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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
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  • 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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• 2000
  • 2000-02-24 WO PCT/US2000/005004 patent/WO2000053757A2/en active IP Right Grant
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  • 2000-02-24 CA CA002361849A patent/CA2361849A1/en not_active Abandoned
  • 2000-02-24 EP EP00912015A patent/EP1159419A1/de not_active Withdrawn
  • 2000-02-24 KR KR1020017011378D patent/KR20010104373A/ko unknown
  • 2000-02-24 KR KR1020017011378A patent/KR100553300B1/ko not_active IP Right Cessation
  • 2000-02-24 JP JP2000603378A patent/JP2003531811A/ja active Pending
• 2003
  • 2003-12-18 JP JP2003420475A patent/JP2004154140A/ja active Pending

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