrite((char *)&dx[ix] . offset, sizeof (dxfix]. offset), 1, fj);
Page 4 of nwsubr.c Table 2A
PRO XXXXXXXXXXXXXXX (Length = 15 ammo acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 ammo acids)
% amino acid sequence identity =
(the number of identically matchmg ammo 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 = 333 %
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 PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent -sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are withm the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech. Inc , South San Francisco, California or may be compiled from the source code provided in Table 1 The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % ammo 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 //v. w w ncbi nlm nih gov or otherwise obtained from the National Institute of Health, Bethesda, MD NCBI- BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occuπences = 10 minimum low complexity length = 1 . '5. multipass e-\ alue = 0 01 , constant for multi-pass = 25. dropoff for final gapped alignment = 25 and scoπng matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given ammo 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 amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A
In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al , Methods in 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) = 11 , and scoring matrix = BLOSUM62 For purposes herein, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison ammo acid sequence of interest (t 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 amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B' the amino acid sequence A is the comparison amino acid sequence of interest and the am o acid sequence B is the amino acid sequence of the PRO polypeptide of interest ' PRO variant polynucleotide" or "PRO variant nucleic acid sequence' means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO pohpeptide, with or without the signal peptide as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein OrdinaπK a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternate elv at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity alternamely at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity alternatively at least about 85% nucleic acid sequence identity , alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity alternatively at least about 90% nucleic acid sequence identity, alternativeh at least about 91 % nucleic acid sequence identity alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Variants do not encompass the native nucleotide sequence Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length, alternatively at least about
60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more
"Percent (%) nucleic acid sequence identity" with respect to the PRO polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST 2, ALIGN, ALIGN 2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuπng 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 ιdentιt\ values are obtained as described below bv using the sequence comparison computer program ALIGN 2, wherein the complete source code for the ALIGN 2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN 2 program is publicly available through Genentech Inc , South San Francisco California or may be compiled from the source code provided in Table 1 The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with or against a given nucleic acid sequence D (which can alternatively be phrased as a C that has or comprises a certain % nucleic acid sequence identity to, with, oi 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 ofthe nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO- DNA"
Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res , 25 3389-3402 ( 1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov or otherwise obtained from the National Institute of Health, Bethesda, MD NCBI- BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10 minimum low complexity length = 15/5, multi- pass e-value = 0 01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence 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 (/ 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, PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) , Figure 10 (SEQ ID NO 10), Figure 12 (SEQ ID NO 12), Fιguιe l4 (SEQ ID NO 14), Figure 16 (SEQ ID NO 16), Figure 18 (SEQ ID NO 18), Figure 20 (SEQ ID NO 20), Figure 22 (SEQ ID NO 22), Figure 24 (SEQ ID NO 24), Figure 26 (SEQ ID NO 26), Figure 28 (SEQ ID NO 28), Figure 30 (SEQ ID NO 30), and Figure 32 (SEQ ID NO 32), respectively PRO variant polypeptides may be those that are encoded by a PRO 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 prefeπed substitution (as defined in Table 3 below) of the amino acid residue of interest
For purposes herein, the % value of positives of a given ammo 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 a polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones and other proteinaceous or non-protemaceous 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 am o 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 ofthe PRO natural environment will not be present Ordinarily, howe\ er isolated polypeptide will be prepared by at least one purification step An "isolated nucleic acid molecule encoding a PRO polvpeptide or an "isolated" nucleic acid molecule encoding an anti-PRO antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PRO-encoding nucleic acid or the natural source of the anti-PRO-encoding nucleic acid Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated An isolated PRO-encoding nucleic acid molecule or an isolated anti-PRO-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated nucleic acid molecules therefore are distinguished from the PRO-encoding nucleic acid molecule or from the anti-PRO-encoding nucleic acid molecule as it exists in natural cells However, an isolated nucleic acid molecule encoding a PRO polypeptide or an isolated nucleic acid molecule encoding an anti PRO antibody includes PRO-nucleic acid molecules or anti-PRO-nucleic acid molecules contained in cells that ordinarily express PRO polypeptides or anti-PRO antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells The term ' control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a πbosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a PRO polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a πbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by hgation at convenient restriction sites If such sites do not exist, the synthetic ohgonucleotide 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 low er temperatures Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hybπdizable sequence, the higher the relative temperature that can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so For additional details and explanation of stringency of hybridization reactions see, Ausubel et al Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995)
' Stringent conditions" or "high stringency conditions' , as defined herein, may be identified by those that (1 ) employ low ionic strength and high temperature for washing, for example, 0 015 M sodium chloπde/0 0015 M sodium cιtrate/0 1 % sodium dodecyl sulfate at 50°C, (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0 1 % bovine serum albumιn/0 1 % Fιcoll/0 1 % polyvιnylpyrrohdone/50mM sodium phosphate buffer at pH 6 5 with 750 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 ιn 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 mM tπsodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50 °C The skilled 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 chimeπc polypeptide comprising a PRO polypeptide fused to a "tag polypeptide" The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity 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 PRO variants refers to form(s) of PRO proteins that retain the biologic and/or lmmunologic activities of a native or naturally-occurring PRO polypeptide
"Biological activity" in the context of a molecule that antagonizes a PRO polypeptide that can be identified by the screening assays disclosed herein (e g , an organic or inorganic small molecule, peptide, etc ) is used to refer to the ability of such molecules to bind or complex with the PRO polypeptide identified herein, or otherwise interfere with the interaction of the PRO 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 melhtus 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 mitogemc 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 mitoge c, angiogenic, oi other biological activity of PRO polypeptides and thus are useful for the treatment of diseases or disorders characterized by undesirable excessive neovasculaπzation, including by way of example tumors and especially solid malignant tumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies retrolental fibroplasia, age related macular degeneration neovascular glaucoma, hemangiomas, thyroid 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 excessiv e vascular permeability, such as edema associated with bra tumors, ascites associated with malignancies, Meigs syndrome lung inflammation nephrotic syndrome, peπcardial effusion (such as that associated with pericarditis), and pleural effusion In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native 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 "lmmunoglobulins" (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 heterotetrameπc glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced intracham 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 ot the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains
The term ' variable' refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody to and for its particular antigen However, the variability is not evenly distributed throughout the variable domains of antibodies It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervaπable regions both in the light-chain and the heavy-chain variable domains The more highly conserved portions of variable domains are called the framework regions (FR) The variable domains of native heavy and light chains each comprise four FR regions largely adopting a β-sheet configuration connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the othei chain, contribute to the formation of the antigen-bindmg site of antibodies See, Kabat et al , NIH Publ No 91 3242, Vol I, pages 647-669 ( 1991 ) The constant domains are not involved directly in binding an antιbod\ 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 v ariable region of the intact antibody Examples of antibody fragments include Fab, Fab', F(ab')-,, 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 (CHI) of the heavy chain Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CHI domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine resιdue(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 ammo 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 ot immunoglobulins are well known
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population ot substantially homogeneous antibodies, i 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 ot 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 Kohlei 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 coπesponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. U.S. Patent No.4,816,567; Morrison etal, Proc. Nati. Acad. Sci. USA, 81; 6851-6855 (1984).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. 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 ofthe 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, Cuπ. Op. Struct. Biol. 2: 593-596 (1992). The humanized antibody includes a PRIMATIZED™ antibody wherein the antigen-binding region ofthe antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv see, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 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 Hollinger et al, Proc. Nati. Acad. Sci. USA. 90: 6444-6448 (1993).
An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues 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 other composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody The label may be detectable by itself (e g , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable Radionuchdes that can serve as detectable labels include, for example, 1-131, 1-123, 1-125, Y-90, Re-188, At-211 , 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 sihcones In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275,149 A "liposome" is a small vesicle composed of various types of hpids, phosphohpids and/or surfactant that is useful for delivery of a drug (such as the PRO polypeptide or antibodies thereto disclosed herein) to a mammal The components of the liposome are commonly aπanged in a bilayer formation similar to the hpid 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 amino acid sequence comprising at least the binding site of a receptor or a hgand 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 PRQ179. PR0238. PR0 64 PRQ844. PRQ846. PR01760. PRO205 PRQ321. PRQ333 PRO840 PRQ877. PRQ878. PRQ879. PRQ882 PRQ885 and PRQ887 Variants In addition to the full length natne sequence PR0179, PR0238, PR0364 PR0844, PR0846, PRO1760,
PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882 PR0885 and PR0887 polypeptides described herein, it is contemplated that PROl 79, PR0238, PR0364, PR0844 PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877 PR0878, PR0879, PR0882, PR0885 and PR0887 variants can be prepared PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840 PR0877, PR0878, PR0879, PR0882, PR0885 and PR0887 variants can be prepared by introducing appropriate nucleotide changes into the PROl 79, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 DNA, and/or by synthesis of the desired PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
Variations m the native full-length sequence PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887, or in various domains ofthe PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 that results in a change in the amino acid sequence of the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 as compared with the native sequence PRO 179, PR0238 PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879 PR0882, PR0885 or PR0887 Optionally the variation is by substitution of at least one am o acid with any other amino acid in one or more of the domains of the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333 PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 Guidance determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence ofthe PRO 179, PR0238 PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and or chemical properties, such as the replacement of a leucine with a serine, i e , conservative amino acid replacements Insertions or deletions may optionally be in the range of about 1 to 5 ammo acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of ammo 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,
Phe (F) leu, val, lie, ala, tyr leu Pro (P) ala ala
Ser (S) thr thr
Trp (W) tyr, phe tyr
Tyr (Y) trp, phe, thr, ser phe Val (V) lie, leu, met, phe, ala, norleucine leu
Substantial modifications in function or immunological identity ofthe PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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, lie,
(2) neutral hydrophihc 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 consen ative 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 alamne scanning, and PCR mutagenesis Site-directed mutagenesis [Carter et al Nucl Acids Res 13 4331 (1986), Zoller etal , Nucl Acids Res , 10 6487 ( 1987)], cassette mutagenesis [Wells et al , Gene, 34 31 (1985)] restriction selection mutagenesis [Wells et al Philos Trans R Soc London SerA, 317 41 ( 1986)1 or other known techniques can be performed on the cloned DNA to produce the PROl 79, PR0238, PR0364 PR0844, PR0846, PROl 760, PRO205, PR0321 PR0333 PRO840, PR0877, PR0878, PR0879 PR0882 PR0885 or PR0887 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 ammo acids Such ammo acids include alanme, glycine, serine, and cysteine Alanine is typically a prefeπed 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 lsoteπc amino acid can be used
B Modifications of PRQ179, PRQ238, PRQ364. PRQ844, PRQ846. PRO1760. PRO205. PRQ321 ,
PRQ333. PRO840. PRQ877. PRQ878. PRQ879. PRQ882. PRQ885 and PRQ887
Covalent modifications of PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 and PR0887 are included within the scope of this invention One type of covalent modification includes reacting targeted amino acid residues of a PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide with an organic deπvatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 Deπvatization with bifunctional agents is useful, for instance, for crosshnking PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 to a water-insoluble support matrix or surface for use in the method for purifying anti- PR0179, antι-PR0238, antι-PR0364, antι-PR0844, antι-PR0846, antι-PRO1760, antι-PRO205, antι-PR0321 , antι-PR0333, antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 orantι-PR0887 antibodies, and vice- versa Commonly used crosshnking agents include, e g J , 1 -bιs(dιazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azιdosalιcylιc acid homobifunctional lmidoesters, including disuccinimidyl esters such as 3,3'-dιthιobιs(succιnιmιdylpropιonate) bifunctional maleimides such as bιs-N-maleιmιdo-l ,8-octane and agents such as methyl-3-[(p-azιdophenyl)dιthιo]propιoιmιdate
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of prohne and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation ofthe α-amino groups of lysine, arginine and histidine side chains [T E Creighton, Proteins Structure and Molecular Properties. W H Freeman & Co San Francisco, pp 79-86 (1983)], acetylation of the N-terminal amine, and amidation ot any C-terminal carboxyl group
Another type of covalent modification of the PROl 79, PR0238, PR0364 PR0844 PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR088 oi PR0887 polypeptide included withm the scope of this invention comprises altering the native glycosylation pattern ot the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PROl 79, PR0238, PR0364, PR0844. PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840. PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 (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 PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887. 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 (for O-linked glycosylation sites). The PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205. PR0321. PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PRO 179, PR0238, PR0364. PR0844. PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al, Arch. Biochem. 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. Enzvmol.. 138:350 ( 1987).
Another type of covalent modification of PR0179, PR0238, PR0364. PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 comprises linking the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333. PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205. PR0321. PR0333, PRO840.
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 ofthe present invention may also be modified in a way to form a chimeric molecule comprising PR0179, PR0238, PR0364, PR0844, PR0846. PRO1760, PRO205, PR0321. PR0333, PRO840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887 fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 The presence of such epitope-tagged forms of the PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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-histidme- glycine (poly-His-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Biol , 8 2159-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al , Molecular and Cellular Biology. 5 3610 3616 (1985)], and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al , Protein Engineering. 3(6) 547-553 (1990)] Other tag polypeptides include the Flag peptide [Hopp et al , BioTechnology. 6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science, 255 192-194 (1992)], an α-tubuhn epitope peptide [Skinner et al , J Biol Chem . 266 15163-15166 (1991)], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Nati Acad Sci USA, 82 6393-6397 (1990)] In an alternative embodiment, the chimeric molecule may comprise a fusion of the PROl 79, PR0238,
PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 with an immunoglobulin or a particular region of an immunoglobulin For a bivalent form of the chimeric molecule (also referred to as an "lmmunoadhesin") such a fusion could be to the Fc region of an IgG molecule The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted oπnactιvated) formofa PRO179, PRO238, PRO364, PRO844, PRO846, PRO1760 PRO205, PRO321 PR0333, PRO840, PR0877, PR0878, PR0879 PR0882, PR0885 or PR0887 polypeptide in place of at least one variable region within an Ig molecule In a particularly preferred embodiment the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge CHI , CH2 and CH3 regions of an IgGl molecule For the production of immunoglobulin fusions see also, US Patent No 5,428,130 issued June 27, 1995
C Preparation of the PRO 179 PRQ238. PRQ364. PRQ844. PRQ846 PROl 760 PRO205 PRQ321.
PRQ333 PRO840. PRQ877 PRQ878 PRQ879. PRQ882. PRQ885 and PRQ887
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0179, PR0238, PR0364, PR0844 PR0846, PRO1760 PRO205, PRO321 , PRO333, PRO840, PRO877 PR0878, PR0879, PR0882 PR0885 or PR0887 In particular, cDNAs encoding PROl 79, PR0238, PR0 64 PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879 PR0882, PR0885 or PR0887 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below It is noted that proteins pioduced in separate expression rounds may be given different PRO numbei s but the UNQ number is unique tor 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 DNA16451 -1388, DNA35600-1 162, DNA47365-1206, DNA59838-1462, DNA44196-1353, DNA76532-1702, DNA30868, DNA34433, DNA41374, DNA53987, DNA58120, DNA58121 , DNA58122, DNA58125, DNA58128, or DNA58130, as well as all further native homologues and variants included in the foregoing definition of PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887, will be refeπed to as "PR0179", "PR0238", "PR0364", "PR0844", "PR0846", "PRO1760", "PRO205", "PR0321 ", "PR0333", "PRO840", "PR0877", "PR0878", "PR0879", "PR0882", "PR0885" or "PR0887", respectively, regardless of their origin or mode of preparation. The description below relates primarily to production of PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptides by culturing cells transformed or transfected with a vector containing nucleic acid encoding PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptides. It is, of course, contemplated that alternative methods that are well known in the art may be employed to prepare PROl 79. PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879. PR0882, PR0885 or PR0887. For instance, the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321, PR0333, PRO840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques. See, e.g. , Stewart etal, Solid-Phase Peptide Synthesis (W.H. Freeman Co.: San Francisco, CA, 1969); 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333. PRO840, PR0877, PR0878, PR0879, PR0882. PR0885 or PR0887 polypeptide.
i. Isolation of DNA Encoding PROl 79. PRQ238. PRQ364. PRQ844. PRQ846. PROl 760. PRQ205. PRQ321. PRQ333. PRO840. PRQ877. PRQ878. PRQ879. PRQ882. PRQ885 or PRQ887
DNA encoding PR0179, PR0238, PR0364, PR0844, PR0846. PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA encoding PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887 and to express it at a detectable level. Accordingly, DNAs encoding human PROl 79, human PR0238. human PR0364, human PR0844, human PR0846, human PRO 1760, human PRO205, human PR0321. human PR0333. human PRO840, human PR0877, human PR0878, human PR0879, human PR0882, human PR0885 or human PR0887 can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in the Examples. The gene encoding PRO 179. PR0238, PR0364, PR0844, PR0846, PRO 1760. PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879. PR0882, PR0885 or PR0887 polypeptide may also be obtained from a genomic library or by ohgonucleotide synthesis
Libraries can be screened with probes (such as antibodies to the PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 etal , supra An alternative means to isolate the gene encoding PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 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 ohgonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized The ohgonucleotide 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 31P-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 tor the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse transcribed into cDNA
π Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors descπbed herein tor PROl 79, PR0238 PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877 PR0878, PR0879 PR0882, PR0885 or PR0887 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 w ithout 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 Butlei , ed (IRL Press, 1991 ) and Sambrook et al , supia
Methods of transfection are known to the ordinarily skilled artisan, for example CaP0 treatment and electroporation Depending on the host cell used, transformation is performed using standaid techniques appropriate to such cells The calcium treatment employing calcium chloride, as described in Sambrook et al , supta, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers Infection with Aξt obacterium tumefaciens is used for transformation of certain plant cells, as descπbed by Shaw et al, Gene, 23: 315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 ( 1978) can be employed. General aspects of mammalian cell host system transformations have been described in U.S. Patent No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al , J. Bact.. 130: 946 (1977) and Hsiao et al, Proc. Nati. Acad. Sci. (USA). 76: 3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene or polyornithine, may also be used. For various techniques for transforming mammalian cells, see, Keown et al, Methods in Enzvmology, 185: 527-537 (1990) and Mansour et al, Nature. 336: 348-352 (1988). Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram- positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325); and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter. Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain 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 kari ; E. co// W31 10 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kari; E. coli W31 10 strain 40B4. which is strain 37D6 with a non- kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature.290: 140 [ 1981 ]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al, Bio/Technology. 9: 968-975 ( 1991 )) such as. e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourtefύ- .J. Bacteriol.J37 [1983]), K. fragilis (ATCC 12.424). K. bulgaricus (ATCC 16,045). K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosopl larum (ATCC 36,906; Van den Berg et al, Bio/Technology. 8: 135 (1990)), K . thermotolerans, and K. marxianus; yarrowia (EP 402,226): Pichia pastoris (EP 183,070; Sreekrishna et al, J. Basic Microbiol.. 28: 265-278 [ 1988]); Candida: Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al. Proc. Nati. Acad. Sci. USA, 76: 5259-5263 [ 1979]): Schwanniomyces such as Schwaniuoi ces occidentals (EP 394,538 published 31 October 1990), and filamentous fungi such as, e g Neurospora, Penicύlium, Tolypocladium (WO 91/00357 published 10 January 1991 ), and Aspergillus hosts such as A mdulans (Ballance et al , Biochem Biophys Res Commun , 1 12 284-289 [1983]. Tilburn et al . Gene. 26 205-221 ri983]. Yelton e/ a/ , Proc Nati Acad Sci USA, 81 1470-1474 [1984]) and A iger (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, Totulopsi , 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 Nati Acad Sci USA. 77 4216 (1980)), mouse sertoli cells (TM4, Mather, Biol Reprod . 23 243-251 (1980)), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), and mouse mammary tumor (MMT 060562, ATCC CCL51 ) The selection of the appropriate host cell is deemed to be within the skill in the art
in Selection and Use of a Rephcable Vector
The nucleic acid (e g , cDNA or genomic DNA) encoding PRO 179, PR0238 PR0364, PR0844, PR0846 PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 may be inserted into a rephcable vector for cloning (amplification ot the DNA) or for expression Various vectors are publicly available The vector may tor example, be in the form ot a plasmid cosmid. viral particle or phage The appropriate nucleic acid sequence may be inserted into the vector by a vaπety of procedures In general, DNA is inserted into an appropriate restriction endonuclease sιte(s) using techniques known in the art Vector components generally include, but are not limited to, one or more ot a signal sequence it the sequence is to be secreted, an origin of replication, one or more marker genes, an enhancei element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more ot these components employs standard hgation techniques that are known to the skilled artisan
The PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760 PRO205 PR0321 , PR0333 PRO840 PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 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 piotein or polypeptide In general, the signal sequence may be a component ofthe vector, oi it may be a part of the DNA encoding PRO 179, PR0238 PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840 PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887 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, penicilhnase lpp or heat-stable enterotoxin II leaders For yeast secretion the signal sequence may be, e g , the yeast invertase leader, alpha factor leadei (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 , ampicilhn, neomycm, methotrexate, or tetracychne, (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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887, 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 Nati 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 e/ α/ , Gene, 7 141 (1979). Tschemper et al , Gene, 10 157 (1980) The trp\ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No 44076 or PEP4-1 Jones, Genetics. 85 12 (1977) Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840 PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 to direct mRNA synthesis Promoters recognized by a variety of potential host cells are well known Promoters suitable tor 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 (trp) promoter system (Goeddel, Nucleic Acids Res . 8 4057 (1980) EP 36,776), and hybrid promoters such as the tac promoter deBoer et al . Proc Nati Acad Sci USA. 80 21-25 (1983) Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S D ) sequence operably linked to the DNA encoding PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333 PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 Examples of suitable promoting sequences for use with yeast hosts include the promoters tor 3- phosphoglycerate kinase (Hitzeman et al , J Biol Chem . 255 2073 ( 1980)) or other glycolytic enzymes (Hess et al , J Adv Enzyme Re .7 149 ( 1968), Holland, Biochemistry, V_ 4900 (1978)), such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase 3-phosphoglycerate mutase, pyruvate kinase, tπosephosphate isomerase, phosphoglucose isomerase and glucokinase
Other yeast promoters that are inducible promoters having the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization Suitable vectors and promoters for use in yeast expression are further described in EP 73,657
PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 nucleic acid transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, and Simian Virus 40 (SV40), by heterologous mammalian promoters, e g , the actin promoter or an immunoglobulin promoter, and by heat-shock promoters, provided such promoters are compatible with the host cell systems
Transcription of a DNA encoding the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) Typically, however, one will use an enhancer from a eukaryotic cell virus Examples include the SV40 enhancer on the late side 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 PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879 PR0882 PR0885 or PR0887, but is preferablv 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 ot transcription and for stabilizing the mRNA Such sequences are commonlv 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 ofthe mRNA encoding PRO 179, PR0238, PR0364 PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840 PR0877, PR0878. PR0879, PR0882, PR0885 or PR0887
Still other methods, vectors, and host cells suitable tor adaptation to the synthesis of PRO 179 PR0238, PR0364 PR0844, PR0846, PROl 760, PRO205, PR0321 PR0333, PRO840, PR0877, PR0878 PR0879, PR0882 PR0885 or PR0887 in recombinant vertebrate cell culture are described in Gething et a! , Nature 293 620-625 (1981 ), Mantei et al , Nature. 281 40-46 (1979) EP 1 17,060, and EP 1 17,058
iv Detecting Gene Amplification/Expression Gene amplification and/or expression may be measured in a sample directly, for example by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc Nati Acad Sci USA, 77 5201 -5205 (1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-prote 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 PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA encoding PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 and encoding a specific antibody epitope
v Purification of Polypeptide
Forms of PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321. PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell-lys g agents It may be desired to purify the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205 PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide from recombinant cell proteins oi polypeptides The following procedures are exemplary of suitable purification procedures by tractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, foi example, Sephadex G-75, protein A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope-tagged forms ofthe PROl 79, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 tor example, on the nature of the production process used and the particular PROl 79. PR0238, PR0364, PR0844. PR0846. PRO 1760, PRO205, PR0321 , PR0333, PRO840. PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 produced D Uses of the PRQ179. PRQ238, PRQ364. PRQ844. PRQ846. PRO1760, PRO205. PRQ321. PRQ333, PRO840. PRQ877. PRQ878, PRQ879. PRQ882. PRQ885 or PRQ887 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 endothelm 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 lodimzed endothelιn-1 binding in a receptor assay, an endothelm receptor binding assay testing for intact cell binding of radiolabeled endothelm- 1 using rabbit renal artery vascular smooth muscle cells, an inositol phosphate accumulation assay where functional activity is determined in Rat-1 cells by measuring intra-cellular levels of second messengers, an arachidonic acid release assay that measures the ability of added compounds to reduce endothehn-stimulated arachidonic acid release in cultured vascular smooth muscles, in vitro (isolated vessel) studies using endothehum 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 endothehum)
Assays for wound-healing activity include, for example, those described in Winter, Epidermal Wound Healing Maibach, HI and Rovee, DT, eds (Year Book Medical Publishers, Inc , Chicago), pp 71-1 12, as modified by the article of Eaglstein and Mertz, J Invest Dermatol , l± 382-384 (1978)
An assay to screen for a test molecule relating to a PRO polypeptide that binds an endothelm Bt (ETB,) receptor polypeptide and modulates signal transduction activity involves providing a host cell transformed with a DNA encoding endothelm B, receptor polypeptide, exposing the cells to the test candidate, and measuring endothelm B, receptoi signal transduction activity, as described, e g , in U S Pat No 5,773,223
There are several cardiac hypertrophy assays In vitro assays include induction of spreading of adult rat cardiac myocytes In this assay, ventricular myocytes are isolated from a single (male Sprague-Dawley) rat, essentially following a modification of the procedure described in detail bv Piper et al "Adult ventricular rat heart muscle cells" in Cell Culture Techniques in Heart and Vessel Research, H M Piper ed (Berlin Spπnger-Verlag 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 F,α (PGF^J have been shown to induce a spreading response in these adult cells The inhibition of myocyte spreading induced by PGF1α or PGF2c, analogs (e g fluprostenol) and phenylephπne by various potential inhibitors ot cardiac hypertrophy is then tested
One example of an in vno assay is a test for inhibiting cardiac hypertiophy 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 PGF2α) It is known that rats with pathologic cardiac hypertrophy induced by myocardial infarction have chronically elevated levels of extractable PGF,α in their myocardium Lai et al , Am J Physiol (Heart Circ Physiol ), 271 H2197 H2208 (1996) Accordingly , factors that can inhibit the effects of tlupiostenol 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 eight 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 w 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) aie treated under anesthesia, and the abdominal aorta of each rat is narrowed down just below the diaphragm Beznak M , Can J Biochem Physiol , 33 985-94 (1955) The aorta is exposed through a surgical incision, and a blunted needle is placed next to the vessel The aorta is constricted with a ligature of silk thread around the needle which is immediately removed and which reduces the lumen of the aorta to the diameter of the needle This approach is described, for example, in Rossi et al , Am Heart J . 124 700-709 (1992) and O'Rourke and Reibel, P S E M B . 200 95-100 (1992)
In yet another in vivo assay, the effect on cardiac hypertrophy following experimentally induced myocardial infarction (MI) is measured Acute MI is induced in rats by left coronary artery 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 etal , supi a 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 g , subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e g colon cancer cells implanted in colonic tissue See, e g , PCT publication No WO 97/33551 published September 18, 1997 Probably the most often used animal species in oncological studies are immunodeficient mice and, in particular, nude mice The observation that the nude mouse with thymic hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose The autoso al recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse including, for example, ASW, A/He, AKR BALB/c, B10 LP, C 17, C3H, C57BL, C57 CBA, DBA DDDJ/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 PiessJnc , 1991 ) The cells introduced into such animals can be derived from known tumor/cancei cell lines, such as any of the above listed tumor cell lines, and, for example, the B 104-1 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene), /αs-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 Karmah et al , Br J Cancer. 48 689-696 (1983)
Tumor cells can be introduced into animals such as nude mice by a variety of procedures The subcutaneous (s c ) space in mice is very suitable for tumor implantation Tumors can be transplanted s c as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions For solid-block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s c space Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously Tumor cells can also be injected as subdermal implants In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s c tissue
Animal models of breast cancer can be generated, for example, by implanting rat neuroblastoma cells (from which the neu oncogene was initially isolated), or raeα-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 etal , Cancer Research. 54 4726-4728 (1994) and Too et al , Cancer Research. 55 681-684 (1995) This model is based on the so-called "METAMOUSE™" sold by AntiCancer, Inc , (San Diego, California)
Tumors that arise in animals can be removed and cultured in vitw 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 aie 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 Palladino et al , J Immunol . 138 4023-4032 (1987) Briefly, tumor cells are propagated in vitro in cell culture Prior to injection into the animals the cell lines are washed and suspended in buffer, at a cell density of about 10x 10'' to 10xl 07 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 investigational tumor model Efficacy 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 tumoi 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 cahper in two or three dimensions The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula However, the measurement of tumor size is very inaccurate The therapeutic effects of a drug candidate can be better described as treatment-induced growth delay and specific growth delay Another important variable in the description of tumor growth is the tumor volume doubling time Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc 6th Int Workshop on Immune-Deficient Animals, Wu and Sheng eds (BaselJ 989), 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 morphometπc method and flow cytometπc analysis
Further, recombinant (transgemc) animal models can be engineered by introducing the coding portion of the PRO gene identified herein into the genome of animals of interest, using standard techniques for producing transgemc animals Animals that can serve as a target for transgemc 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 micro jection (U S Patent No 4,873,191), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al , Proc Nati Acad Sci USA. 82 6148-615 (1985)), gene targeting in embryonic stem cells (Thompson etal , Cell.56 313-321 ( 1989)), electroporation of embryos (Lo, Mol Cell Biol .3 1803- 1814 ( 1983)), and sperm-mediated gene transfer Lavitrano et al , Cell, 57 717-73 (1989) For a review, see for example, U S Patent No 4,736,866
For the purpose of the present invention, transge c 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 etal , Proc Nati Acad Sci USA, 89 6232-636 (1992) The expression ofthe transgene in transgemc 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 PRO polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the PRO polypeptide and altered genomic DNA encoding the same polypeptide intioduced into an embryonic cell of the animal For example, cDNA encoding a particular PRO polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques A portion of the genomic DNA encoding a particular PRO polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vectoi See, e g , Thomas and Capecchi, Cell 5j_ 503 ( 1987) for a description of homologous recombination vectors The vector is introduced into an embryonic stem cell line (e g , bv 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 Teratocarc omas 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 PRO 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 subhngual oral squamous cell tumors are excluded from the study The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor, the animals may not be able to feed themselves Each cat is treated repeatedly, over a longer period of time Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck After treatment each cat undergoes another CT scan CT scans and thoracic radiograms are evaluated every 8 weeks thereafter The data are evaluated for differences in survival, response, and toxicity as compared to control groups Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span
In addition, other spontaneous animal tumors, such as fibrosarcoma, adenocarcinoma, lymphoma, chondroma, or leiomyosarcoma of dogs, cats, and baboons can also be tested Of these mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those m humans However, the use of this model is limited by the rare occurrence of this type of tumor in animals
Other in vitto 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 ot mRNA (Thomas, Proc Nati Acad Sci USA.77 5201 5205 ( 1980)), dot blotting (DNA analysis) or in situ hybi idization, using an appropriately labeled probe, based on the sequences provided herein Alternativeh 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 lmmunohistochemical staining of 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 PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO DNA and encoding a specific antibody epitope General techniques for generating antibodies, and special protocols for in situ hybridization are provided hereinbelow
m Antibody Binding Studies The results of the cardiovascular, endothelial, and angiogenic study can be further verified by antibody binding studies, in which the ability of anti-PRO antibodies to inhibit the effect ofthe PRO polypeptides on endothelial cells or other cells used in the cardiovascular, endothelial, and angiogenic assays is tested Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and lmmunoprecipitation 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-immunoglobuhn antibody that is labeled with a detectable moiety (indirect sandwich assay ) For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme
For immunohistochemistry, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example
iv Cell-Based Tumor Assays
Cell-based assays and animal models for cardiovascular, endothelial, and angiogenic disorders, such as tumoi s 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 , tumoi cells, can be tested by using cells or cells lines that have been identified as being stimulated or inhibited by the PRO polypeptide herein Such cells include, for example, those set forth in the Examples below
In a different approach, cells of a cell type known to be involved in a particular cardiovascular, endothelial, and angiogenic disorder are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessiv e 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 B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and /as-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 transgemc 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 transge c animals are well known in the art See, e g , Small et al , Mol Cell Biol . 5 642-648 (1985)
v Gene Therapy The PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846. PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide is required i e , the site of synthesis of the PRO 179, PR0238, PR0364, PR0844, PR0846. PRO 1760, PRO205, PR0321 , PR0333, PRO840. PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide, if known, and the site (e g , wound) where biological activity of PR0179, PR0238, PR0364, PR0844 PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840 PR0877, PR0878, PR0879, PR0882 PR0885 orPR0887 polypeptide is needed For ew o treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient (see, e 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 vitio, or transferred in o in the cells of the intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in xitio include the use of liposomes, electroporation, micro jection, 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 prefeπed in vivo nucleic acid transfer techniques include transtection with viral or non-viral vectors (such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) and hpid-based systems (useful hpids for hpid-mediated transfer ot the gene are, for example, DOTMA, DOPE, and DC-Choi, see, e g , Tonk son 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide from a host cell in which it is placed Preferably the signal sequence for this purpose is a mammalian signal sequence, most preferably the native signal sequence for the PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 hgand for a receptor on the target cell, etc Where liposomes are employed, proteins that bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e 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 ot receptor-mediated endocytosis is described, for example, by Wu et al , J Biol Chem . 262 4429-4432 (1987), and W agner et al Proc Nati Acad Sci USA, 82 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 PRO 179 PR0238 PR0364, PR0844, PR0846 PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877 PR0878 PR0879, PR0882, PR0885 or PR0887 polypeptide as a diagnostic Detection of a mutated form of the PRO 179 PR0238 PR0364, PR0844 PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 polypeptide will allow a diagnosis of a cardiovascular, endothelial and angiogenic disease or a susceptibility to a cardiovascular, endothelial, and angiogenic disease, such as a tumoi , since mutations in the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 PR0333 PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may cause tumors
Individuals carrying mutations in the genes encoding a human PRO 179, human PR0238, human PR0364, human PR0844, human PR0846, human PRO] 760, human PRO205, human PR0321 , human PR0333, human PRO840, human PR0877, human PR0878, human PR0879, human PR0882, human PR0885 or human PR0887 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 purpose As an example, PCR pπmers complementary to the nucleic acid encoding the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide can be used to identify and analyze PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide, or alternatively, radiolabeled antisense DNA sequences encoding the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 S I protection or the chemical cleavage method, for example, Cotton et al , Proc Nati Acad Sci USA, 85 4397-4401 (1985)
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage direct DNA sequencing, or the use ot restriction enzymes, e g restriction fragment length polymorphisms (RFLP) and Southern blotting of genomic DNA
vn 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 neovascularization associated with tumor formation, since an altered level of this PRO polypeptide may be indicative of such disorders.
A competition assay may be employed wherein antibodies specific to the PRO polypeptide are attached to a solid support and the labeled PRO polypeptide and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of the PRO polypeptide in the sample.
viii. 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 cuπent need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis for the 3'- untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene coπesponding 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 ohgonucleotide 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 PROl 79, PR0238, PR0364, PR0844, PR0846. PRO 1760, PRO205, PR0321 , PR0333, PRO840. PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region is then identified through linkage analysis (coinheritance of physically adjacent genes). Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease
With cuπent 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)
ιx Screening Assays for Drug Candidates
This invention encompasses methods of screening compounds to identify those that mimic the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide (agonists) or prevent the effect of the PROl 79, PR0238, PR0364 PR0844, PR0846, PRO 1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide (antagonists) Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates
The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art All assays for antagonists are common in that they call for contacting the drug candidate with a PRO 179,
PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877 PR0878, PR0879 PR0882, PR0885 or PR0887 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 oi detected in the reaction mixture In a particular embodiment, the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760 PRO205, PR0321. PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO 1760 PRO205. PR0321 , PR0333 PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide and drying Alternatively, an immobilized antibody, e g , a monoclonal antibody, specific for the PRO 179 PR0238 PR0364, PR0844 PR0846, PRO1760, PRO205, PR0321 , PR0333 PRO840, PR0877, PR0878. PR0879 PR0882, PR0885 or PR0887 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 suiface 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-immobihzed component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex
If the candidate compound interacts with but does not bind to a particular PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 etal , Proc Nati Acad Sci USA. 88 9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc Nati Acad Sci USA. 89 5789-5793 (1991 ) Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription- activation domain The yeast expression system described in the foregoing publications (generally refeπed 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-actιvated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions
Compounds that interfere with the interaction of a gene encoding a PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878. PR0879, PR0882, PR0885 or PR0887 polypeptide identified herein and other mtra- 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 reactιon(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 ot 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 H-thymidine and harvested onto glass fiber filters (phD, Cambridge Technology, Watertown, MA) Mean ' H- thymidine incorporation (cpm) of triplicate cultures is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA) Significant 3 (H) thymidine incorporation indicates stimulation of endothelial cell proliferation
To assay for antagonists, the assay described above is performed, however, in this assay the PRO polypeptide is added along with the compound to be screened and the ability of the compound to inhibit 3 (H)thymιdιne incorporation 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 Cohgan 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 lodination or inclusion of a recognition site for a site-specific protein kinase Following fixation and incubation, the slides are subjected to autoradiographic analysis Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor
As an alternative approach for receptor identification, the labeled PRO polypeptide can be photoaffinity- hnked 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 ohgonucleotide 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, tπple-hehx molecules, etc , that inhibit the expression and/or activity of the target gene product
More specific examples of potential antagonists include an ohgonucleotide 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 ldiotypic 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 tπple-hehx formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature PRO polypeptides herein, is used to design an antisense RNA ohgonucleotide of from about 10 to 40 base pairs in length A DNA ohgonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see, Lee etal , 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 ofthe PRO polypeptide The antisense RNA ohgonucleotide 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, oligodeoxyπbonucleotides derived from the translation-initiation site, e g , between about -10 and +10 positions of the target gene nucleotide sequence, are preferred
Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases 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 activ ity of the PRO polypeptide Examples of small molecules include, but are not limited to, small peptides or peptide-hke 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 turthei details see, e g , Rossi Current Biology. 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18 1997)
Nucleic acid molecules in tπple-hehx formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides The base composition of these oligonucleotides is designed such that it promotes tπple-hehx formation via Hoogsteen base-pairing rules, which generally require sizeable stretches ot purines or pyπmidines on one strand of a duplex For further details see, e g , PCT publication No WO 97/33551 supia
These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other scieemng techniques well known for those skilled in the art x Types of Cardiovascular, Endothelial. and Angiogenic Disorders to be Treated
The PRO polypeptides, or agonists or antagonists thereto, that have activity in the cardiovascular, angiogenic, and endothelial assays described herein, and/or whose gene product has been found to be localized to the cardiovascular system, are likely to have therapeutic uses in a variety of cardiovascular, endothelial, and angiogenic disorders including systemic disorders that affect vessels, such as diabetes melhtus 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 lschemic 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 cardiovascularization, genesis of endothelial cells or angiogenesis or inhibits these conditions For example, if the molecule promotes angiogenesis, an antagonist thereof would be useful for treatment of disordei s where it is desired to limit or prevent angiogenesis Examples of such disorders include vascular tumors such as haemangioma, tumor angiogenesis, neovascularization in the retina choroid or cornea associated with diabetic retinopathy or premature infant ret opathy or macular degeneration and proliferative vitreoretinopathy, rheumatoid arthritis Crohn s disease, atherosclerosis, ovarian hyperstimulation, psoriasis endometπosis associated with neovascularization, restenosis subsequent to balloon angioplasty, scar tissue overproduction, for example, that seen in a keloid that forms after surgery, fibrosis after myocardial infarction, or fibrotic lesions associated with pulmonary tibrosis
If, however, the molecule inhibits angiogenesis, it would be expected to be used directly tor 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 vascuhtides, 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 mtimal 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 vascuhtides include giant cell arteπtis, Takayasu's arteπtis, polyarteπtis nodosa (including the microangiopathic form), Kawasaki's disease, microscopic polyangiitis, Wegener's granulomatosis, and a vaπety 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 abnoi mal prohtei ation and growth of cellular elements of the vascular system For example, lymphangiomas are benign tumors ot the lymphatic system that are congenital, often cystic, malformations of the lymphatics that usually occui in newborns Cystic tumors tend to grow into the adjacent tissue Cystic tumors usually occur in the cervical and axillary legion 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 Gπener et al , Lymphology. 4 140-144 (1971 )
Another use for the PRO polypeptides herein or antagonists thereto is in the prevention of tumor angiogenesis, which involves vasculaπzation 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, aπhenoblastomas, cervical carcinomas, endometπal carcinoma, endometπal hyperplasia, endometπosis, fibrosarcomas, choπocarcinoma, 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, oligodendroghoma, medulloblastoma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma, abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome
Age-related macular degeneration (AMD) is a leading cause of severe visual loss in the elderly population The exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment Because choroidal neovascularization is associated with a dramatic worsening in prognosis, the PRO polypeptide or 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 no\ o 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 oi regeneration of other tissues, such as organs (including, for example, pancreas, liver intestine, kidney, skin, oi endothehum), muscle (smooth, skeletal or cardiac), and vascular (including vascular endothehum) tissue or foi 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 precursoi tissues oi cells, or for inhibiting the growth of tissues described above
A PRO polypeptide or antagonist thereto may also be used in the treatment of peπodontal 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/hgament-hke 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/hgament-hke 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 othei 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, carpal tunnel syndrome, and other tendon or ligament defects The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art
The PRO polypeptide or its antagonist may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, ; e , for the treatment of central and peripheral nervous system disease and neuropathies as well as mechanical and traumatic disorders, that involve degeneration, death, 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 injuiies, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndiome 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-repertusion injury
Rheumatoid arthritis is a further indication Blood vessel growth and targeting ot inflammatory cells through the vasculature is an important component in the pathogenesis of rheumatoid and seio-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 ot 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 atπal fibrillation, which develops in a substantial portion of patients diagnosed with hypertrophic cardiomyopathy
Further indications include angina, myocardial infarctions such as acute myocardial infarctions, and heart failure such as congestive heart failure Additional non-neoplastic conditions include psoriasis, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, peπcardial 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 earners, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate citrate, and other organic acids, antioxidants including ascorbic acid and methionme, preservatives (such as octadecy ldimethylbenzyl 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, hydrophihc polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine asparagine, histidine, arginine, or lysine, monosacchaπdes, disacchaπdes, and other carbohydrates including glucose, mannose, ordextπns, chelating agents such as EDTA, sugars such as sucrose manmtol, trehalose or sorbitol, salt-forming counter-ions such as sodium, metal complexes (e g , Zn-protein complexes), and/or non- lo c 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 protamme sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts colloidal silica, magnesium tπsihcate, poly vinyl pyrrohdone, cellulose-based substances, and polyethylene glvcol Carriers for topical or gel-based forms of antagonist include polysacchaπdes such as sodium caiboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols For all admimsttations, conventional depot forms are suitably used Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, subhngual tablets, and sustained-release preparations The PRO polypeptides or agonists or antagonists will typically be formulated in such vehicles at a concentration of about 0 1 mg/ml to 100 mg/ml
Another formulation comprises incorporating a PRO polypeptide or 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 systemtcally 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 halide e g , chloride, and f) water
If the detergent employed is non-ionic, it may, for example, be polysorbates (e g , POLYSORBATE™
(TWEEN™) 20, 80, etc ) or 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 lsotomcity of a liquid composition of the PRO polypeptide or antagonist thereto, and includes polyhydπc sugar alcohols, preferably tπhydπc or higher sugar alcohols, such as glycerin, erythπtol, arabitol, xyhtol sorbitol, and manmtol These sugar alcohols can be used alone or in combination Alternatively, sodium chloride or other appropriate inorganic salts may be used to render the sol utions lsotonic
The buffer may, for example be an acetate, citrate succinate, or phosphate butter depending on the pH desired The pH of one type of liquid formulation of this invention is buffeted in the range of about 4 to 8, preferably about physiological pH
The preservatives phenol, benzvl 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 (1 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 . L5 167-277 ( 1981 ) and Langer, Chem Tech , Y2 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-glycohc acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde 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 CC, resulting in a loss of biological activity and possible changes in lmmunogemcity 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, lyophihzing 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 pei se DE 3,218, 121 , Epstein et al Proc Nati Acad Sci USA. 82 3688-3692 (1985), Hwang et al Proc Nati Acad Sci USA, 77 4030-4034 (1980), EP 52,322, EP 36,676, EP 88,046, EP 143,949, EP 142 641 , Japanese patent application 83-1 18008, U S Patent Nos 4,485,045 and 4,544,545, and EP 102,324 Ordinarily the liposomes are ot the small (about 200-800 Angstroms) unilamellar type in which the hpid 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, vaiy depending on such factors as the pathological condition to be treated (including pievention). the method ot administration, the type of compound being used for treatment, any co-therapy involved the patient s age, weight, general medical condition, medical history, etc , and its determination is well with the skill of a practicing physician Accordingly, it will be necessary for the therapist to titer the dosage and modify the route ot administration as required to obtain the maximal therapeutic effect It the PRO polypeptide has a naπow host range, tor 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 withm 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, pyrogemcity, 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 in the pharmaceutical composition For example, the addition of other known growth factors, such as IGF-I, to the final composition may also affect the dosage Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations, and tetracychne labeling
The route of PRO polypeptide or antagonist or agonist administration is in accord with known methods e g , by injection or infusion by intravenous, intramuscular, intracerebral, intraperitoneal, intracerobrospinal, subcutaneous, intraocular, intraarticular, intrasynovial, mtrathecal, 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, intralesional, or peπlesional routes, to exert local as well as systemic therapeutic effects The intraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors
If a peptide or small molecule is employed as an antagonist or agonist, it is preferably administered orally or non-orally in the form of a liquid or solid to mammals
Examples of pharmacologically acceptable salts of molecules that form salts and are useful hereunder include alkali metal salts (e g , sodium salt, potassium salt), alkaline earth metal salts (e g , calcium salt, magnesium salt), ammonium salts organic base salts (e g , pyridine salt, tπethylamine salt), inorganic acid salts (e g , hydrochloride, sulfate, nitrate), and salts of organic acid (e g , acetate, oxalate, p-toluenesulfonate) For compositions herein that are useful for bone, cartilage, tendon, or ligament regeneration the therapeutic method includes administering the composition topically systemically, or locally as an implant or dev ice 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 ot 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 biocompatibihty , biodegradability, mechanical properties, cosmetic appearance, and interface properties The particular application of the compositions will define the appropπate 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 glycohc 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 oi 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 catiomc salts of carboxymethylcellulose (CMC) Other prefeπed sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and poly(vιnyl alcohol) The amount of sequestering agent useful herein is 0 5-20 wt%, preferably 1 -10 wt%, based on total formulation weight, which represents the amount necessary to prevent desorption of the polypeptide (or its antagonist) from the polymer matπ x and to provide appropriate handling ofthe composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide (or its antagonist) the opportunity to assist the osteogenic activity of the progenitor cells
xπ Combination Therapies The effectiveness of the PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 ,
PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or an agonist or antagonist thereof in preventing or treating the disorder in question may be improved by administering the active agent serially or in combination with another agent that is effective for those purposes, either in the same composition or as separate compositions For example, for treatment of cardiac hypertrophy . PRO polypeptide therapy can be combined with the administration of inhibitors of known cardiac myocyte hypertrophy factors, e g , inhibitors of α-adrenergic agonists such as phenylephπne, endothelm- 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
For treatment of cardiac hypertrophy associated with hypertension, the PRO polypeptide can be administered in combination with β adrenergic receptor blocking agents, e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol, ACE inhibitors, e g , quinapπl, captopπl, enalapπl, ramipπl, benazepπl, fosmopπl, or lisinopπl, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, or mdapamide, 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), 5 Ith Edition
Preferred candidates for combination therapy m the treatment of hypertrophic cardiomyopathy are β- adrenergic-blocking drugs (e g , propranolol, timolol, tertalolol, carteolol, nadolol betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol), verapamil, difedipine, or diltiazem Treatment of hypertrophy associated with high blood pressure may require the use of antihypertensive drug therapy, using calcium channel blockers, e g , diltiazem, nifedipine, verapamil, or nicardipine, β-adrenergic blocking agents, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, or mdapamide, and or ACE-inhibitors, e g , quinapπl, captopπl, enalapπl, ramipπl, benazepπl, fosinopπl, or lisinopπl 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- or TGF-β, IGF, FGF, and CTGF
In addition, PRO polypeptides or their antagonists used to treat cancer mav be combined with cytotoxic, chemotherapeutic, or growth-inhibitory agents as identified above Al so, 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 hypei or hypotension, renal impairment, etc The dose will additionally depend on such factors as the type ot 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
xin Articles of Manufacture An article of manufacture such as a kit containing PRO 179, PR0238, PR0364 PR0844, PR0846, PRO 1760,
PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882 PR0885 or PR0887 polypeptide or agonists 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 v ials 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 PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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.
i. 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 intraperitoneal injections. The immunizing agent may include the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A or synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
ii. Monoclonal Antibodies The anti-PROl 79, anti-PR0238, anti-PR0364, anti-PR0844. anti-PR0846. anti-PRO 1760, anti-PRO205. anti-PR0321, anti-PR0333. anti-PRO840, anti-PR0877, anti-PR0878. anti-PR0879, anti-PR0882, anti-PR0885 or anti-PR0887 antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 ( 1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include the PROl 79, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell Goding, Monoclonal Antibodies Principles and Practice (New York Academic Press, 1986), pp 59-103 Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin Usually, rat or mouse myeloma cell lines are employed The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells For example, if the parental cells lack the enzyme hypoxanthine guamne 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
Prefeπed 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, tor instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies Kozbor, J Immunol , 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications (Marcel Dekker. Inc New York, 1987) pp 51-63
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 assavs are known in the art The binding affinity of the monoclonal antibody can, for example, be determined bv the Scatchaid analysis of Munson and Pollard, Anal Biochem . 107 220 (1980)
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods Goding, supra Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures 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 ohgonucleotide 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 prefeπed source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U S Patent No 4,816,567, Morrison et al , supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobuhn 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 chimeric bivalent antibody The antibodies may be monovalent antibodies Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosshnking Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosshnking In vitro methods are also suitable for preparing monovalent antibodies Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art
in Human and Humanized Antibodies
The antι-PR0179, antι-PR0238, antι-PR0364, antι-PR0844, antι-PR0846, antι-PRO1760. antι-PRO205, antι-PR0321, antι-PR0333, antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882 antι-PR0885 or antι-PR0887 antibodies may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobulins immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab 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 ofthe human immunoglobulin are replaced by corresponding non-human residues Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions coπespond 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 compi tse at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin Jones et l Nature 321 522 525 (1986), Riechmann et al , Nature. 332 323-329 (1988), Presta Curr Op Struct Biol 2 593 596 ( 1992) Methods for humanizing non-human antibodies are well known in the art Generally a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an ' import' v aπable 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 coπesponding sequences of a human antibody Accordingly, such "humanized" antibodies are chimeric antibodies (U S Patent No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the coπesponding 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 etal . Monoclonal Antibodies and Cancer Therapy. Alan R Lιss, p 77 (1985) and Boerner etal , J Immunol . 147(1 ) 86-95 (1991) Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgemc 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 reaπangement, 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, andιn thefollowιng scιentιfιc publιcatιons Marks etal . Bio/Technology. 10 779-783 (1992), Lonberg etal . Nature, 368 856-859 (1994), Morrison. Nature. 368 12- 1 (1 94) . Fishwild etal . Nature Biotechnology, |4 845-851 (1996), Neuberger. NatureBιotechnology. 14 826 (1996). Lonberg and Huszar. Intern Rev Immunol , __ 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 PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit Methods for making bispecific antibodies are known in the art Traditionally the recombinant production of bispecific antibodies is based on the co expression of two immunoglobulin heavy-chain/hght-cham pairs, wheie the two heavy chains have different specificities Milstein and Cuello. Natuie, 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 coπect bispecific structure The purification ofthe correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker etal , EMBO J , __ 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 pref eπed to have the first heavy-chain constant region (CHI ) containing the site necessary for light-chain binding present in at least one of the fusions DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co transfected into a suitable host organism For further details of generating bispecific antibodies, vee, for example, Suresh et al . Methods in Enzvmology. 121 210 (1986)
v Heteroconiugate Antibodies
Heteroconjugate antibodies are composed ot 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 crosshnking agents For example, immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include lminothiolate and methyl-4- mercaptobutyπmidate and those disclosed, for example, in U S Patent No 4,676,980
vi Effector Function Engineering
It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e g , the effectiveness of the antibody in treating cancer For example, cysteine resιdue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region The homodimeπc antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See, Caron et al , J Exp Med , 126 1 191 - 1 195 ( 1992) and Shopes, J Immunol , M8 2918-2922 ( 1992) Homodimeπc antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al , Cancer Research, 53 2560-2565 (1993) Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See, Stevenson et al , Anti-Cancer Drug Design, 3 219-230 (1989)
vn Immunoconi u gates
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 activ e fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeiuginosa), ricin A chain, abπn A chain, modeccin A chain, alpha-sarcm, Aleut ites fordu proteins, dianthin proteins, Plntolaca ameucana proteins (PAPI PAPII, and PAP-S), momordica charantia inhibitor, curcm, crotin sapaonaπa officinahs inhibitoi, gelo n mitogelhn, restπctocin, phenomycin, enomycin, and the tπcothecenes A v aπety of radionuchdes are available for the production of radioconjugated antibodies Examples include -1 Bi "'I π'In, l,Y, and I 6Re
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succιmmιdyl-3-(2-pyπdyldιthιol) propionate (SPDP) lminothiolane (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), dusocyanates (such as tolyene 2,6 diisocyanate), and bis- active fluorine compounds (such as 1.5 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 tπaminepentaacetic 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 g , a radionucleotide)
vni Immunohposomes
The antibodies disclosed herein may also be formulated as immunohposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein etal , Proc Nati Acad Sci USA. 82 3688 (1985), Hwang et al , Proc Nati Acad Sci USA. 77 4030 (1980), and U S Pat Nos 4,485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5,013,556
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a hpid composition comprising phosphatidylcholine, cholesterol, and PEG-deπvatized phosphatidylethanolamme (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-mterchange 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)
ιx Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760 PRO205, PR0321 , PR0333. PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbetore, can be administered for the treatment of various disorders as noted above and below in the form of pharmaceutical compositions If the PROl 79, PR0238, PR0364, PR0844. PR0846 PROl 760, PRO205, PR0321 , PR0333 PRO840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred However, hpofections 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/oi produced by recombinant DNA technology See, e g , Marasco et al , Proc Nati Acad Sci USA 90 7889-7893 ( 1993)
The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each othei Alternatively , or in addition, the composition may comprise an agent that enhances its function, such as. for example a cytotoxic agent, cytokme, chemotherapeutic agent, or growth-inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interf acial 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(vιnylalcohol)), polylactides (U S Pat No 3,773,919), copolymers of L-glutamic acid and γ ethyl-L glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid glycohc acid copolymer and leuprohde acetate), and poly-D-(-)-3-hydroxybutyπc acid While polymers such as ethylene-vinyl acetate and lactic acid glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 CC, resulting in a loss of biological activity and possible changes in lmmunogenicity 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, lyophihzing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matnx compositions
x Methods of Treatment using the Antibody
It is contemplated that the antibodies to a PR0179, PR0238, PR0364, PR0844 PR0846, PROl 760 PRO205, PR0321 , PR0333, PRO840, PR0877 PR0878, PR0879, PR0882, PR0885 or PR0887 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, lntracerobrosp al, subcutaneous, intra-articular, intrasynovial, mtrathecal 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 tor such chemotherapeutic agents may be used according to manufacturers instructions or as determined empirically by the skilled practitionei 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 onapπstone (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, 01 VEGF receptor(s) These also include the agents set forth above Also, the antibody is suitably administered serially or combination with radiological treatments, whether involving lπadiation 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 coadministered 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, vasculaπzation 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, antι-HER2 antibody, hereguhn. anti-hereguhn antibody, D-factor, interleukin- 1 (IL-1), ιnterleukιn-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 purposes previous therapy, the patient s clinical history and response to the antibody, and the discretion ot the attending physician The antibody is suitably administered to the patient at one time or over a series of treatments
For example, depending on the type and severity of the disorder, about 1 g/kg to 50 mg/kg (e g , 0 1 -20 mg/kg) of antibody is an initial candidate dosage foi administration to the patient whethei for example, by one or more separate administrations, or by continuous infusion A typical daily or weekly dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above For repeated administrations over several days or longer depending on the condition, the treatment is repeated or sustained until a desired suppression of disorder symptoms occurs However, other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging
xi Articles of Manufacture with Antibodies
An article of manufacture containing a container with the antibody and a label is also provided Such articles are described above, wherein the active agent is an anti-PROl 79, antι-PR0238, antι-PR0364, antι-PR0844, anti- PR0846, antι-PRO1760, antι-PRO205, antι-PR0321 , antι-PR0333, antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 or antι-PR0887 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 g , 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 immunofluorescence or immunoelectron microscopy For this purpose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample This procedure also allows for determining the distribution of 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 purposes 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 incorporated by reference in their entirety
EXAMPLES
Commercially av ailable 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 heremabove 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)Jnnιs /α/ , PCR Protocols A Guide to Methods and Applications (Academic Press, Inc N Y , 1990), Harlow et al , Antibodies A Laboratory Manual (Cold Spring Harbor Press Cold Spring Harbor, 1988). Gait. Ohgonucleotide Synthesis (IRL Press Oxford, 1984), Freshnev. Animal Cell Culture, 1987, Cohgan etal , Current Protocols in Immunology, 1991
EXAMPLE 1 Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNA Encoding Therefor The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases The EST databases included public databases (e g , GenBank), and proprietary databases (e g , LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) The search was performed using the computer program BLAST or BLAST-2 [Altschul etal , 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 with a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington Seattle, Washington)
Using this extracellular domain homology screen, consensus DNA sequences were assembled relative to other identified EST sequences using phrap In addition, the consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above Based upon the consensus sequences obtained as described above, oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length The probe sequences are typically 40-55 bp in length In some cases additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp In order to screen several libraries for a full-length clone DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supia with the PCR pnmer pair A positive library was then used to isolate clones encoding the gene of interest using the probe ohgonucleotide and one of the primer pairs
The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen San Diego, CA The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site see, Holmes et al , Science 253 1278 1280 (1991 )) in the unique Xhol and Notl sites EXAMPLE 2 Isolation of cDNA clones bv Amylase Screening 1 Preparation of oligo dT primed cDNA library mRNA was isolated from a human tissue of interest using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2) This RNA was used to generate an oligo dT primed cDNA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System) In this procedure, the double stranded cDNA was sized to greater than 1000 bp and the Sall/Notl linkered cDNA was cloned into Xhol/Notl cleaved vector pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restriction enzyme site preceding the Xhol/Notl cDNA cloning sites
2 Preparation of random primed cDNA library
A secondary cDNA library was generated in order to preferentially represent the 5' ends of the primary cDNA clones Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY 0 using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above) In this procedure the double stranded cDNA was sized to 500- 1000 bp, linkered with blunt to Notl adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector pSST- AMY 0 is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites Thus, cDNAs cloned into this vector that are fused in frame with am ny\lase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies
3 Transformation and Detection
DNA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml) The bacteria and vector mixture was then electroporated as recommended by the manufacturer Subsequently. SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37 °C for 30 minutes The transformants were then plated onto 20 standard 150 mm LB plates containing ampicilhn and incubated for 16 hours (37°C) Positive colonies were scraped oft the plates and the DNA was isolated from the bacterial pellet using standard protocols, e g , CsCl-gradient The purified DNA was then earned on to the yeast protocols below
The yeast methods were divided into three categories (1 ) Transformation of yeast with the plasmid/cDNA combined vector, (2) Detection and isolation of yeast clones secreting amylase, and (3) PCR amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis
The yeast strain used was HD56-5A (ATCC-90785) This strain has the following genotype MAT alpha, ura3-52 leu2-3, leu2-l 12, hιs3-l 1 , hιs3-15, MAL+, SUC+, GAL+ Preferably, yeast mutants can be employed that have deficient post-translational pathways Such mutants may have translocation deficient alleles in vec71 , vet72, sec62. with truncated sec71 being most preferred Alternatively, antagonists (including antisense nucleotides and/oi ligands) which interfere with the normal operation of these genes, other proteins implicated in this post translation pathw ay (e g , SECόlp, SEC72p, SEC62p, SEC63p, TDJlp or SSA lp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressmg yeast Transformation was performed based on the protocol outlined by Gietz et al , Nucl Acid Res , 20 1425 (1992) Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and grown overnight at 30 °C The YEPD broth was prepared as described in Kaiser et al , Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994) The overnight culture was then diluted to about 2 x 106 cells/ml (approx ODwm=0 1 ) into fresh YEPD broth (500 ml) and regrown to 1 x 107 cells/ml (approx ODax O 4-0.5)
The cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tπs-HCl, 1 mM EDTA pH 7 5, 100 mM Lι2OOCCH3), and resuspended into LiAc/TE (2 5 ml)
Transformation took place by mixing the prepared cells ( 100 μ 1) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 μg, vol < 10 μl) in microfuge tubes The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tπs-HCl, 1 mM EDTA, 100 mM Lι2OOCCH3, pH 7 5) was added This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes The cells were then heat shocked at 42°C for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 μl, 10 mM Tπs-HCl, 1 mM EDTA pH 7 5) followed by recentπfugation The cells were then diluted into TE (1 ml) and ahquots (200 μl) were spread onto the selective media previously prepared in 150 mm growth plates (VWR) Alternatively, instead of multiple small reactions, the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly
The selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al , Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p 208- 210 (1994) Transformants were grown at 30 °C for 2-3 days The detection of colonies secreting amylase was performed by including red starch in the selective growth media Starch was coupled to the red dye (Reactive Red- 120, Sigma) as per the procedure described by Biely et al , Anal Biochem . 172 176-179 (1988) The coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0 15% (w/v), and was buffered with potassium phosphate to a pH of 7 0 (50-100 mM final concentration) The positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies Well isolated single colonies positive for amylase secretion were detected by direct incorporation ot red starch into buffered SCD-Ura agar Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly
4 Isolation of DNA by PCR Amplification When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile watei
(30 μl) in a 96 well plate At this time, the positive colonies were either frozen and stored for subsequent analysis or immediately amplified An aliquot of cells (5 μl) was used as a template for the PCR reaction in a 25 u\ volume containing 0 5 μl Klentaq (Clontech Palo Alto, CA), 4 Oμl l O mM dNTP s (Perkin Elmer-Cetus), 2 5 μl Klentaq buffer (Clontech), 0 25 μl forward oligo 1 , 0 25 μl reverse oligo 2. 12 5 μl distilled water The sequence of the forward ohgonucleotide 1 was
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID NO 33)
The sequence of the reverse ohgonucleotide 2 was
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID NO 34)
PCR was then performed as follows a Denature 92°C, 5 minutes b 3 cycles of Denature 92°C, 30 seconds
Anneal 59 °C, 30 seconds
Extend 72°C, 60 seconds c 3 cycles of Denature 92°C, 30 seconds
Anneal 57°C, 30 seconds
Extend 72°C, 60 seconds d 25 cycles of Denature 92°C, 30 seconds
Anneal 55°C, 30 seconds
Extend 72°C, 60 seconds
Hold 4°C
The underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY 0 when no insert was present Typically, the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers
Thus, the total product of the PCR reaction from an empty vector was 343 bp However, signal sequence-fused cDNA resulted in considerably longer nucleotide sequences
Following the PCR, an aliquot of the reaction (5 μl) was examined by agarose gel electrophoresis in a 1 % agarose gel using a Tπs-Borate-EDTA (TBE) buffering system as described by Sambrook et al , supra Clones resulting in a single stiong PCR product larger than 400 bp weie further analyzed by DNA sequencing after purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc . Chatsworth CA)
EXAMPLE 3
Isolation of cDNA Clones Using Signal Algorithm Analysis
Various polypeptide-encoding nucleic acid sequences were identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc , (South San Francisco. CA) upon ESTs as well as clustered and assembled EST fragments from public (e g GenBank) and or private (LIFESEQ®, Incyte
Pharmaceuticals, Inc , Palo Alto, CA) databases The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides suπounding the first and optionally the second methionme codon(s)
(ATG) at the 5 -end of the sequence or sequence fragment under consideration The nucleotides following the first ATG must code for at least 35 unambiguous ammo acids without any stop codons It the first ATG has the required amino acids, the second is not examined If neither meets the requirement the candidate sequence is not scored
In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation paiameters) known to be associated with secretion signals Use of this algorithm resulted in the identification of numerous polypeptide-encoding nucleic acid sequences
EXAMPLE 4
Isolation of cDNA Clones Encoding Human PRO 179 A cDNA sequence was isolated in a screen as described in Example 2 above The cDNAsequence isolated in the above screen was found, by BLAST and FastA sequence alignment, to have sequence homology to a nucleotide sequence encoding various angiopoietin proteins This cDNA sequence is herein designated DNA 10028 and/or DNA25250
Based on the sequence homology, ohgonucleotide probes were then generated from the sequence of the DNA10028 molecule and used to screen a human fetal liver (LIB6) library prepared as described in paragraph 1 of Example 2 above The cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the
Sfil site, see, Holmes etal , Science. 253 1278-1280 (1991)) in the unique Xhol and Notl sites, and the cDNA size cut was less than 2800 bp
DNA sequencing of the clones isolated as described above gave the full length DNA sequence for PRO 179 and the derived protein sequence for PROl 79 The entire nucleotide sequence of DNA16451-1388 is shown in Figure 1 (SEQ ID NO 1) Clone DNA16451 -1388 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 37-39 and ending at the stop codon at nucleotide positions 1417- 1419 (Figure 1 ) The predicted polypeptide precursor is 460 amino acids long (Figure 2, SEQ ID NO 2) The full length PRO 179 protein shown in Figure 2 has an estimated molecular weight of about 53,637 daltons and a pi of about 661
Analysis of the full-length PRO 179 sequence shown in Figure 2 (SEQ ID NO 2) evidences the presence ot a variety 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 PROl 79 sequence (Figure 2, SEQ ID NO 2), evidences the presence ofthe following a signal peptide from about amino acid 1 to about amino acid 16, leucine zipper patterns from about amino acid 120 to about amino acid 142 and from about amino acid 127 to about amino acid 149, N-glycosylation sites from about amino acid 23 to about amino acid 27, from about amino acid 1 15 to about amino acid 1 19, from about amino acid 296 to about amino acid 300, and from about amino acid 357 to about amino acid 361 , and fibπnogen beta and gamma chains C-terminal domains from about amino acid 271 to about amino acid 310, from about amino acid 312 to about amino acid 322 from about amino acid 331 to about amino acid 369, and from about amino acid 393 to about amino acid 424
Clone DNA16451-1388 has been deposited with the ATCC on April 14, 1998 and is assigned ATCC deposit no 209776 Regarding the sequence, it is understood that the deposited clone contains the correct sequence and the sequences provided herein are based on known sequencing techniques
Analysis of the amino acid sequence of the full-length PRO 179 polypeptide suggests that it possesses significant similarity to the angiopoietin family of proteins, thereby indicating that PRO 179 may be a novel angiopoietin family member More specifically, an analysis ofthe Dayhoff database (version 35 45 SwissPiot 35) evidenced significant homology between the PRO 179 amino acid sequence and the following Dayhoff sequences
AF004326J P_R94605, HSU83508 1 , P_R94603, P R94317 AF025818_1 , HSY 16132_1 , P_R65760 137391 and HUMRSC192_l
EXAMPLE 5 Isolation of cDNA Clones Encoding Human PRQ238 A consensus DNA sequence was assembled relative to other EST sequences using phrap as descπbed in Example 1 above This consensus sequence is herein designated DNA30908 Based on the DNA30908 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 PR0238 PCR primers (forward and reverse) were synthesized based upon the DNA30908 sequence Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA30908 sequence In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supia, with the PCR primer pair A positive library was then used to isolate clones encoding the PR0238 gene using the probe ohgonucleotide and one of the PCR primers
The ohgonucleotide sequences used in the above procedure were the following forward PCR primer 1
5'-GGTGCTAAACTGGTGCTCTGTGGC-3' (SEQ ID NO 35) forward PCR primer 2
5'-CAGGGCAAGATGAGCATTCC-3' (SEQ ID NO 36) reverse PCR primer 5'-TCATACTGTTCCATCTCGGCACGC-3' (SEQ ID NO 37) hybridization probe 5'-AATGGTGGGGCCCTAGAAGAGCTCATCAGAGAACTCACCGCTTCTCATGC-3* (SEQ ID NO 38)
RNA for construction of the cDNA libraries was isolated from human fetal liver tissue The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with 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 ot 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 tor PR0238 [herein designated as DNA35600-1 162] (Figure 3, SEQ ID NO 3) and the deπved protein sequence for PR0238
The entire nucleotide sequence of DNA35600- 1 162 is shown in Figure 3 (SEQ ID NO 3) Clone DNA35600-
1 162 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 134-
136 and ending at the stop codon at nucleotide positions 1064- 1066 (Figure 3) The predicted polypeptide precursor is 310 amino acids long (Figure 4, SEQ ID NO 4), and has an estimated molecular weight ot about 33.524 daltons and a pi of about 9 55
Analysis of the full-length PR0238 sequence shown in Figure 4 (SEQ ID NO 4) evidenced the presence of a variety 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 PR0238 sequence (Figure 4, SEQ ID NO 4) evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 21 , transmembrane domains from about amino acid 102 to about amino acid 1 19 and from about amino acid 278 to about amino acid 292, an N-glycosylation site from about amino acid 228 to about ammo acid 232, a glycosaminoglycan attachment site from about amino acid 47 to about amino acid 51, a tyrosine kinase phosphorylation site from about amino acid 145 to about amino acid 153, N-myπstoylation sites from about amino acid 44 to about amino acid 50, from about amino acid 105 to about ammo acid 11 1 , from about amino acid 238 to about amino acid 244, from about amino acid 242 to about amino acid 248, and from about amino acid 291 to about amino acid 297, an amidation site from about ammo acid 265 to about amino acid 269, and a prokaryotic membrane lipoprotein lipid attachment site from about amino acid 6 to about amino acid 17 Clone DNA35600- 1162 has been deposited with ATCC on October 16, 1997 and is assigned ATCC deposit no 209370
Analysis ofthe amino acid sequence of the full-length PR0238 polypeptide suggests that portions of it possess significant homology to reductase, particularly oxidoreductase, thereby indicating that PR0238 may be a novel reductase
EXAMPLE 6
Isolation of cDNA Clones Encoding Human PRQ364 An expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) was searched and an Incyte EST sequence (Incyte EST No 3003460) was identified that showed homology to members of the tumor necrosis factor receptor (TNFR) family of polypeptides A consensus DNA sequence was then assembled relative to the Incyte 3003460 EST and other EST sequences using repeated cycles of BLAST (Altshul etal , Methods in Enzvmology. 266 460-480 (1996)) and "phrap" (Phil
Green, University of Washington, Seattle Washington) The consensus sequence is herein designated
"<consen01>" and is also herein designated as DNA44825
Ohgonucleotide probes based upon the DNA44825 and "<consen01>" consensus sequences were then synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) tor use as probes to isolate a clone ofthe full-length coding sequence for PR0364 Forward and rev erse PCR primers generally range from 20-30 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 order to screen several libraries tor a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Cuπent 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 ohgonucleotide and one of the primer pairs
Pairs of PCR primers (forward and reverse) were synthesized forward PCR primer (44825 f 1 )
5'-CACAGCACGGGGCGATGGG-3' (SEQ ID NO 39) forward PCR primer (44825 f2)
5'-GCTCTGCGTTCTGCTCTG-3' (SEQ ID NO 40) forward PCR primer (44825 GITR f)
5'-GGCACAGCACGGGGCGATGGGCGCGTTT-3' (SEQ ID NO 41 ) reverse PCR primer (44825 rl )
5'-CTGGTCACTGCCACCTTCCTGCAC-3' (SEQ ID NO 42) reverse PCR primer (44825 r2)
5'-CGCTGACCCAGGCTGAG-3' (SEQ ID NO 43) reverse PCR primer (44825 GITR r)
5'-GAAGGTCCCCGAGGCACAGTCGATACA-3 (SEQ ID NO 44)
Additonally, synthetic ohgonucleotide hybridization probes were constructed from the consensus DNA44825 sequence which had the following nucleotide sequences hybridization probe (44825 pi ) 5'-GAGGAGTGCTGTTCCGAGTGGGACTGCATGTGTGTCCAGC-3* (SEQ ID NO 45) hybridization probe (44825 GITR p) 5'-AGCCTGGGTCAGCGCCCCACCGGGGGTCCCGGGTGCGGCC-3' (SEQ ID NO 46)
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 primer pairs identified above A positive library was then used to isolate clones encoding the PR0364 gene using the probe oligonucleotides and one of the PCR primers
RNA for construction of the cDNA libraries was isolated from human bone maπow tissue The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with oligo dT containing a Notl site, 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 cDNA clone was identified and sequenced in entirety The entire nucleotide sequence of DN A47365 1206 is shown in Figure 5 (SEQ ID NO 5) Clone DNA47365-1206 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 121-123 and a stop codon at nucleotide positions 844 846 (Figure 5, SEQ ID NO 5) The predicted polypeptide precursor is 241 amino acids long and has a predicted molecular weight of approximately 26,000 daltons and an estimated pi of about 6 34 The full length PR0364 protein is shown in Figure 6 (SEQ ID NO 6)
Analysis of the full-length PR0364 sequence shown in Figure 6 (SEQ ID NO 6) evidences the presence ot 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 PR0364 sequence (Figure 6 SEQ ID NO 6) evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 25 a potential transmembrane domain from about amino acid 162 to about ammo acid 180, an N-glycosylation site from about amino acid 146 to about amino acid 150, N-myπstovlation sites from about amino acid 5 to about amino acid 1 1 , from about amino acid 8 to about amino acid 14, from about amino acid 25 to about amino acid 31 , from about ammo acid 30 to about amino acid 36, from about ammo acid 33 to about ammo acid 39, from about amino acid 118 to about amino acid 124 from about amino acid 122 to about amino acid 128, and from about amino acid 1 6 to about ammo acid 162 a prokaryotic membrane lipoprotein lipid attachment site from about amino acid 166 to about amino acid 177 and a leucine zipper pattern from about amino acid 171 to about am o acid 193 Clone DNA47365-1206 has been deposited with ATCC on November 7, 1997 and is assigned ATCC deposit no 209436 It is understood that the deposited clone has the actual correct sequence rather than the representations provided herein
Analysis of the amino acid sequence of the full-length PR0364 polypeptide suggests that portions of it possess homology to members of the tumor necrosis factor receptor family, thereby indicating that PR0364 may be a novel member of the tumor necrosis factor receptor family The intracellular domain of PR0364 contains a motif (in the region of amino acids 207-214) similar to the minimal domain within CD30 receptor shown to be required for TRAF2 binding and which is also present within TNFR2 There are three apparent extracellular cysteine-πch domains characteristic of the TNFR family (see, Naismith and Sprang, Trends Biochem Sci , 23 74-79 (1998)), of which the third CRD has 3 rather than the more typical 4 or 6 cysteines of the TNFR family As compared to the mouse GITR (described below) the PR0364 amino acid sequence has 8 cysteines in the CRD1 relative to 5 cysteines in CRD1 of mouse GITR, and the presence of one potential N-hnked glycosylation site in the ECD as compared to 4 potential N-hnked glycosylation sites in mouse GITR
A detailed review of the putative amino acid sequence of the full-length native PR0364 polypeptide and the nucleotide sequence that encodes it evidences sequence homology with the mouse GITR (mGITR) protein reported by Nocentini et al , Proc Nati Acad Sci USA. 94 6216-6221 (1997) It is possible, therefore, that PR0364 represents the human counterpart or ortholog to the mouse GITR protein reported by Nocentini et al
EXAMPLE 7 Isolation of cDNA Clones Encoding Human PRQ844
An expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST was identified that showed sequence identity with a LP Based on the information and discoveries provided herein, the clone for this EST, Incyte clone 2657496 from a cancerous lung library (309- LUNGTUT09) was further examined A cDNA clone was identified and sequenced in entirety The entire nucleotide sequence of DNA59838- 1462 is show n in Figure 7 (SEQ ID NO 7) Clone DNA59838-1462 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 5-7, and a stop codon at nucleotide positions 338-340 (Figure 7, SEQ ID NO 7) The predicted polypeptide precursor is 1 1 1 amino acids long and has a predicted molecular weight of approximately 12,050 daltons and an estimated pi of about 5 45 The full-length PR0844 protein is shown in Figure 8 (SEQ ID NO 8)
Analysis of the full-length PR0844 sequence shown in Figure 8 (SEQ ID NO 8) evidences the presence of important polypeptide domains as shown in Figure 8 wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full length PR0844 sequence (Figure 8 SEQ ID NO 8) evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 19 N-myπstoylation sites from about amino acid 23 to about am o acid 29, from about amino acid 27 to about ammo acid 33 from about amino acid 32 to about amino acid 38 and from about amino acid 102 to about amino acid 108, and a WAP-type 'four-disulfide core' domain signatuie from about amino acid 49 to about amino acid 63
Clone DNA59838-1462 has been deposited with ATCC on June 16, 1998 and is assigned ATCC deposit no 209976 It is understood that the deposited clone has the actual correct sequence rathei than the representations provided herein
Analysis of the amino acid sequence of the full-length PR0844 polypeptide suggests that it possesses significant similarity to serine protease inhibitors, thereby indicating that PR0844 may be a novel proteinase inhibitor More specifically, an analysis of the Dayhoff database (version 35 45 SwissProt 35) evidenced significant homology between the PR0844 amino acid sequence and at least the following Dayhoff sequences ALK1_HUMAN, P_P82403, P_P82402, ELAF_HUMAN and PJP60950
EXAMPLE 8 Isolation of cDNA Clones Encoding Human PRQ846 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described in
Example 1 above This consensus sequence is herein designated DNA39949 and "<consenl 322> Based on the DNA39949 consensus sequence and the "<consenl322>" sequnece, 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 PR0846 PCR primers (forward and reverse) were synthesized based upon the DNA39949 and "<consen 1322>" consensus sequences Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA30908 sequence
In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Cureent Protocols in Molecular Biology, supia, with the PCR primer pair A positive library was then used to isolate clones encoding the PR0846 gene using the probe ohgonucleotide and one of the PCR primers
The ohgonucleotide sequences used in the above procedure were the following forward PCR primer (39949 f 1 )
5'-CCCTGCAGTGCACCTACAGGGAAG-3' (SEQ ID NO 47) reverse PCR primer (39949 rl ) 5 -CTGTCTTCCCCTGCTTGGCTGTGG-3' (SEQ ID NO 48)
Additionally, a synthetic ohgonucleotide hybridization probe was constructed from the consensus DNA39949 sequence which had the following nucleotide sequence hybridization probe (39949 pi )
5 GGTGCAGGAAGGGTGGGATCCTCTTCTCTCGCTGCTCTGGCCACATC-3' (SEQ ID NO 49)
RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB227) The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed ith Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoiesis 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 etal . 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 PR0846 [herein designated as DNA44196-1 53] (Figure 9, SEQ ID NO 9) and the derived protein sequence for PR0846 The entire nucleotide sequence of DN A44196- 1353 is shown m Figure 9 (SEQ ID NO 9) Clone DN A44196 1353 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 25-27 and ending at the stop codon at nucleotide positions 1021-1023 (Figure 9) The predicted polypeptide precursor is 332 amino acids long and has an estimated molecular weight of approximately 36,143 daltons and pi of about 5 89 (Figure 10, SEQ ID NO 10)
Analysis of the full-length PR0846 sequence shown in Figure 10 (SEQ ID NO 10) evidenced the presence of a variety of important polypeptide domains as shown in Figure 10, wherein the locations given for those important polypeptide domains are approximate as described above Analysis of the full-length PR0846 sequence (Figure 10, SEQ ID NO 10) evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 17, a transmembrane domain from about amino acid 248 to about amino acid 269, an N glycosylation site from about amino acid 96 to about amino acid 100, a fibπnogen beta and gamma chains C terminal domain from about amino acid 104 to about amino acid 1 14, and a Ig like V-type domain from about amino acid 13 to about amino acid 128 Clone DNA44196-1353 has been deposited with ATCC on May 6, 1998 and is assigned ATCC deposit no 209847
EXAMPLE 9 Isolation of cDNA Clones Encoding Human PRO 1760 Use of the signal sequence algorithm descπbed in Example 3 above allowed identification of an EST cluster sequence from the Incyte database This EST cluster sequence was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies One or more ofthe ESTs was derived from a prostate tumor library The homology search was pei formed using the computer program BLAST or BLAST2 (Altshul ef α/ Methods in Enzvmology, 266 460-480 (1996)) 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 a consensus DNA sequence with the program "phrap" (Phil Green University of Washington Seattle, Washington) The consensus sequence obtained therefrom is herein designated DNA58798
In light of an observed sequence homology between the DNA58798 consensus sequence and the Incyte EST 3358745, the clone including this EST was purchased and the cDNA insert was obtained and sequenced It was found herein that that insert encoded a full-length protein The sequence of this cDNA insert is shown in Figure 11 (SEQ ID NO 11 ) and is herein designated DNA76532 1702
Clone DNA76532-1702 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 60-62 and ending at the stop codon at nucleotide positions 624-626 (Figure 1 1 ) The predicted polypeptide precursor is 188 ammo acids long (Figure 12, SEQ ID NO 12) The full-length PRO1760 protein shown Figure 12 has an estimated molecular weight of about 21 ,042 daltons and a pi of about 5 36 Analysis of the full-length PRO 1760 sequence shown in Figui e 12 (SEQ ID NO 12) evidences the presence of a variety of important polypeptide domains as shown in Figure 12, wherein the locations given for those important polypeptide domains are approximate as described above Analysis ofthe full-length PRO 1760 sequence evidences the presence of the follo ing features a signal peptide from about amino acid 1 to about amino acid 20, N-glycosylation sites from about ammo acid 121 to about amino acid 125 and from about amino acid 171 to about amino acid 175, a tyrosine kinase phosphorylation site from about amino acid 25 to about amino acid 32, and N myπstoylation sites from about am o acid 54 to about amino acid 60 and from about amino acid 160 to about amino acid 166 Clone DNA76532-1702 has been deposited with ATCC on November 17, 1998 and is assigned ATCC deposit no 203473
An analysis ofthe Dayhoff database (version 35 45 SwissProt 35), using a WU-BLAST2 sequence alignment analysis ofthe full-length sequence shown in Figure 12 (SEQ ID NO 12), evidenced sequence identity between the PRO1760 amino acid sequence and the following Dayhoff sequences CELT07F12_2, T22J18_16, ATF1C12_3, APE3_YEAST, P_W22471 , S AU56908_ 1 , SCPA_STRPY, ATAC00423817, S APURCLUS_2 and AF041468_9
EXAMPLE 10
Stimulation of Heart Neonatal Hypertrophy (Assay 1 ) This assay is designed to measure the ability of PRO polypeptides to stimulate hypertrophy of neonatal heart PRO polypeptides testing positive in this assay are expected to be useful for the therapeutic treatment of various cardiac insufficiency disorders Cardiac myocytes from 1-day old Harlan Sprague Dawley rats were obtained Cells (180 μl at 7 5 x lOVml, serum <0 1 %, freshly isolated) are added on day 1 to 96-well plates previously coated with DMEM/F12 + 4% FCS Test samples containing the test PRO polypeptide or growth medium only (negative control) (20 μl/well) are added directly to the wells on day 1 PGF (20 μl/well) is then added on day 2 at a final concentration of 106 M The cells are then stained on day 4 and visually scored on day 5, wherein cells showing no increase in size (as compared to negative controls) are scored 0 0, cells showing a small to moderate increase in size (as compared to negative controls) are scored 1 0 and cells showing a large increase in size (as compared to negative controls) are scored 2 0 A positive result in the assay is a score of 1 0 or greater
PR0882 tested positive in this assay as shown in TABLE 4 below
TABLE 4
PRO # Concentration/Dilution Relative Increase in Size (Compared to Negative Control)
PR0882 0 01% 3
PR0882 0 01 % 3
PR0882 0 10% 4
PR0882 0 10% 4
PR0882 1 00% 6
PR0882 1 00% 6
PR0882 0 01 %
PR0882 0 10% 4 5
PR0882 1 00% 6
PR0882 1 00% 5 5
PR0882 2 6 nM 5 75
16 EXAMPLE
Inhibition of Vascular Endothelial Growth Factor (VEGF) Stimulated Proliferation of Endothelial Cell Growth
(Assay 9) The ability of various PRO polypeptides to inhibit VEGF stimulated proliferation of endothelial cells was tested. Polypeptides testing positive in this assay are useful for inhibiting endothelial cell growth in mammals where such an effect would be beneficial, e.g., for inhibiting tumor growth.
Specifically, bovine adrenal cortical capillary endothelial cells (ACE) (from primary culture, maximum of 12-
14 passages) were plated in 96- well plates at 500 cells/well per 100 microliter. Assay media included low glucose
DMEM, 10% calf serum, 2 mM glutamine, and IX penicillin/streptomycin/fungizone. Control wells included the following: (1 ) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGF plus
5 ng/ml LIF. The test samples, poly-his tagged PRO polypeptides (in 100 microliter volumes), were then added to the wells (at dilutions of 1%, 0.1% and 0.01%, respectively). The cell cultures were incubated for 6-7 days at
37°C/5% C02. After the incubation, the media in the wells was aspirated, and the cells were washed IX with PBS. An acid phosphatase reaction mixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1 % Triton X-100. 10 mM p-nitrophenyl phosphate) was then added to each well. After a 2 hour incubation at 37 °C, the reaction was stopped by addition of 10 microliters IN NaOH. Optical density (OD) was measured on a microplate reader at 405 nm.
The activity of PRO polypeptides was calculated as the percent inhibition of VEGF (3 ng/ml) stimulated proliferation (as determined by measuring acid phosphatase activity at OD 405 nm) relative to the cells without stimulation. TGF-beta was employed as an activity reference at 1 ng/ml, since TGF-beta blocks 70-90% of VEGF- stimulated ACE cell proliferation. The results, as shown in TABLE 5 below, are indicative of the utility ofthe PRO polypeptides in cancer therapy and specifically in inhibiting tumor angiogenesis. The numerical values (relative inhibition) shown in TABLE 5 are determined by calculating the percent inhibition of VEGF stimulated proliferation by the PRO polypeptides relative to cells without stimulation and then dividing that percentage into the percent inhibition obtained by TGF-β at 1 ng/ml which is known to block 70-90% of VEGF stimulated cell proliferation. The results are considered positive if the PRO ploypeptide exhibits 30% or greater inhibition of
VEGF stimulation of endothelial cell growth (relative inhibition 30% or greater).
TABLE 5 Inhibition of VEGF Stimilated Endothelial Cell Growth
PRO Name PRO Concentration Relative % Inhibition
PR0333 0 01% 97 0 PR0333 0 10% 90 0
PR0333 1 00% 63 0
PR0877 0 01 % 101 0 PR0877 0 01 % 108 0 PR0877 0 10% 86 0 PR0877 0 10% 99 0 PR0877 1 00% 46 0 PR0877 1 00% 46 0
PR0879 0 01 % 100 0 PR0879 0 01 % 105 0 PR0879 0 10% 97 0 PR0879 0 10% 101 0 PR0879 1 00% 55 0 PR0879 1 00% 66 0
PR0882 0 01 % 96 0 PR0882 0 10% 86 0 PR0882 1 00% 70 0
PR0885 0 01 % 100 0 PR0885 0 10% 93 0 PR0885 1 00% 64 0
EXAMPLE 12
Induction of c-fos in Endothelial Cells (Assay 34) This assay is designed to determine whether PRO polypeptides show the ability to induce c-fos in endothelial cells PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of conditions or disorders where angiogenesis would be beneficial including, for example wound healing, and the like (as would agonists of these PRO polypeptides) Antagonists of the PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of cancerous tumors
Human venous umbilical vein endothelial cells (HUVEC Cell Systems) in growth media (50% Ham s F12 w/o GHT low glucose, and 50% DMEM without glycine with NaHC03, 1 % glutamme, 10 mM HEPES, 10% FBS, 10 ng/ml bFGF) were plated on 96-well microtiter plates at a cell density of 1 x 10 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 1 O mM HEPES J 40 mM NaCl 4% (w/v) mannitol pH 6 8) The cells were incubated for 30 minutes at 37 °C in 5%- CO, The samples were removed and the first part of the bDNA kit protocol (Chiron Diagnostics, cat #6005 037) was followed where each capitalized reagent/buffer listed below was available from the kit Briefly, the amounts of the TM Lysis Buffei and Probes needed for the tests were calculated based on information prov ided 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 ofthe 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 CC 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). The results are shown in TABLE 6 below, and are considered positive if the PRO polypeptide exhibits at least a two-fold value over the negative control. Negative control = 1.00 RLU at 1.00% dilution. Positive control = 8.39 RLU at 1.00% dilution.
TABLE 6
Induction of c-fos in Endothelial Cells
PRO Name PRO Concentration RLU values
PR0321 001 1 nM 1 51 PR0321 0 1 1 nM 1 07
PR0321 1 1 nM 2 11
PR0321 0 01 1 nM 2 13 PR0321 0 1 1 nM 2 27 PR0321 1 10 nM 2 65
PRO840 244 nM 1 85 PRO840 24 4 nM 2 21 PRO840 244 nM 3 04
PRO840 244 nM 2 82
PRO840 24 4 nM 2 90 PRO840 244 nM 1 01
PR0878 0 01 % 2 43 PR0878 0 10% 2 71 PR0878 1 00% 1 39
PR0878 0 01% 248 PR0878 0 10% 245 PR0878 1 00% 1 89
PR0879 0 01 % 1 23 PR0879 0 10% 1 33 PR0879 1 00% 2 54
PR0879 0 01 % 2 06 PR0879 0 10% 1 65 PR0879 1 00% 2 25
EXAMPLE 13 Enhancement of Heart Neonatal Hypertrophy Induced bv F2a (Assay 37) This assay is designed to measure the ability of PRO polypeptides to stimulate hypertrophy of neonatal heart
PRO polypeptides testing positive in this assay are expected to be useful for the therapeutic treatment of various cardiac insufficiency disorders
Cardiac myocytes from 1 -day old Harlan Sprague Daw ley rats were obtained Cells (180 μl at 7 5 x 1 4/ml serum <0 1 %, freshly isolated) are added on day 1 to 96-well plates previously coated with DMEM/F12 + 4% FCS Test samples containing the test PRO polypeptide (20 μl/well) are added directly to the wells on day 1 PGF (20 μl/well) is then added on day 2 at a final concentration of 10 " M The cells are then stained on day 4 and visually scored on day 5 Visual scores are based on cell size, wherein cells showing no increase in size as compared to negative controls are scored 0 0, cells showing a small to moderate increase in size as compared to negative controls are scored 1 0 and cells showing a large increase in size as compared to negative controls are scored 2 0 A score of 1 0 or greater is considered positive No PBS is included, since calcium concentration is critical for assay response Plates are coated with
DMEM/F12 plus 4% FCS (200 μl/well) Assay media included DMEM/F12 (with 2 44 gm bicarbonate), 10 μg/ml transferπn, 1 μg/ml insulin, 1 μg/ml aprotinin, 2 mmol/L glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin Protein buffer containing mannitol (4%) gave a positive signal (score 3 5) at 1/10 (0 4%) and 1/100 (0 04%), but not at 1/1000 (0 004%) Therefore the test sample buffer containing mannitol is not run
PRO205, PR0882 and PR0887 polypeptides tested positive in this assay
EXAMPLE 14 Inhibition of Heart Adult Hypertrophy (Assay 42) This assay is designed to measure the inhibition of heart adult hypertrophy PRO polypeptides testing positive in this assay may find use in the therapeutic treatment of cardiac disorders associated with cardiac hypertrophy
Ventricular myocytes are freshly isolated from adult (250g) Harlan Sprague Dawley rats and the cells are plated at 2000/well in 180 μl volume On day two, test samples (20 μl) containing the test PRO polypeptide are added On day five, the cells are fixed and then stained An increase in ANP message can also be measured by PCR from cells after a few hours Results are based on a visual score of cell size 0 = no inhibition, -1 =small inhibition, -2 = large inhibition A score of less than 0 is considered positive Activity reference corresponds to phenylephπn (PE) at 0 1 mM, as a positive control Assay media included Ml 99 (modιfιed)-glutamιne free,
NaHCOj, phenol red, supplemented with 100 nM insulin, 0 2% BSA, 5 mM creatine, 2 mM L-carmtine, 5 mM tauπne, 100 U/ml penicillin G, 100 μg/ml streptomycin (CCT medium) Only inner 60 wells are used in 96 well plates Of these, 6 wells are reserved for negative and positive (PE) controls
PR0878 polypeptide provided a score of less than 0 in the above assay
EXAMPLE 15 Induction of Endothelial Cell Apoptosis (Assay 73) The ability of PRO polypeptides to induce apoptosis in endothelial cells was tested in human v enous umbilical vein endothelial cells (HUVEC, Cell Systems) A positiv e test in the assay is indicative of the usefulness of the polypeptide in therapeutically treating tumors as well as vascular disordei s where inducing apoptosis of endothelial cells would be beneficial
The ability of PRO polypeptides to induce apoptosis in endothelial cells was tested in human venous umbilical vein endothelial cells (HUVEC, Cell Systems), using a 96-w ell format in 0% serum media supplemented with 100 ng/ml VEGF (As HUVEC cells are easily dislodged from the plating surface, all pipetting in the wells must be done as gently as practicable )
The medium was aspirated and the cells washed once with PBS 5 ml of 1 x trypsin was added to the cells in a T-175 flask and the cells were allowed to stand until thev were released from the plate (about 5-10 minutes)
Trypsimzation was stopped by adding 5 ml of growth media The cells were spun at 1000 rpm for 5 minutes at 4 °C The media was aspirated and the cells were resuspended in 10 ml of 10% serum complemented medium (Cell
Systems), 1 x penicillin/streptomycin
The cells were plated on 96-well microtiter plates (Amersham Lite Science cytostar-T scintillating microplate, RPNQ160 sterile tissue culture treated, individually wrapped) in 10% serum (CSG medium Cell Systems) at a density of 2 x 104 cells per well in a total volume of 100 μl Test PRO polypeptide samples were added in triplicate at dilutions of 1 %, 0 33% and 0 1 1 % Wells without cells were used as a blank and wells with cells only were used as a negative control As a positive control 1 3 serial dilutions of 50 μl of a 3x stock of staurospoπne were used The ability of the test PRO polypeptide to induce apoptosis was determined using Annexm-V, a member of the calcium and phosphohpid binding proteins, to detect apoptosis
0 2 ml Annexin V - Biotin stock solution (100 μg/ml) were diluted in 4 6 ml 2 x Ca2+ binding buffer and 2 5% BSA (1 25 dilution) 50 μls of the diluted Annexin V - Biotm solution were added to each well (except controls) to a final concentration of 1 0 μg/ml The samples were incubated for 10-15 minutes with Annexin-Biotin prior to direct addition of 35S-Streptavιdιn 35S-Streptavιdιn was diluted in 2x Ca2+ Binding buffer, 2 5% BSA and was added to all wells at a final concentration of 3 x 104 cpm/well The plates were then sealed, centrifuged at 1000 rpm for 15 minutes and placed on orbital shaker for 2 hours The analysis was performed on 1450 Mιcrobeta Tπlux (Wallac) The results are shown in TABLE 7 below where percent above background represents the percentage amount of counts per minute above the negative controls Percents greater than or equal to 30% above background are considered positive PR0333, PR0364 and PR0879 scored positive results in the above described assay
TABLE 7
Induction of Endothelial Cell Apoptosis
PRO Name PRO Concentration Percent Above Background
PR0333 0 11 % 61 7 %
PR0333 0 33% 37 6 %
PR0364 299 nM 19 3 %
PR0364 8 99 nM 6 9 %
PR0364 27 23 nM 31 5 %
PR0879 1 00% 64 2 %
PR0879 0 1 1 % 65 5%
PR0879 0 33% 14 7%
EXAMPLE 16
Inhibition of Heart Neonatal Hypertroj 3hy Induced by LIF plus Endothelm- 1 (ET 1 ) (Assay 74)
This assay is designed to determine whether PRO polypeptides of the present invention show the ability to inhibit neonatal heart hypertrophy induced by LIF and endothelιn-1 (ET-1 ) A test compound that provides a positiv e response in the present assay would be useful for the therapeutic treatment of cardiac insufficiency diseases or disorders characterized or associated with an undesired hypertrophy of the cardiac muscle
Cardiac myocytes from 1 -day old Harlan Sprague Dawley rats ( 180 μl at 7 5 \ 104/ml serum <0 1 freshly isolated) are introduced on day 1 to 96-well plates previously coated with DMEM/F12 + 4%FCS Test PRO polypeptide samples or growth medium alone (negative control) are then added directly to the wells on day 2 in 20 μl volume LIF + ET-1 are then added to the wells on day 3 The cells are stained attei an additional 2 days in culture and are then scored visually the next day A positive in the assay occurs when the PRO polypeptide treated myocytes are visually smaller on the average or less numerous than the untreated myocytes PR0238 and PRO 1760 polypeptides tested positive in this assay
EXAMPLE 17 Stimulation of Endothelial Tube Formation - Sprout formation (Assay 86) This assay is designed to determine whether PRO polypeptides show the ability to promote endothelial vacuole and lumen formation in the absence of exogenous growth factors PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of disorders where endothelial vacuole and/or lumen formation would be beneficial including, for example, where the stimulation of pinocytosis, ion pumping, vascular permeability and/or junctional formation would be beneficial HUVEC cells (passage <8 from primary) are mixed with type I rat tail collagen (final concentration 2 6 mg/ml) at a density of 6x 105 cells per ml and plated at 50 μl per well of M 199 culture media supplemented with 1 % FBS and 1 μM 6-FAM-FITC dye to stain the vacuoles while they are forming and in the presence of the PRO polypeptide The cells are then incubated at 37°C/5% CO, for 48 hours, fixed with 3 7% formalin at room temperature for 10 minutes, washed 5 times with Ml 99 medium and then stained with Rh-Phalloidin at 4°C overnight followed by nuclear staining with 4 μM DAPI A positive result in the assay is equal to or less 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]
PRO 179 polypeptide tested positive in this assay
EXAMPLE 18 Induction of Endothelial Cell Apoptosis (ELISA) (Assay 109)
The ability of PRO polypeptides to induce apoptosis in endothelial cells was tested in human venous umbilical vein endothelial cells (HUVEC, Cell Systems) using a 96-well format, in 0% serum media supplemented with 100 ng/ml VEGF, 0 1 % BSA, lX penn/strep A positive result in this assay indicates the usefulness of the polypeptide for therapeutically treating any of a variety of conditions associated with undesired endothelial cell growth including, for example, the inhibition of tumor growth The 96-well plates used were manufactured by Falcon (No 3072) Coating of 96 well plates were prepared by allowing gelati zation to occur foi >30 minutes with 100 μl of 0 2% gelatin in PBS solution The gelatin mix was aspirated thoroughly before plating HUVEC cells at a final concentration of 2 x 104 cells/ml in 10% serum containing medium - 100 μl volume per well The cells were grown for 24 hours before adding test samples containing the PRO polypeptide of interest To all wells, 100 μl of 0% serum media (Cell Systems) complemented with 100 ng/ml VEGF, 0 1 % BSA, 1 X penn/strep was added Test samples containing PRO polypeptides were added in triplicate at dilutions of 1 %, 0 33% and 0 1 1 % Wells without cells were used as a blank and wells with cells only were used as a negative control As a positive control, 1 3 serial dilutions of 50 μl of a 3x stock of staurospoπne were used The cells were incubated tor 24 to 35 hours prior to ELISA ELISA was used to determine levels of apoptosis preparing solutions according to the Boehπnger Manual
[Boehπnger, Cell Death Detection ELISA plus, Cat No 1 920685] Sample preparations 96 well plates were spun down at 1 krpm for 10 minutes (200g), the supernatant was removed by fast inversion, placing the plate upside down on a paper towel to remove residual liquid To each well, 200 μl of IX Lysis buffer was added and incubation allowed at room temperature for 30 minutes without shaking The plates were spun down for 10 minutes at 1 krpm, and 20 μl of the lysate (cytoplasmic fraction) was transferred into streptavidin coated MTP 80 μl of immunoreagent mix was added to the 20 μl lystate in each well The MTP was covered with adhesive foil and incubated at room tempearature for 2 hours by placing it on an orbital shaker (200 rpm) After two hours, the supernatant was removed by suction and the wells rinsed three times with 250 μl of IX incubation buffer per well (removed by suction) Substrate solution was added (100 μl) into each well and incubated on an orbital shaker at room temperature at 250 rpm until color development was sufficient for a photometric analysis (approx after 10-20 minutes) A 96 well reader was used to read the plates at 405 nm, reference wavelength, 492 nm The levels obtained for PIN 32 (control buffer) was set to 100% Samples with levels >130% were considered positive for induction of apoptosis
PR0846, and PR0844 polypeptides tested positive in this assay
EXAMPLE 19
In situ Hybridization In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations It 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-generated33P-labeledπboprobes Bnefly, 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 (" 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 "P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed-vacuum dried To each tube containing dried 3P-UTP, the following ingredients were added 2 0 μl 5x transcription buffer 1 O μΙ DTT (lOO mM) 2 0 μl NTP mix (2 5 mM 10 μl each of 10 mM GTP, CTP & ATP + 10 μl H,0)
1 0 μl UTP (50 μM) 1 0 μl RNAsin 1 0 μl DNA template ( 1 μg) 1 0 μl H,0 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 I 0 μl RQ1 DNase was added, followed by incubation at 37 °C for 15 minutes A total of 90 μl TE (10 mM Tris pH 7 6/1 mM EDTA pH 8 0) was added and the mixture was pipetted onto DE81 paper The remaining solution was loaded in a MICROCON 50 ultrafiltration unit, and spun using program 10 (6 minutes) The filtration unit was inverted over a second tube and spun using program 2 (3 minutes) After the final recovery spin, a total of 100 μl TE was added, then 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR II™
The probe was 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-Hybπdization A Pretreatment of frozen sections
The slides were removed from the freezer, placed on aluminum trays, and thawed at room temperature for 5 minutes The trays were placed in a 55 °C incubator for five minutes to reduce condensation The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0 5 x SSC for 5 minutes at room temperature (25 ml 20 x SSC + 975 ml SQ H,0) After deproteination in 0 5 μg/ml proteinase K for 10 minutes at 37°C (12 5 μl of 10 mg/ml stock m 250 ml prewarmed RNAse-free RNAse buffer), the sections were washed in 0 5 x SSC for 10 minutes at room temperature The sections were dehydrated in 70%, 95%, and 100% ethanol, 2 minutes each
B Pretreatment of paraffin-embedded sections
The slides were deparaffinized, placed in SQ H20, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml
RNase-free RNase buffer, 37 °C, 15 minutes) for human embryo tissue, or 8 x proteinase K ( 100 μl in 250 ml Rnase buffer 37 °C, 30 minutes) for formalin tissues Subsequent rinsing in 0 5 x SSC and dehydration ere performed as described above
C Preh ybt idization 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 sulf te + 6 ml SQ H-,0) v ortexed, and heated in the microwave for 2 minutes with the cap loosened After cooling on ice, 18 75 ml formamide, 3 75 ml 20 x SSC, and 9 ml SQ H20 were added, and the tissue was vortexed well and incubated at 42 °C tor 1 -4 hours
D Hxbridtzation 1 0 x 10' cpm probe and 1 0 μl tRNA (50 mg/ml stock) per slide were heated at 95 °C for 3 minutes The slides w ere cooled on ice, and 48 μl hybridization buffer was added per slide After vortexing, 50 / 1 P mix was added to 50 μl prehybπdization on the slide The slides were incubated overnight at 55 °C
E Washes
\\ ashing was done for 2x 10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0 25 M EDTA V,=4L) followed by RNAseA treatment at 37 °C for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer = 20 μg/ml), The slides were washed 2 x10 minutes with 2 x SSC, EDTA at room temperature The stringency wash conditions were as follows 2 hours at 55 °C 0 1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA,
V,=4L F Oligonucleotides
In situ analysis was performed on three of the DNA sequences disclosed herein The oligonucleotides employed for these analyses are as follows
(1 ) DNA47365-1206 (PRQ364) (TNF receptor homolog) pi
5'-GGATTCTAATACGACTCACTATAGGGCAACCCGAGCATGGCACAGCAC-3'(SEQIDNO 50) p2
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA TCT CCC AGC CGC CCC TTC TC 3' (SEQ ID NO 51 )
(2) DNA30868 (PRO205) (follistat homolog) pi
5'-GGATTCTAATACGACTCACTATAGGGCAGAGACAGGGCAAGCAGAATG-3*(SEQIDNO 52) p2
5'-CTATGAAATTAACCCTCACTAAAGGGAGAAGGGGATGACTGGAGGAAC 3'(SEQIDNO 53)
(3) DNA41374 (PRQ333) (CD33 homolog) pi
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC CTC CAC AGA ACC TCG CCA TCA-3' (SEQ ID NO 54) p2
5'-CTATGAAATTAACCCTCACTAAAGGGATGGGGCAAGACTCACAAGCAG-3'(SEQIDNO 55)
G Results In situ analysis was performed on the above three DNA sequences disclosed herein The results from these analyses are as follows
(1 ) DNA47365-1206 (PRQ364) (TNF receptor homolog)
In the fetus, there was expression in the fascia lining the anterior surface of the v ertebi al body There was also expression over the fetal retina However there was low level expression over the fetal neurones All other tissues were negative
(2) DNA30868 (PRO205) (follistatm homolog)
In fetal tissue, there was expression in the spinal cord, autonomic ganglia, enteric nerves, sacral plexus, peripheral and cranial nerves All other fetal and adult tissues were negative
Fetal tissues (12-16 weeks) examined included placenta, umbilical cord, liv er kidney adrenals thyroid, lungs, heart, great vessels, oesophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord body wall, pelvis and lower limb
Adult tissues included liver, kidney adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung and (3) DNA41374 (PRQ333) (CD33 homolog)
This molecule has been shown to be immunostimulatory (enhances T lymphocyte proliferation in the one-way mixed lymphocyte reaction in T lymphocyte co stimulation assays) The distribution pattern of this molecule was evaluated with a limited tissue screen comprising tissues available at the initiation of this study In numerous tissues evaluated, a weak diffuse expression was detected in thymic T lymphocytes (spleen and lymph node were not evaluated) This result was confirmed in the subsequent in situ hybridization study The results of that study showed similar low level expression in non-human primate thymus and m human tonsil in T lymphocyte specific regions The limited distribution pattern suggests expression by T lymphocytes or cells closely associated with T lymphocytes such as antigen presenting cells (dendritic cell populations, etc) In inflamed human tissue with significant lymphocytic inflammation and presence of reactive follicle formation (in inflammatory bowel disease and chronic lymphocytic interstitial pneumonia/bronchitis) there was no detectable expression in areas which contained significant numbers of T lymphocytes
The distribution discrepancy (; e expression in thymic and tonsillar T lymphocyte areas but not in areas with T lymphocytic inflammation) suggests the following possibilities 1 That there is selective/restricted expression in a T lymphocyte subset population present in thymus and tonsillar lymph node but not inflamed tissue Immature, non-committed T lymphocytes are present in both tonsil and thymus but likely would not be a major population in chronically inflamed tissues
2 That expression in T lymphocytes is weak and differences in detection in tissues with T lymphocytes is a reflection of RNA quality in those tissues sections rather than a reflection of different T lymphocyte cell population types
3 That expression in thymus and tonsil is not in lymphocytes but rather in a specific cell population closely associated with T lymphocytes, that is not present in the inflamed lung and bowel evaluated One such possibility is a dendritic cell subpopulation
In a non-human primate there was weak diffuse expression in thymic lymphocytes In a subsequent study, the following results were reported
Inflammed lung (chronic lymphocytic and granulomatous pneumo tis) a weak to negative signal was observed in the interstitium compared to the control sense probe, there was weak expression in the normal chimp thymus (human thymus not available) and in the human tonsil In the later, the expression was predominantly in T lymphocyte areas of this structure including the peπfollicular marginal zone and in the paracortex There was no detectable expression in the following human tissues lnflammatoiy bowel disease (8 patient specimens), chronically inflamed and normal lung (6 patient specimens) chronic sclerosing nephritis ( 1 patient specimen), chronically and acutely inflammed and cirrhotic liver (10 specimen multiblock), normal and psoπatic skin, and peripheral lymph node (non-reactive)
EXAMPLE 20 Use of PROl 79. PRQ238. PRQ364. PRQ844. PRQ846. PROl 760. PRO205 PRQ321. PRQ333. PRO840
PRQ877. PRQ878 PRQ879. PRQ882. PRQ885 or PRQ887 as a Hybridization Probe The following method describes use of a nucleotide sequence encoding PRO 179 PR0238, PR0364, PR0844, PRQ846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878 PR0879, PR0882, PR0885 oi PR0887 as a hybridization probe.
DNA comprising the coding sequence of full-length or mature PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 (as shown in Figures 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, 27, 29, and 31 , respectively, SEQ ID NOS: 1, 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, and 31 , respectively) or a fragment thereof is employed as aprobe to screen for homologous DNAs (such as those encoding naturally-occuπing variants of PROl 79, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887) 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 0.1 x SSC and 0.1 % SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art.
EXAMPLE 21
Expression of Nucleic Acid Encoding PROl 79, PRQ238, PRQ364. PRQ844. PRQ846. PROl 760. PRO205. PRQ321. PRQ333. PRO840. PRQ877. PRQ878. PRQ879. PRQ882. PRQ885 or PRQ887 in E. coli
This Example illustrates preparation of an unglycosylated form of PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760. PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 by recombinant expression in E. coli.
The DNA sequence encoding PROl 79, PR0238, PR0364. PR0844, PR0846. PROl 760, PRO205. PR0321 , PR0333, PRO840. PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 (SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1, 13, 15, 17, 19, 21 , 23, 25, 27, 29, or 31 , 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 ρBR322 (derived from E. coli; see Bolivar etal. , Gene.2: 95 (1977)), which contains genes for ampicilhn 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 trp promoter, a poly-His leader (including the first six STII codons, poly-His sequence, and enterokinase cleavage site), the region encoding PROl 79. PR0238, PR0364, PR0844. PR0846, PRO 1760. PRO205, PR0321. PR0333, PRO840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887, lambda transcriptional terminator, and an argU gene.
The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al, supra. Transformants are identified by their ability to grow on LB plates and antibiotic-resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing
Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight 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 cultuπng the cells for several more hours, the cells can be harvested by centπfugation The cell pellet obtained by the centπfugation can be solubihzed using various agents known in the art, and the solubihzed PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide can then be purified using a metal-chelating column under conditions that allow tight binding of the polypeptide PR0238, PR0364 and PRO 1760 were successfully expressed in £ cob m a poly-His tagged form by the above procedure
EXAMPLE 22
Expression of Nucleic Acid Encoding PRQ179. PRQ238. PRQ364. PRQ844. PRQ846. PRO1760. PRO205.
PRQ321. PRQ333, PRO840. PRQ877. PRQ878. PRQ879. PRQ882. PRQ885 or PRQ887 in Mammalian Cells This Example illustrates preparation of a potentially glycosylated form of PROl 79, PR0238, PR0364,
PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 by recombinant expression in mammalian cells
The vector, pRK5 (see, EP 307,247, published March 15, 1989), is employed as the expression vector Optionally, the PRO 179, PR0238, PR0364, PR0844, PR0846. PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 oi PR0887 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the DNA encoding PRO 179. PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 using ligation methods such as described in Sambrook et al , supia The resulting vector is called pRK5-(DNA encoding PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760 PRO205, PR0321. PR0333, PRO840 PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887)
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, nutπent components and or antibiotics About 10 μg DNA of pRK5-(DNA encoding PRO 179, PR0238, PR0364, PR0844 PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879. PR0882, PR0885 or PR0887) is mixed with about 1 μg DNA encoding the VA RNA gene (Thimmappaya et al , C l, 3J 543 (1982)) and dissolved in 500 μl of 1 mM Tπs-HCl, 0 1 mM EDTA, 0 227 M CaCl2 To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7 35), 280 mM NaCl 1 5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25°C The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37"C The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added tor 30 seconds The 293 cells are then washed with serum-free medium, fresh medium is added, and the cells are incubated tor 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 "S-cv steine and 200 μCi/ml After a 12- hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 1 % SDS gel The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of the PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Nati Acad Sci , 12 7575 ( 1981 ) 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-(DNA encoding PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887) is added The cells are first concentrated from the spinner flask by centπfugation and washed with PBS The DNA-dextran precipitate is incubated on the cell pellet for four hours The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re- mtroduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin, and 0 1 μg/ml bovine transfemn After about four days, the conditioned media is centrifuged and filtered to remove cells and debris
The sample containing the expressed gene encoding the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760,
PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography
In another embodiment, the gene encoding PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 can be expressed in CHO cells The pRK5-(DNA encoding PR0179, PR0238, PR0364, PR0844 PR0846, PRO1760, PRO205 PRO321 , PRO333, PRO840, PRO877, PRO878, PRO879, PRO882 PR0885 or PR0887) 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) oi medium containing a radiolabel such as 'S-methιomne Aftei determining the presence of PROl 79 PR0238 PR0364, PR0844, PR0846 PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879 PR0882, PR0885 or PR0887 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 PR0179 PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 can then be concentrated and purified by any selected method
Epitope-tagged gene encoding the PROl 79, PR0238, PR0364, PR0844, PR0846 PRO 1760 PRO205,
PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may also be expressed host CHO cells The gene encoding PROl 79, PR0238, PR0364 PR0844 PR0846, PROl 760,
PRO205 PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR088 or PR0887 may be subcloned out of the pRK5 vectoi 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-[PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0 21 PR0333, PRO840, PR0877 PR0878, PR0879, PR0882, PR0885 or PR0887] can then be subcloned into a SV40- driven vectoi containing a selection marker such as DHFR for selection of stable clones Finally the CHO cells can be transfected (as described above) with the S V40-driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed gene encoding the poly-His-tagged-[PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887] can then be concentrated and purified by any selected method, such as by Ni2+-chelate affinity chromatography.
PROl 79, PR0364, PRO840, PR0844, PR0846 and PRO205 were stably expressed in CHO cells by the above described method. In addition, PR0364 and PR0846 were expressed in CHO cells by a transient procedure.
EXAMPLE 23 Expression of Nucleic Acid Encoding PRQ179, PRQ238. PRQ364. PRQ844. PRQ846. PRO1760. PRO205, PRQ321. PRQ333. PRO840. PRQ877. PRQ878. PRQ879. PRQ882. PRQ885 or PRQ887 in Yeast The following method describes recombinant expression of the gene encoding PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 in yeast. First, yeast expression vectors are constructed for intracellular production or secretion of PRO 179, PR0238,
PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 from the ADH2/GAPDH promoter. DNA encoding PR0179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the gene encoding PRO 179, PR0238, PR0364. PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887. For secretion, DNA encoding PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PROl 79, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887. 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 ofthe gels with Coomassie Blue stain.
Recombinant PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760. PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879. PR0882, PR0885 or PR0887 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 PRO 179, PR0238. PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 may further be purified using selected column-chromatography resins. EXAMPLE 24 Expression of Nucleic Acid Encoding PRQ179, PRQ238 PRQ364, PRQ844, PRQ846. PRO1760. PRO205. PRQ321. PRQ333. PRO840. PRQ877. PRQ878. PRQ879. PRQ882. PRQ885 or PRQ887 in Baculovirus- infected Insect Cells The following method describes recombinant expression in Baculovirus-infected insect cells
The sequence coding for PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321. PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 or the desired portion of the coding sequence of PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 [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 incorporate 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 hpofectin (commercially available from GIBCO-BRL) After 4 - 5 days of incubation at 28 °C, the released viruses are harvested and used for further amplifications Viral infection and protein expression are performed as described by O'Reilley et al , Baculovirus
Expression Vectors A Laboratory Manual (Oxford Oxford University Press (1994))
Expressed poly-His taggedJPRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887] 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 9J2 5 mM MgCl2, 0 1 mM EDTA, 10% glycerol, 0 1 % NP-40, 0 4 M KC1), and sonicated twice for 20 seconds on ice The sonicates are cleared by centπfugation, 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 A280 with loading buffer, at which point fraction collection is started Next, the column is washed w ith a secondary wash buffer (50 mM phosphate. 300 mM NaCl, 10% glycerol, pH 6 0), which elutes non-specifically- bound protein After reaching A2 0 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-[PRO 179, PR0238. PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333 PRO840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887] respectively, are pooled and dialyzed against loading buffei Alternatively, purification of the IgG-tagged (or Fc tagged)- [PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887] can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography While expression was actually performed in a 0 5-2 L scale, it can be readily scaled up for larger (e g , 8 L) preparations The proteins were expressed as an IgG construct (lmmunoadhesin), in which the protein extracellular region was fused to an IgGl constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms
Following PCR amplification, the respective coding sequences were subcloned into a baculovirus expression vector (pb PH IgG for IgG fusions and pb PH His c for poly-His tagged proteins), and the vector and Baculogold® baculovirus DNA (Pharmingen) were co transfected into 105 Spodoptera frugψerda ("Sf9") cells (ATCC CRL 171 1 ) using Lipofectin (Gibco BRL) pb PH IgG and pb PH His are modifications of the commercially available baculov IΓUS expression vector pVLl 393 (Pharmingen), with modified polyhnker regions to include the His or Fc tag sequences The cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells were incubated for 5 days at 28 °C The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink s TNM FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10 Cells were incubated for 3 days 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 Protem-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 St9 cells grown in ESF 921 medium (Expression Systems LLC) at an approximate MOI of 0 1 Cells were incubated for 3 days at 28 °C The supernatant was harvested and filtered Batch binding and SDS PAGE analysis were repeated, as necessary until expression of the spinner culture was confirmed
The conditioned medium from the transfected cells (0 5 to 3 L) was harvested by centπfugation to remove the cells and filtered through 0 22 micron filters For the poly-His tagged constructs, the protein construct was purified using a Ni 1+-NTA column (Qiagen) Before purification, imidazole was added to the conditioned media to a concentration of 5 mM The conditioned media was pumped onto a 6 ml Ni 9+ NTA column equilibrated in 20 mM Hepes pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4 5 ml/mm at 4°C After loading the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8 with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C lmmunoadhesin (Fc containing) constructs of proteins were pui lfied from the conditioned media as follows
The conditioned media was pumped onto a 5 ml Protein A column (Phaimacia) which had been equilibrated in 20 mM a 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 bv collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer pH 9 The highly purified protein as 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
PRO205, PR0321 , PRO840, PR0846, PR0885 and PR0887 were successfully expressed in Baculovirus- infected insect Sf9 cells by the above procedure
Alternatively, a modified baculovirus procedure may be used incorporating 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 pIEl-1 (Novagen) The pIEl-1 and pIE 1-2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus lei promoter in stably-transformed insect cells The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lei -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element pIEl-1 and pIEl-2 include the translation initiation site and can be used to produce fusion proteins Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) was amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incorporate flanking (selected) restriction enzyme sites The product was then digested with those selected restriction enzymes and subcloned into the expression vector For example, derivatives of pIEl - 1 can include the Fc region of human IgG (pb PH IgG) or an 8 histidine (pb PH His) tag downstream (3 -of) the desired sequence Preferably, the vector construct is sequenced for confirmation
Hιgh-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no CO,, 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 CellFECTTN, 30 ml of fresh Ex -Cell media was added and the cells are incubated tor 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 Protem-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining
The conditioned media from the transfected cells (0 5 to 3 L) was harvested by centπfugation to remove the cells and filtered through 0 22 micron filters For the poly-His tagged constructs, the protein comprising the sequence is purified using a Ni 2+-NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media was pumped onto a 6 ml Ni 2+-NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 48 °C After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein was then subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80 °C lmmunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows The conditioned media was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5 The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9 The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity of the sequence was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary
PR0179, PRO205, PR0321, PR0333, PR0364, PR0844, PR0846, PR0877, PR0879, PR0882, PR0885 and PRO 1760 were expressed in high 5 cells by the above described method
EXAMPLE 25
Preparation of Antibodies that Bind PROl 79, PRQ238, PRQ364. PRQ844 PRQ846. PROl 760. PRO205. PRQ321. PRQ333. PRO840, PRQ877. PRQ878, PRQ879. PRQ882. PRQ885 or PRQ887 This Example illustrates preparation of monoclonal antibodies that can specifically bind PRO 179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supia Immunogens that may be employed include purified PROl 79 PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 PR0333, PRO840, PR0877, PR0878 PR0879, PR0882, PR0885 or PR0887 fusion proteins containing PROl 79,PR0238, PR0364, PR0844, PR0846 PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 and cells expressing the gene encoding PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205 PR0321 , PR0333, PRO840 PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 on the cell surface Selection ot the immunogen can be made by the skilled artisan without undue experimentation
Mice, such as Balb/c, are immunized with the PRO 179, PR0238, PR0364 PR0844, PR0846, PRO 1760, PRO205, PR0321 PR0333, PRO840, PR0877, PR0878, PR0879, PR0882 PR0885 or PR0887 immunogen emulsified in complete Freund s adjuvant and injected subcutaneously or lntrapeπtoneally in an amount from 1 to 100 micrograms Alternatively, the immunogen is emulsified in MPL TDM adjuvant (Ribi Immunochemical Reseaich, 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 w ith additional immunization injections Serum samples may be periodically obtained from the mice by retro orbital bleeding for testing in ELISA assays to detect anti PRO 179, antι-PR0238, anti- PR0364, antι-PR0844, antι-PR0846, antι-PRO1760, antι-PRO205, antι-PR0321 antι-PR0333, antι-PRO840 antι-PR0877, anti PR0878, anti PR0879, antι-PR0882, anti PR0885 or anti PR0887 antibodies
After a suitable antibody titer has been detected, the animals positive toi antibodies can be injected with a final intravenous injection of PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 is within the skill in the art
The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti PR0179, antι-PR0238, antι-PR0364, antι-PR0844, antι-PR0846, anti-PROl 760, anti PRO205, antι-PR0321 , antι-PR0333, antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 or antι-PR0887 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-sulfate precipitation, followed by gel-exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed
Deposit of Material The following mateπal(s) has/have been deposited with the American Type Culture Collection, 10801
University Blvd , Manassas, VA 201 10-2209, USA (ATCC)
Material ATCC Den No Deposit Date
DNA16451-1388 209776 April 14, 1998
DNA35600 1 162 209370 October 16, 1997 DNA47365-1206 209436 November 7, 1997
DNA59838-1462 209976 June 16, 1998
DNA44196- 1353 209847 May 6, 1998
DNA76532-1702 203473 November 17, 1998
DNA34433 209719 March 31 , 1998 DNA53987 209858 May 12, 1998
This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty ) This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc , and ATCC, which assures permanent and unrestiicted 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 detei mined 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 material(s) on deposit should die or be lost or destroyed when cultivated under suitable conditions, the material(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 ofthe invention, including the best mode thereof, nor is it to be construed as limiting the scope ofthe claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS
1 A composition comprising a PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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-PRO 179, antι-PR0238, antι-PR0364, anti- PR0844, antι-PR0846, antι-PRO1760, antι-PRO205, antι-PR0321 , antι-PR0333, antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 or antι-PR0887 antibody
4 The composition of Claim 1 , wherein the antagonist is an anti-PROl 79, antι-PR0238, antι-PR0364, anti- PR0844, antι-PR0846, antι-PRO1760, antι-PRO205, antι-PR0321 , antι-PR0333, antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 or antι-PR0887 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 PRO 179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or agonist or antagonist thereof, with a pharmaceutically acceptable carrier
7 An article of manufacture comprising
(1 ) a composition comprising (a) a PR0179 PR0238, PR0364, PR0844 PR0846, PRO1760, PRO205 PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide, (b) an agonist of a PR0179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205. PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide, or (c) an antagonist of a PROl 79, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333. PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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, refemng to the use of said composition, in the treatment of a cardiovascular, endothelial, and angiogenic disordei
8 The article of manufacture of Claim 7, wherein said agonist is an anti-PRO 179. antι-PR0238, anti- PR0364, antι-PR0844, antι-PR0846, anti-PRO 1760, antι-PRO205, antι-PR0321 antι-PR0333, antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 or antι-PR0887 antibody
9 The article ot manufacture of Claim 7, wherein said antagonist is an anti-PRO 179. antι-PR0238, anti- PR0364, anti PR0844, antι-PR0846, antι-PRO1760, antι-PRO205, antι-PR0321 , antι-PR0333, antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 or antι-PR0887 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 earner
1 1 A method for identifying an agonist of a PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PROl 79 PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PROl 79, PR02 8, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877. PR0878 PR0879, PR0882, PR0885 oi PR0887 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 PRO 179, PR0238. PR0364, PRQ844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877 PR0878, PR0879, PR0882, PR0885 or PR0887 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 PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide
18 An antagonist of a PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide
19 A compound that inhibits the expression of a PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide in a mammalian cell which expresses said polypeptide
20 The compound of Claim 19, wherein said compound is an antisense ohgonucleotide
21 An isolated antibody that binds to a PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 ofthe 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 PRO 179 PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877. PR0878 PR0879, PR0882, PR0885 or PR0887 polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a contiol sample of known normal tissue cells of the same cell type, wherein a higher or lowei expression level in the test sample as compared to the control sample is indicative of the presence of a cardiovasculai , 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 ofthe 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-PR0179, anti-PR0238, anti-PR0364, anti-PR0844, anti-PR0846, anti-PRO1760, anti- PRO205, anti-PR0321 , anti-PR0333, anti-PRO840, anti-PR0877, anti-PR0878, anti-PR0879, anti-PR0882, anti- PR0885 or anti-PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0179, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321, PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide in a sample comprising contacting a sample suspected of containing said polypeptide with an anti-PRO 179, anti- PR0238, anti-PR0364, anti-PR0844, anti-PR0846, anti-PRO1760, anti-PRO205, anti-PR0321. anti-PR0333, anti-PRO840, anti-PR0877, anti-PR0878, anti-PR0879, anti-PR0882, anti-PR0885 or anti-PR0887antibody and determining binding of said antibody to a component of said sample.
30. A cardiovascular, endothelial or angiogenic disorder diagnostic kit comprising an anti-PRO 179, anti- PR0238, anti-PR0364, anti-PR0844. anti-PR0846, anti-PRO1760, anti-PRO205, anti-PR0321 , anti-PR0333, anti-PRO840, anti-PR0877, anti-PR0878, anti-PR0879, anti-PR0882. anti-PR0885 or anti-PR0887 antibody and a caπier 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 PROl 79, PR0238, PR0364. PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882. PR0885 or PR0887 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 PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide is administered together with a cardiovascular, endothelial or angiogenic agent
37 The method of Claim 34, wherein the PROl 79, PR0238, PR0364, PR0844, PR0846, PROl 760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 anti-PRO 179, antι-PR0238, antι-PR0364, anti- PR0844, antι-PR0846, antι-PRO1760, antι-PRO205, antι-PR0321 , antι-PR0333, antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 or antι-PR0887 antibody
41 The method of Claim 31 wherein said antagonist is an anti-PRO 179, antι-PR0238, antι-PR0364, anti- PR0844, antι-PR0846, antι-PRO1760, antι-PRO205, antι-PR0321 , antι-PR0333 antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 or antι-PR0887 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 PROl 79 PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or agonist or antagonist thereof
43 The method of Claim 42 wherein said agonist is an anti-PRO 179, antι-PR0238, antι-PR0364, anti- PR0844, antι-PR0846, anti-PRO 1760. antι-PRO205, antι-PR0321 , antι-PR0333 antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882 antι-PR0885 or antι-PR0887 antibody
44 The method of Claim 42 wherein said antagonist is an anti-PRO 179, antι-PR0238, antι-PR0364, anti- PR0844. antι-PR0846, anti-PRO 1760, antι-PRO205, antι-PR0321 , antι-PR0333 antι-PRO840, antι-PR0877, antι-PR0878, antι-PR0879, antι-PR0882, antι-PR0885 or antι-PR0887 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 PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333 PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 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 PR0333, PR0364, PR0877, PR0879, PR0882 or PR0885 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 PR0179, PR0321 , PRO840, PR0844, PR0846, PR0878 or PR0879 polypeptide or agonist thereof, wherein endothelial cell growth in said mammal is stimulated
51 A method ot inhibiting endothelial cell growth in a mammal comprising administering to the mammal an antagonist of a PRO 179, PR0321 , PRO840, PR0844, PR0846, PR0878 oi PR0879 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 PR0333, PR0364, PR0877, PR0879, PR0882 or PR0885 polypeptide, wherein endothelial cell growth in said mammal is stimulated
53 A method for inducing cardiac hypertrophy in a mammal comprising administering to the mammal a PRO205, PR0882 or PR0887 polypeptide or agonist thereof, wherein cardiac hypertrophy in said mammal is induced
54 A method of reducing cardiac hypertrophy in a mammal comprising administering to the mammal a PR0238, PR0878 or PRO 1760 polypeptide or agonist thereof, wherein cardiac hvpei trophy in said mammal is reduced 55 A method of inducing cardiac hypertrophy in a mammal comprising administering to the mammal an antagonist of a PR0238, PR0878 or PRO 1760 polypeptide, wherein cardiac hypertrophy in said mammal is reduced
56 A method of reducing cardiac hypertrophy in a mammal comprising administering to the mammal an antagonist of a PRO205, PR0882 or PR0887 polypeptide, wherein cardiac hypertrophy in said mammal is reduced
57 A method for inhibiting angiogenesis induced by a PR0179, PR0321 , PRO840, PR0844, PR0846, PR0878 or PR0879 polypeptide in a mammal comprising administering a therapeutically effective amount of an anti-PRO 179, antι-PR0321, antι-PRO840, antι-PR0844, antι-PR0846, antι-PR0878 or antι-PR0879 antibody to the mammal, wherein said angiogenesis is inhibited
58 A method for stimulating angiogenesis induced by a PRO 179, PR0321 , PRO840, PR0844, PR0846, PR0878 or PR0879 polypeptide in a mammal comprising administering a therapeutically effective amount of said polypeptide to the mammal, wherein said angiogenesis is stimulated
59 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8), Figure 10 (SEQ ID NO 10), Figure 12 (SEQ ID NO 12), Figure 14 (SEQ ID NO 14), Figure 16 (SEQ ID NO 16), Figure 18 (SEQ ID NO 18), Figure 20 (SEQ ID NO 20), Figure 22 (SEQ ID NO 22), Figure 24 (SEQ ID NO 24), Figure 26 (SEQ ID NO 26), Figure 28 (SEQ ID NO 28), Figure 30 (SEQ ID NO 30), and Figure 32 (SEQ ID NO 32)
60 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 3), Figure 5 (SEQ ID NO 5), Figure 7 (SEQ ID NO 7), Figuie 9 (SEQ ID NO 9), Figuie 1 1 (SEQ ID NO 1 1 ), Figure 13 (SEQ ID NO 1 ), Figure 15 (SEQ ID NO 15), Figure 17 (SEQ ID NO 17), Figure 19 (SEQ ID NO 19), Figure 21 (SEQ ID NO 21 ), Figure 23 (SEQ ID NO 23) Figure 25 (SEQ ID NO 25), Figure 27 (SEQ ID NO 27), Figure 29 (SEQ ID NO 29), and Figure 1 (SEQ ID NO 31 )
61 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 3) Figuie 5 (SEQ ID NO 5), Figure 7 (SEQ ID NO 7), Figure 9 (SEQ ID NO 9), Figure 1 1 (SEQ ID NO 1 1), Figure 13 (SEQ ID NO 13), Figure 15 (SEQ ID NO 15), Figure 17 (SEQ ID NO 17), Figure 19 (SEQ ID NO 19), Figure 21 (SEQ ID NO 21 ) Figure 23 (SEQ ID NO 23), Figure 25 (SEQ ID NO 25), Figure 27 (SEQ ID NO 27), Figure 29 (SEQ ID NO 29), and Figure 31 (SEQ ID NO 31 )
62 Isolated nucleic acid having at least 80% nucleic acid sequence identity to the full-length coding sequence of the DNA deposited under ATCC accession numbei 209776, 209370, 209436, 209976, 209847 203473, 209719, or 209858
63 A vector comprising the nucleic acid of any one of Claims 59 to 62
64 The vector of Claim 63 operably linked to control sequences recognized by a host cell transformed with the vector
65 A host cell comprising the vector of Claim 63
66 The host cell of Claim 65, wherein said cell is a CHO cell
67 The host cell of Claim 65, wherein said cell is an E coli
68 The host cell of Claim 65, wherein said cell is a yeast cell
69 The host cell of Claim 65, wherein said cell is a Baculovirus infected insect cell
70 A process for producing a PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PRO205, PR0321 , PR0333, PRO840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide comprising cultuπng the host cell of Claim 65 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture
71 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 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8), Figure 10 (SEQ ID NO 10), Figure 12 (SEQ ID NO 12) Figure 14 (SEQ ID NO 14), Figure 16 (SEQ ID NO 16) Figure 18 (SEQ ID NO 18), Figure 20 (SEQ ID NO 20) Figure 22 (SEQ ID NO 22), Figure 24 (SEQ ID NO 24), Figure 26 (SEQ ID NO 26) Figure 28 (SEQ ID NO 28), Figure 30 (SEQ ID NO 30), and Figure 32 (SEQ ID NO 32)
72 An isolated polypeptide scoring at least 80% positives when compared to an ammo acid sequence selected from the group consisting of the ammo acid sequence shown in Figure 2 (SEQ ID NO 2) Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8), Figure 10 (SEQ ID NO 10), Figure 12 (SEQ ID NO 12) Figure 14 (SEQ ID NO 14), Figure 16 (SEQ ID NO 16) Figui e 18 (SEQ ID NO 18), Figure 20 (SEQ ID NO 20) Figure 22 (SEQ ID NO 22), Figure 24 (SEQ ID NO 24) Figure 26 (SEQ ID NO 26), Figuie 28 (SEQ ID NO 28) Figure 30 (SEQ ID NO 30), and Figure 32 (SEQ ID NO 32)
73 An isolated polypeptide having at least 80% ammo acid sequence identity to an amino acid sequence encoded by the full-length coding sequence ofthe DNA deposited under ATCC accession number 209776 209370, 209436 209976 209847, 203473, 209719, or 209858 74 A chimeric molecule comprising a polypeptide according to any one of Claims 71 to 73 fused to a heterologous amino acid sequence
75 The chimeric molecule of Claim 74, wherein said heterologous amino acid sequence is an epitope tag sequence
76 The chimeric molecule of Claim 74, wherein said heterologous ammo acid sequence is a Fc region of an immunoglobulin
77 An antibody which specifically binds to a polypeptide according to any one of Claims 71 to 73
78 The antibody of Claim 77, wherein said antibody is a monoclonal antibody, a humanized antibody or a single chain antibody
79 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 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8), Figure 10 (SEQ ID NO 10), Figure 12 (SEQ ID NO 12), Figure 14 (SEQ ID NO 14), Figure 16 (SEQ ID NO 16), Figure 18 (SEQ ID NO 18), Figure 20 (SEQ ID NO 20), Figure 22 (SEQ ID NO 22), Figure 24 (SEQ ID NO 24), Figure 26 (SEQ ID NO 26), Figure 28 (SEQ ID NO 28) Figure 30 (SEQ ID NO 30), or Figure 32 (SEQ ID NO 32), lacking its associated signal peptide,
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2) Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) Figure 10 (SEQ ID NO 10), Figure 12 (SEQ ID NO 12), Figure 14 (SEQ ID NO 14), Figure 16 (SEQ ID NO 16) Figure 18 (SEQ ID NO 18), Figure 20 (SEQ ID NO 20), Figure 22 (SEQ ID NO 22), Figure 24 (SEQ ID NO 24) Figure 26 (SEQ ID NO 26), Figure 28 (SEQ ID NO 28), Figure 30 (SEQ ID NO 30), or Figure 32 (SEQ ID NO 32) with its associated signal peptide or
(c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2) Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) Figure 10 (SEQ ID NO 10) Figure 12 (SEQ ID NO 12), Figure 14 (SEQ ID NO 14), Figure 16 (SEQ ID NO 16) Figuie 18 (SEQ ID NO 18), Figure 20 (SEQ ID NO 20), Figure 22 (SEQ ID NO 22), Figure 24 (SEQ ID NO 24) Figure 26 (SEQ ID NO 26) Figure 28 (SEQ ID NO 28), Figure 30 (SEQ ID NO 30), or Figure 32 (SEQ ID NO 32) lacking its associated signal peptide
80 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 4) Figure 6 (SEQ ID NO 6) Figure 8 (SEQ ID NO 8), Figure 10 (SEQ ID NO 10), Figure 12 (SEQ ID NO 12) Figure 14 (SEQ ID NO 14) Figure 16 (SEQ ID NO 16), Figure 18 (SEQ ID NO 18), Figure 20 (SEQ ID NO 20) Figure 22 (SEQ ID NO 22), Figure 24 (SEQ ID NO 24), Figure 26 (SEQ ID NO 26), Figure 28 (SEQ ID NO 28) Figuie 30 (SEQ ID NO 30), or Figure 32 (SEQ ID NO 32) lacking its associated signal peptide, (b) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NOJ), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), or Figure 32 (SEQ ID NO:32), 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 NOJ), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), or Figure 32 (SEQ ID NO:32), lacking its associated signal peptide.
EP00912015A 1999-03-08 2000-02-24 Promotion or inhibition of angiogenesis and cardiovascularization Withdrawn EP1159419A1 (en)

Applications Claiming Priority (29)

Application Number Priority Date Filing Date Title
WOPCT/US99/05028 1999-03-08
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US12395799P 1999-03-12 1999-03-12
US123957P 1999-03-12
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US144758P 1999-07-20
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WOPCT/US99/20111 1999-09-01
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PCT/US1999/021090 WO2000015796A2 (en) 1998-09-16 1999-09-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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PCT/US1999/028409 WO2000032778A2 (en) 1998-12-01 1999-11-30 Methods and compositions for inhibiting neoplastic cell growth
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PCT/US1999/028313 WO2000032221A2 (en) 1998-12-01 1999-11-30 Promotion or inhibition of angiogenesis and cardiovascularization
WOPCT/US99/28565 1999-12-02
PCT/US1999/028565 WO2000037638A2 (en) 1998-12-22 1999-12-02 Methods and compositions for inhibiting neoplastic cell growth
WOPCT/US00/00219 2000-01-05
PCT/US2000/000219 WO2000053753A2 (en) 1999-03-08 2000-01-05 Promotion or inhibition of angiogenesis and cardiovascularization
WOPCT/US00/04342 2000-02-18
PCT/US2000/004342 WO2000078961A1 (en) 1999-06-23 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/004341 WO2000053756A2 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
WOPCT/US00/04341 2000-02-18
WOPCT/US00/04414 2000-02-22
PCT/US2000/004414 WO2001004311A1 (en) 1999-07-07 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/005004 WO2000053757A2 (en) 1999-03-08 2000-02-24 Promotion or inhibition of angiogenesis and cardiovascularization

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