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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
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
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  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • 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
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• • A—HUMAN NECESSITIES
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
  • A61P9/10—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
• • 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
  • A61P9/12—Antihypertensives
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to compositions and methods useful for promoting or inhibiting angiogenesis and/or cardiovascula ⁇ zation 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 hospitaiization for people age 65 and older in the United States Recent advances in the management of acute cardiac diseases, including acute myocardial infarction, are resulting in an expanding patient population that will eventually develop chronic heart failure From 1979 to 1995 hospitalizations for congestive heart failure (CHF) rose from 377 000 to 872 000 (a 130 percent increase) and CHF deaths increased 1 16 percent
• CHF is a syndrome characterized by left ventricular dysfunction, reduced exercise tolerance, impaired quality of life and markedly shortened life expectanc>
• 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 bodv'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 vasoconst ⁇ ction increased heart rate increased cardiac contractility and increased plasma volume These effects are mediated primarily by the sympathetic nervous system and the renm-angiotensm system See, Eichhorn, American Journal of Medicine.
• angiotensin II may also have directly deleterious effects on the heart by promoting myocyte necrosis (impairing systolic function) and intracar ac fibrosis (impairing diasto c 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 Morgan and Baker Circulation. 83 13-25 ( 1991)
• This hypertrophic response is frequently associated with a variety of distinct pathological conditions such as hypertension aortic stenosis myocardial infarction, cardiomyopathv valvular regurgitation. and intracardiac shunt all of which result in chronic hemodynamic overload
• Hypertrophy is generally defined as an increase in size of an organ or structure independent of natural growth that does not involve tumor formation Hypertrophy of the heart is due either to an increase in the mass of __ the individual cells (myocytes). or to an increase in the number of cells making up the tissue (hyperplasia), or both While the enlargement of an embryonic heart is largely dependent on an increase in myocyte number (which continues until shortly after birth), post-natal cardiac myocytes lose their proliferative capacity Further growth occurs through hypertrophy of the individual cells
• non-myocytes are primarily fibroblast/mesenchymal cells, they also include endothe al 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
• non-myocytes are primarily fibroblast/mesenchymal cells, they also include endothe al 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
• 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 at ⁇ al nat ⁇ uretic peptide (ANP) Chien et al .
• APN at ⁇ al nat ⁇ uretic peptide
• parac ⁇ ne factors produced by non-myocyte supporting cells may additionally be involved in the development of cardiac hypertrophy, and various non-myocyte derived hypertrophic factors, such as. leukocyte inhibitory factor (LIF) and endothe n, have been identified Metcalf, Growth Factors, 7 169- 173 (1992).
• LIF leukocyte inhibitory factor
• endothe n have been identified Metcalf, Growth Factors, 7 169- 173 (1992).
• LIF leukocyte inhibitory factor
• endothe n have been identified Metcalf, Growth Factors, 7 169- 173 (1992).
• LIF leukocyte inhibitory factor
• endothe n have been identified Metcalf, Growth Factors, 7 169- 173 (1992).
• LIF leukocyte inhibitory factor
• endothe n have been identified Metcalf, Growth Factors, 7 169- 173 (1992).
• LIF leukocyte inhibitory factor
• endothe n have been identified Met
• cardiac hypertrophy vanes depending on the underlying cardiac disease
• Catecholamines, adrenocorticosteroids, angiotensin. prostaglandins, LIF, endothehn (including endothel ⁇ n-1. -2, and -3 and big endothehn), and CT-1 are among the factors identified as potential mediators of hypertrophy
• beta-adrenergic receptor blocking drugs beta-blockers, e g , propranolol. timolol. tertalolol, carteolol, nadolol, betaxolol, penbutolol. acetobutolol. atenolol.
• beta-blockers have been used — extensively in the treatment of hypertrophic cardiomyopathy
• the beneficial effects of beta-blockers on symptoms (eg , chest pain) and exercise tolerance are largely due to a decrease in the heart rate with a consequent prolongation of diastole and increased passive ventricular filling Thompson et al , Br Heart J . 44 488-98 (1980),
• Nifedipine and diltiazem have also been used occasionally in the treatment of hypertrophic cardiomyopathy Lorell et al , Circulation 65 499-507 (1982). Betocchi et al . Am J Cardiol , 78 451-457 (1996) However, because of its potent vasodilating properties, nifedipine may be harmful, especially m patients with outflow obstruction Disopyramide has been used to relieve symptoms by virtue of its negative inotropic properties Pollick, N Engl J Med , 307 997-999 ( 1982) In many patients, however, the initial benefits decrease with time Wigle et al , Circulation, 92 1680- 1692 ( 1995) Antihypertensive drug therapy has been reported to have beneficial effects on cardiac hypertrophy associated with elevated blood pressure Examples of drugs used in antihypertensive therapy, alone or in combination, are calcium antagonists, e g , nitrendipine, adrenergic receptor blocking agents, e g , those
• Beta-adrenergic receptor blockers have recently been advocated for use in heart failure Evidence from clinical trials suggests that improvements in cardiac function can be achieved without increased mortality, though documented improvements patient survival have not yet been demonstrated See also U S Pat Nos 5 935,924 5,624,806. 5,661.122.
• Endothehn is a vasoconst ⁇ cting peptide comprising 21 amino acids, isolated from swine arterial endothe al culture supernatant and structurally determined Yanagisawa et al , Nature, 332 41 1-415 ( 1988) Endothehn was later found to exhibit various actions, and endothehn antibodies as endothehn antagonists have- proven effective in the treatment of myocardial infarction, renal failure, and other diseases Since endothehn is present in live bodies and exhibits vasoconst ⁇ cting action, it is expected to be an endogenous factor involved m the regulation of the circulatory system, and may be associated with hypertension, cardiovascular diseases such as myocardial infarction, and renal diseases such as acute renal failure Endothe
• ACE angiotensm-converting enzyme
• ACE inhibitors has been limited For example, while prolonging survival in the setting of heart failure. ACE inhibitors appear to slow the progression towards end-stage heart failure, and substantial numbers of patients on ACE inhibitors have functional class III heart failure
• thrombolytic agents e g , ⁇ ⁇ streptokinase.
• urokinase and in particular tissue plasminogen activator (t-PA) have significantly increased the survival of patients who suffered myocardial infarction
• t-PA tissue plasminogen activator
• t-PA may also be administered as a single bolus, although due to its relatively short half-life, it is better suited for infusion therap ⁇ Tebbe et al , Am J Cardiol .
• TNK t-PA a T103N I 17Q, KHRR(296-299)AAAAt-PA variant, Keyt etal Proc Nati Acad Sci USA, 9J . 3670-3674 ( 1994)
• TNK t-PA a T103N I 17Q, KHRR(296-299)AAAAt-PA variant, Keyt etal Proc Nati Acad Sci USA, 9J . 3670-3674 ( 1994)
• TNK t-PA a T103N I 17Q, KHRR(296-299)AAAAt-PA variant, Keyt etal Proc Nati Acad Sci USA, 9J . 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 factors
• PD-ECGF platelet-derived endotheiialcell growth factor
• VEGF vascular endothelial growth factor
• hVEGF human VEGF
• hVEGF-related proteins The 121-am ⁇ no acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 1 16 and 159 in hVEGF
• the 189-am ⁇ no acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 1 16 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF)
• hVPF human vascular permeability factor
• the 206-am ⁇ no acid protein differs from hVEGFby virtue of an insertion of 41 amino acids at residue 1 16 in hVEGF Houck et al
• angiogenesis which involves the formation of new blood vessels from preexisting endothelium. is implicated in the pathogenesis of a variety of disorders These include solid tumors and metastasis, atherosclerosis, retrolental fibropiasia. hemangiomas. chronic inflammation, intraocular neovascular syndromes such as prohferative retinopathies, e , diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis Folkman etal J Biol Chem .267 10931-10934 (1992).
• the search for positive regulators of angiogenesis has yielded many candidates, including aFGF, bFGF, TGF- ⁇ , TGF- ⁇ , HGF, TNF-cc. angiogenin, IL-8, etc Folkman et al J B C , supra and Klagsbrun et al , supra
• the negative regulators so far identified include thrombospondin (Good et ⁇ l , Proc Nati Acad Sci USA , 87 6624- 6628 (1990)), the 16-kilodalton N-terminal fragment of prolactin (Clapp et ⁇ l Endocrinology, 133 1292-1299 (1993)), angiostatin (O'Reilly er a/ , Cell, 79 315-328 (1994)), and endostatin O'Reilly et l Cell, 88 277-285
• VEGF has been shown to be a key mediator of neovascula ⁇ zation associated with tumors and intraocular disorders Ferrara et ⁇ l , Endocr Rev , supr
• the VEGF mRNA is overexpressed by the majority of human tumors examined Berkman ef ⁇ / J Clin Invest , 91 153-159 (1993). Brown et al . Human Pathol , 26 86-91 (1995), Brown et al Cancer Res . 53 4727-4735 (1993), Mattern e/ al Brit J Cancer, 73 931-934 (1996), Dvorak et al , Am J Pathol . 146 1029-1039 (1995)
• VEGF vascular endothelial growth factor
• concentration levels of VEGF in eye fluids are highly correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies Aiello et al , N_ Engl J Med , 331 1480-1487 (1994)
• recent studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by AMD Lopez et al Invest Ophthalmol Vis Sci . 37 855-868 (1996)
• Anti- VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in nude mice (Kim e/ ⁇ / Nature. 362 841-844 ( 1993), Warren et al J Clin Invest . 95 1789- 1797 (1995). Borgstrom et al Cancer Res , 56 4032-4039 ( 1996) Melnvk et al Cancer Res 56 921 -924 ( 1996)) and also inhibit intraocular angiogenesis in models of lschemic retinal disorders Adamis et al Arch Ophthalmol .
• anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of solid tumors and various intraocular neovascular disorders
• Such antibodies are described, for example, in EP 817,648 published January 14 1998 and in PCT/US 98/06724 filed April 3. 1998
• cvr 61 which is rapidlv activated bv serum- or platelet-derived growth factor (PDGF) (O'Brien et al . Mol Cell Biol Jj) 3569-3577 ( 1990).
• PDGF platelet-derived growth factor
• CTGF human connective tissue growth factor
• TGF- ⁇ transforming growth factor beta
• IGFBPs insulin-like growth factor binding proteins
• IGFBP insulin growth factor
• the present invention concerns compositions and methods for promoting or 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
• 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 PRO230 PR0216 or PRO302 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,
• 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 PRO230, PR0216 or PRO302 polypeptide with a pharmaceutically acceptable carrier
• the present invention provides a composition comprising an agonist or antagonist of a PRO230. PR0216 or PRO302 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
• PRO230, PR0216 or PRO302 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 PRO230 PR0216 or PRO302 polypeptide agonist or antagonist, and might, therefore, be suitable for repeated use
• the present invention provides a method for preparing such a composition usefuifor the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of a PRO230, PR0216 or PRO302 polypeptide agonist or antagonist with a pharmaceutically acceptable carrier
• the present invention concerns a composition
• a composition comprising an ant ⁇ -PRO230. anti- PR0216 or ant ⁇ -PRO302 antibody in admixture with a pharmaceutically acceptable carrier
• the composition comprises a therapeutically effective amount of the ant ⁇ bod ⁇
• the composition comprises a further active ingredient, namelv.
• the composition is sterile
• the composition may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended ⁇ ⁇ storage stability
• Preserved liquid pharmaceutical formulations might contain multiple doses of the ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody, and might, therefore be suitable for repeated use
• the antibody is a monoclonal antibody, an antibody fragment, a humanized antibody, or a single- chain antibody
• the present invention provides a method for preparing such a composition useful for the treatment of a cardiovascular, endothelial or angiogenic disorder comprising admixing a therapeutically effective amount of an ant ⁇ -PRO230.
• ant ⁇ -PR0216 or ant ⁇ -PRO302 antibodv with a pharmaceuticallv acceptable carrier
• the present invention provides an article of manufacture comprising
• composition of matter comprising a therapeutically effective dosage of a PRO230, PR0216 or PRO302 polypeptide
• the present invention provides an article of manufacture comprising
• composition of matter comprising a therapeutically effective dosage of a PRO230.
• PR0216 or PRO302 polypeptide agonist or antagonist a composition of matter comprising a therapeutically effective dosage of a PRO230.
• PR0216 or PRO302 polypeptide agonist or antagonist a composition of matter comprising a therapeutically effective dosage of a PRO230.
• PR0216 or PRO302 polypeptide agonist or antagonist a composition of matter comprising a therapeutically effective dosage of a PRO230. PR0216 or PRO302 polypeptide agonist or antagonist
• the present invention provides an article of maufacture comprising
• composition of matter comprising a therapeutically effective dosage of an ant ⁇ -PRO230.
• ant ⁇ -PR0216 or ant ⁇ -PR0302 antibody (a) a composition of matter comprising a therapeutically effective dosage of an ant ⁇ -PRO230.
• ant ⁇ -PR0216 or ant ⁇ -PR0302 antibody (a) a composition of matter comprising a therapeutically effective dosage of an ant ⁇ -PRO230.
• ant ⁇ -PR0216 or ant ⁇ -PR0302 antibody (a) a composition of matter comprising a therapeutically effective dosage of an ant ⁇ -PRO230.
• ant ⁇ -PR0216 or ant ⁇ -PR0302 antibody (a) a composition of matter comprising a therapeutically effective dosage of an ant ⁇ -PRO230.
• ant ⁇ -PR0216 or ant ⁇ -PR0302 antibody (a) a composition of matter comprising a therapeutically effective dosage of an ant ⁇ -PRO230.
• ant ⁇ -PR0216 or ant ⁇ -PR0302 antibody
• the present invention provides a method for identifying an agonist of a PRO230, PR0216 or PRO302 polypeptide comprising
• the present invention provides a method for identifying an agonist of a PRO230. — PR0216 or PRO302 polypeptide comprising
• the invention provides a method for identifying a compound that inhibits the activity of a PRO230 PR0216 or PRO302 polypeptide comprising contacting a test compound with a PRO230. PR0216 or PRO302 polypeptide under conditions and for a time sufficient to allow the test compound and polypeptide to interact and determining whether the activity of the PRO230, PR0216 or PRO302 polypeptide is inhibited
• either the test compound or the PRO230, PR0216 or PRO302 polypeptide is immobilized on a solid support
• the non-immobihzed component carries a detectable label
• this method comprises the steps of (a) contacting cells and a test compound to be screened in the presence of PRO230, PR0216 or PRO302 polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO230, PR0216 or PRO302 polypeptide, and
• this process comprises the steps of
• the invention provides a method for identifying a compound that inhibits the expression of a PRO230, PR0216 or PRO302 polypeptide in cells that normally expresses the polypeptide. wherein the method comprises contacting the ceils with a test compound and determining whether the expression of the PRO230, PR0216 or PRO302 polypeptide is inhibited In a preferred aspect, this method comprises the steps of
• the invention provides a compound that inhibits the expression of a PRO230, PR0216 or PRO302 polypeptide.
• a compound that is identified b ⁇ the methods set forth above such as a compound that is identified b ⁇ the methods set forth above
• Another aspect of the present invention is directed to an agonist or an antagonist of a PRO230.
• PR0216 or PRO302 polypeptide which may optionally be identified by the methods described above
• the invention provides an isolated antibody that binds a PRO230.
• PR0216 or PRO302 polypeptide is a monoclonal antibody, which preferably has non-human complementarity-determining — region (CDR) residues and human framework-region (FR) residues
• CDR non-human complementarity-determining — region
• FR human framework-region
• the antibody may be labeled and may be immobilized on a solid support
• the antibody is an antibody fragment a single-chain antibody, or a humanized antibody
• the antibody specifically binds to the polypeptide
• the present invention provides a method for diagnosing a disease or susceptibility to a disease which is related to a mutation in a PRO230.
• 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 PRO230,
• PR0216 or PRO302 polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher oi 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
• PR0216 or PRO302 polypeptide may optionally be accomplished bv measuring the level of mRNA or polypeptide in the test sample as compared to the control sample
• the present invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal which comprises detecting the presence or absence of a PRO230, PR0216 or PRO302 polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of said PRO230, PR0216 or PRO302 polypeptide in said test sample is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in said mammal
• the invention provides a method of diagnosing a cardiovascular, endothelial or angiogenic disorder in a mammal comprising (a) contacting an ant ⁇ -PRO230. ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the PRO230.
• PR0216 or PRO302 polypeptide in the test sample wherein the formation of said complex is indicative of the presence of a cardiovascular, endothelial or angiogenic disorder in the mammal
• the detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells ot the same cell tvpe A larger or smaller quantity of complexes formed in the test sample indicates the presence of a cardiovascular, endothelial or angiogenic dysfunction in the mammal from which the test tissue cells were obtained
• the antibody preferably carries a detectable label Complex formation can be monitored for example, by light microscopy flow cytometry, fluo ⁇ metry, or other techniques known in the art
• the test sample is usually obtained from an individual suspected to have a cardiovascular, endothelial or angiogenic disorder
• the invention provides a method for determining the presence ot a PRO230, PR0216 — or PRO302 polypeptide in a sample comprising exposing a sample suspected of containing the PRO230, PR0216 or PRO302 polypeptide to an ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody and determining binding of said antibody to a component of said sample
• the sample comprises a cell suspected of containing the PRO230, PR0216 or PRO302 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 comp ⁇ singan ant ⁇ -PRO230.
• ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody and a carrier in suitable packaging Preferably, such kit further comprises instructions for using said antibody to detect the presence of the PRO230.
• the carrier is a buffer, for example
• the cardiovascular, endothelial or angiogenic disorder is cancer
• the invention provides an article of manufacture, comprising a container, a label on the container, and a composition comprising a PRO230, PR0216 or PRO302 polypeptide contained within the container, wherein the label on the container indicates that the composition can be used for treating cardiovascular, endothelial or angiogenic disorders
• the invention provides an article of manufacture, comprising a container, a label on the container, and a composition comprising a PRO230, PR0216 or PRO302 polypeptide agonist or antagonist contained within the container, wherein the label on the container indicates that the composition can be used for treating cardiovascular, endothelial or angiogenic disorders
• the invention provides an article of manufacture, comprising a container, a label on the container, and a composition comprising an ant ⁇ -PRO230. ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody contained within the container, wherein the label on the container indicates that the composition can be used for treating cardiovascular endothelial or angiogenic disorders
• 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 PRO230, PR0216 or PRO302 polypeptide
• the disorder is cardiac hype ⁇ rophy trauma such as wounds or burns, or a type of cancer
• the mammal is further exposed to angioplastv 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
• the cardiac hypertrophy may be induced by myocardial infarction, wherein preferably the __ administration of PRO230.
• PR0216 or PRO302 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 PRO230, PR0216 or PRO302 polypeptide is administered together with a cardiovascular, endothelial or angiogenic agent
• the preferred cardiovascular, endothelial or angiogenic agent for this purpose is selected from the group consisting of an antihypertensive drug, an ACE inhibitor, an endothehn receptor antagonist and a thrombolytic agent If a thrombolvtic agent is administered, preferablv the PRO230, PR0216 or PRO302 polypeptide is administered following administration of such agent More preferably, the thrombolytic agent is
• the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy and the PRO230, PR0216 or PRO302 polypeptide is administered following primary angioplasty for the treatment of acute myocardial infarction, preferably wherein the mammal is further exposed to angioplasty or a cardiovascular, endothelial, or angiogenic agent
• the cardiovascular, endothelial or angiogenic disorder is a cancer and the PRO230, PR0216 or PRO302 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 m a mammal comprising administering to the mammal an effective amount of an agonist of a PRO230, PR0216 or PRO302 polypeptide
• the cardiovascular, endothelial or angiogenic disorder is cardiac hypertrophy, trauma, a cancer, or age-related macular degeneration
• the mammal is human, and where an effective amount of an angiogenic or angiostatic agent is administered in conjunction with the agonist
• the invention concerns a method for treating a cardiovascular, endothelial or angiogenic disorder in a mammal comprising administering to the mammal an effective amount of an antagonist of a PRO230.
• 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 ant ⁇ -PRO230, ant ⁇ -PR0216orant ⁇ -PRO302ant ⁇ body
• 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 PRO230.
• PR0216 or PRO302 polypeptide (b) an agonist of a PRO230. - PR0216 or PRO302 polypeptide or (c) an antagonist of a PRO230, PR0216 or PRO302 polypeptide. wherein said agonist or antagonist may be an ant ⁇ -PRO230. ant ⁇ -PR0216 or ant ⁇ -PRO302 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 PRO230. PR0216 or PRO302 polypeptide. (b) an agonist polypeptide of a PRO230. PR0216 or PRO302 polypeptide. or (c) an antagonist polypeptide of a PRO230.
• the retroviral vector is in association with retroviral structural proteins
• the signal sequence is from a mammal, such as from a native PRO230, PR0216 or PRO302 polypeptide
• the invention supplies an ex vivo producer cell comprising a nucleic acid construct that expresses retroviral structural proteins and also comprises a retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PRO230, PR0216 or PRO302 polypeptide, (b) an agonist polypeptide of a PRO230, PR0216 or PRO302 polypeptide or (c) an antagonist polypeptide of a PRO230. PR0216 or PRO302 polypeptide. and a signal sequence for cellular secretion of the polypeptide.
• the invention provides a method for inhibiting endothelial cell growth in a mammal comprising administering to the mammal (a) a PR0216 polypeptide. (b) an agonist of a PR0216 polypeptide, (c) an antagonist of a PR0216 polypeptide.
• the invention provides a method for stimulating endothelial cell growth in a mammal comprising administering to the mammal (a) a PRO230 polypeptide (b) an agonist of a PRO230 polypeptide (c) an antagonist of a PRO230 polypeptide.
• the mammal is human in yet another embodiment, the invention provides a method for inhibiting angiogenesis induced by a PRO302 polypeptide in a mammal comprising administering a therapeutically effective amount of an ant ⁇ -PRO302 antibody to the mammal
• the mammal is a human, and more preferably the mammal has a tumor or a retinal disorder
• Figures 1 A through I B show a nucleotide sequence (SEQ ID NO 1 ) ot a native sequence PRO230 cDNA. wherein SEQ ID NO 1 is a clone designated herein as DNA33223-1 136"
• Figure 2 shows the amino acid sequence (SEQ ID NO 2) derived from the coding sequence of SEQ ID NO 1 shown in Figures I A through I B
• Figure 3 shows a nucleotide sequence (SEQ ID NO 6) of a native sequence PR0216 cD A wherein SEQ ID NO 6 is a clone designated herein as DNA33087" ___
• Figure 4 shows the amino acid sequence (SEQ ID NO 7) derived from the coding sequence of SEQ ID NO 6 shown in Figure 3
• Figure 5 shows a nucleotide sequence (SEQ ID NO 1 1 ) of a native sequence PRO302 cDNA wherein SEQ
• ID NO 1 1 is a clone designated herein as "DNA40370- 1217"
• Figure 6 shows the amino acid sequence (SEQ ID NO 12) derived from the coding sequence of SEQ ID NO 11 shown in Figure 5
• Figures 7 A through 7D show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity ( Figures 7A-B) and % nucleic acid sequence identity ( Figures 7C-D) using the
• ALIGN-2 sequence comparison computer program wherein 'PRO " ' represents the amino acid sequence of a hypothet ⁇ calPRO230, PR0216 or PRO302 polypeptide of interest, "Comparison Protein "” represents the ammo acid sequence of a polypeptide against which the "PRO” polypeptide of interest is being compared '"PRO-DNA” represents a hypothetical PRO230-, PR0216- or PRO302-encod ⁇ ng 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
• Figures 8A through 8Q provide the complete source code for the ALIGN-2 sequence comparison computer program This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN- 2 sequence comparison computer program
• cardiovascular, endothelial and angiogenic disorder cardiac endothelial and angiogenic disorder
• cardiac endothelial and angiogenic dysfunction cardiac endothelial or angiogenic disorder
• 'cardiovascular, endothelial or angiogenic disorder cardiac endothelial or angiogenic disfunction
• Such disorders include, for example, arterial disease, such as atherosclerosis, hypertension, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, and arterial restenosis.
• venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema
• other vascular disorders such as peripheral vascular disease, cancer such as vascular tumors, e g , hemangioma (capillary and cavernous) glomus tumors telangiectasia bacillary angiomatosis hemangioendothelioma angiosarcoma haemangiope ⁇ cytoma Kaposi s sarcoma, lymphangioma and lymphangiosarcoma, tumor angiogenesis.
• 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 ceils (true hypertrophy), or to an increase in the number of cells making up the tissue (hyperplasia), or both Certain organs, such as the heart, lose the ability to divide shortly after birth Accordingly,
• 'cardiac hypertrophy is defined as an increase in mass of the heart, which, in adults, is characterized by an increase in myocyte cell size and contractile protein content without concomitant cell division The character of the stress responsible for inciting the hypertrophy, (e g , increased preload, increased afterload, loss of myocytes.
• cardiac hypertrophy is usually characterized morphologically by increases m the size ot 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 At a more advanced stage ofcardiac hypertrophy, there are preferential increases in the size or number of specific organelles, such as mitochondria, and new contractile elements are added in localized areas of the cells, in an irregular manner Cells subjected to long-standing hypertrophy show more obvious disruptions in cellular organization, including markedly enlarged nuclei with highly lobulated membranes, which displace adjacent myofib ⁇ ls and cause breakdown of normal Z-band registration
• cardiac hypertrophy is used to include all stages of the progression of this condition, characterized by various degrees of structural damage of the heart muscle, regardless of the underlying cardiac disorder.
• Heart failure refers to an abnormality of cardiac function where the heart does not pump blood at the rate needed for the requirements of metabolizing tissues
• the heart failure can be caused by a number of factors, including lschemic. congenital, rheumatic, or ldiopathic forms
• CHF Congestive heart failure
• Myocardial infarction generally results from atherosclerosis of the coronary arteries, often with superimposed coronary thrombosis It may be divided into two major types transmural infarcts. in which myocardial necrosis involves the full thickness of the ventricular wall, and subendocardial (nontransmuraL) infarcts, in which the necrosis involves the subendocardium, the intramural myocardium, or both, without extending all the way through the ventricular wall to the epicardium Vlyocardial infarction is known to cause both a change in hemodynamic effects and an alteration in structure in the damaged and healthy zones of the heart Thus, for example, myocardial infarction reduces the maximum cardiac output and the stroke volume of the heart Also associated with myocardial infarction is a stimulation of the DNA synthesis occurring in the interstice as well as an increase in the formation of collagen in the areas of the heart not affected
• hypertrophic 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), Spi ⁇ to et al N Engl J Med , 320 749-755 (1989), Louie and Edwards, Prog Cardiovasc Pis , 36 275-308 ( 1994), Wigle et al , Circulation, 92 1680- 1692 ( 1995)), the heterogeneity of which is accentuated by the fact that it afflicts patients of all ages Spi ⁇ to er ⁇ / N Engl J Med .
• Supravalvular "aortic stenosis ' ' is an inherited vascular disorder characterized by narrowing of the ascending aorta, but other arteries, including the pulmonary arteries, may also be affected
• Untreated aortic stenosis may lead to increased intracardiac pressure resulting in myocardial hypertrophy and eventually heart failure and death The pathogenesis of this disorder is not fully understood, but hypertrophy and possibly hyperplasia of medial smooth muscle are prominent features of this disorder It has been reported that molecular variants of the elastm gene are involved in the development and pathogenesis of aortic stenosis U S Patent No 5.650,282 issued July 22, 1997
• Valvular regurgitation occurs as a result of heart diseases resulting in disorders of the cardiac valves
• Various diseases like rheumatic fever, can cause the shrinking or pulling apart of the valve orifice, while other diseases may result in endocarditis an inflammation of the endocardium or iin ing membrane of the at ⁇ ovent ⁇ cular orifices and operation of the heart Defects such as the narrowing of the valve stenosis or the defective closing of the valve result in an accumulation ot blood in the heart cavity or regurgitation of blood past the valve If uncorrected, prolonged valvular stenosis or insufficiency may result in cardiac hypertrophy and associated damage to the heart muscle, which may eventually necessitate valve replacement
• cancer refers to or describe the physiological condition in mammals that is typically characterized bv 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 sandnon-Hodgkin s lymphoma, pancreatic cancer, g oblastoma. 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 neck cancer
• the preferred cancers for treatment herein are breast, colon, lung, melanoma, ovarian, and others involving vascular tumors as noted above
• 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 ( g ,
• chemotherapeutic agent is a chemical compound useful in the treatment of cancer
• examples of chemotherapeutic agents include alkylating agents, folic acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics py ⁇ midine analogs, 5-fluorourac ⁇ l. cisplatin, pu ⁇ ne nucleosides. amines, amino acids. t ⁇ azol nucleosides or corticosteroids Specific examples include Ad ⁇ amycm. Doxorubicin. 5-Fluorourac ⁇ l,
• Cytosine arabinoside (“Ara-C"), Cyclophosphamide. Thiotepa, Busulfan. Cytoxin, Taxol, Toxotere Methotrexate Cisplatin. Melphalan. Vinblastine. Bleomycin. Etoposide. Ifosfamide. Mitomycin C. Mitoxantrone, Vincreistine, Vmorelbine, Carboplat , Teniposide. Daunomycin, Carminomycin, Aminopte ⁇ n, Dactinomycin. Mitomycins. Esperamicms (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 vitro or in vivo
• the growth-inhibitory agent is one which significantly reduces the percentage of malignant cells in S phase
• growth-inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G 1 arrest and M-phase arrest
• Classical M-phase blockers include the vincas (vinc ⁇ stine and vinblastine). taxol, and topo II inhibitors such as doxorubicin, daunorubicin. etoposide, and bleomycin Those agents that arrest G 1 also spill over into S-phase arrest, for example.
• DNA alkylating agents such as tamoxifen, prednisone. dacarbazine, mechlorethamine. cisplatin. methotrexate. 5-fluorourac ⁇ l. and ara-C Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds , Chapter 1. entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al (WB Saunders Philadelphia, 1995), especially p 13 Additional examples include tumor necrosis factor (TNF).
• TNF tumor necrosis factor
• 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 ischemic insults, such as myocardial infarction
• Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial effect, such as an anti-hypertrophic effect, for an extended period of time
• mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, pigs, etc Preferably, the mammal is human
• Administration in combination with one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order
• 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
• angiotensm-II receptor antagonists include angiotensm-II receptor antagonists, endothehn 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 longer half-life and very high fibrin specificity.
• TNK t-PA (a Tl 03N. N 1 17Q. KHRR(296-299)AAAA t-PA variant, Keyt et al , Proc Nati Acad Sci USA 91 , 3670-3674 (1994)
• inotropic or hypertensive agents such as digoxigenin and ⁇ -adrenergic receptor blocking agents, e g , propranolol. timolol. tertalolol, carteolol, nadolol.
• 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, hvdroflumethazide. methvlchlothiazide benzthiazide. dichlo ⁇ henamide acetazolamide. and indapamide.
• ACE angiotensin converting enzyme
• Angiogenic agents and "endothelial agents” are active agents that promote angiogenesis and/or endothelial cell growth, or. if applicable, vasculogenesis This would include factors that accelerate wound healing, such as growth hormone, insulin-like growth factor-I (IGF-I). VEGF, VIGF, PDGF, epidermal growth factor (EGF). CTGF _ and members of its family. FGF, and TGF- ⁇ and TGF- ⁇
• Angiostatic agents are active agents that inhibit angiogenesis or vasculogenesis or otherwise inhibit or prevent growth of cancer cells
• examples include antibodies or other antagonists to angiogenic agents as defined above, such as antibodies to VEGF They additionally include cytotherapeutic agents such as cytotoxic agents, chemotherapeutic agents, growth-inhibitory agents, apoptotic agents, and other agents to treat cancer, such as anti-
• HER-2 HER-2, ant ⁇ -CD20. and other bioactive and organic chemical agents
• a "therapeutically effective amount" of an active agent such as a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto or an anti-
• PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody refers to an amount effective in the treatment of a cardiovascular, endothelial or angiogenic disorder in a mammal and can be determined empirically
• an "effective amount" of an active agent such as a PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto or an ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody, refers to an amount effective for carrying out a stated pu ⁇ ose, wherein such amounts may be determined empirically for the desired effect
• PRO230 Asusedherein.the terms "PRO230". " PR0216”, “PRO302”, “PRO230 polypeptide” "PR0216 polypeptide” or “PRO302 polypeptide” when used herein encompass native-sequence PRO230. PR0216 or PRO302 polypeptides and PRO230. PR0216 or PRO302 polypeptide variants (which are further defined herein) The PRO230. PR0216 or PRO302 polypeptide may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods
• a “native sequence” PRO230, PR0216 or PRO302 polypeptide comprises a polypeptide having the same ammo acid sequence as a PRO230, PR0216 or PRO302 polypeptide derived from nature Such native sequence PRO230, PR0216 or PRO302 polypeptide can be isolated from nature or can be produced by recombinant and/or synthetic means
• the term "native sequence" PRO230, PR0216 or PRO302 polypeptide specifically encompasses naturally-occurring truncated or secreted forms (e g , an extracellular domain sequence), naturally-occur ⁇ ngva ⁇ ant forms (e g , alternatively spliced forms), and naturally-occurring allelic variants of the PRO230, PR0216 and PRO302 polypeptide
• the native-sequence PRO230, PR0216 or PRO302 polypeptide is a mature or full-length native sequence PRO230.
• PR0216 or PRO302 polypeptide comprising the amino acid sequence of Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7) or Figure 6 (SEQ ID NO 12).
• Fragments of the respective native polypeptides herein include, but are not limited, to polypeptide variants from which the native N-termmal signal sequence has been fully or partially deleted or replaced by another sequence. and extracellulardomains of the respective native sequences, regardless whether such truncated (secreted) forms occur in nature Fragments are preterablv sufficient in length for the production ot an antibody specifically binding the corresponding native "PRO" polypeptide
• PRO230 variant polypeptide means an active PRO230 polypeptide (other than a native sequence PRO230 polypeptide) as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2).
• PR0216 variant polypeptide means an active PR0216 polypeptide (other than a native sequence PR0216 polypeptide) as defined below having at least about 80% amino acid sequence identity with the am o acid sequence of (a) residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO 7) or (b) another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 7)
• PRO302 variant polypeptide means an active PRO302 polypeptide (other than a native sequence PRO302 polypeptide) as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of (a) residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12).
• PRO230, PR0216 or PRO302 variant polypeptides include, for instance, PRO230, PR0216 or PRO302 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C-termmus, as well as withm one or more internal domains, of the sequence of Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7) or Figure 6 (SEQ ID NO 12), respectively
• a PRO230 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% ammo acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% ammo acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 9
• a PR0216 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% ammo acid sequence identity, more preferably at least about
• amino acid sequence identity more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% am o acid __ sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% ammo acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% ammo acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with (a) residues 1 to 421 of the PR0216 polypeptide shown in Figure 4
• a PRO302 variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% ammo acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about
• ammo acid sequence identity more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% ammo acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preterablv at least about 92% ammo acid sequence identity, more preferably at least about 93% am o acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% ammo acid sequence identity with (a) residues 1 or about 26 to 452 of the PRO302 polypeptide shown in Figure 6 (SEQ ID NO 12), (b) X to 452 of the PRO30
• Variants do not encompass the native PRO230.
• PR0216 or PRO302 polypeptide sequence Ordinarily.
• PR0216 and PRO302 variant polypeptides are at least about 10 amino acids in length, often at least about
• amino acids in length more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 ammo acids in length, more often at least about 60 amino acids in
• Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with the ammo acid residues in a PRO230.
• PR0216 or PRO302 sequence after aligning the __ sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity
• Alignment for pu ⁇ oses of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST. BLAST-2, ALIGN, ALIGN-2 or Megahgn (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 pu ⁇ oses 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-
• the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc , and the source code shown in Figures 8A-Q has been filed with user documentation in the U.S Copyright Office, Washington D C, 20559, where it is registered under U S Copyright Registration No TXU510087
• the ALIGN-2 program is publicly available through Genentech, Inc . South San Francisco, California or may be compiled from the source code provided in Figures 8A-Q
• the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
• % amino acid sequence identity of a given amino acid sequence A to. with, or against a given amino acid sequence B is calculated as follows
• % amino acid sequence identity used herein are obtained as described above using the ALIGN-2 sequence comparison computer program
• % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al . Nucleic Acids Res . 25 3389-3402 ( 1997))
• NCBI-BLAST2 sequence comparison program mav be downloaded from http //www ncbi nlm nih gov
• % ammo acid sequence identity of a given amino acid sequence A to. with, or against a given amino acid sequence B is calculated as follows
• NCBI-BLAST2 in that program ' s alignment of A and B. and where Y is the total number of ammo acid residues in B
• % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzv ology, 266 460-480 (1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values, ; e .
• a % ammo acid sequence identitv 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 (/ 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 ammo acid residues of the PRO polypeptide of interest
• the am o acid sequence identitv 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 (/ 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 ammo acid residues of the PRO
• A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest
• PRO230 variant polynucleotide or 'PRO230 variant nucleic acid sequence means a nucleic acid molecule which encodes an active PRO230 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 164 o the
• PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes amino acids X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2). wherein X is any ammo acid residue from 17 to 26 of Figure 2 (SEQ ID NO 2) or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2)
• a PRO230 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity more preferably at least about 82% nucleic acid sequence identity more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about __ 88%
• nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), (b) a nucleic acid sequence which encodes amino acids X to 164 of the PRO230 polypeptide shown in Figure 2 (SEQ ID NO 2), wherein X is any ammo acid residue from 17 to 26 of Figure 2 (SEQ ID NO 2) or (c) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID NO 2) PRO230 polynucleotide variants do not encompass the native PRO230
• PR0216 variant polynucleotide or "PR0216 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PR0216 polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO 7) or (b) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 7)
• a PR0216 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence
• nucleic acid sequence identity with either (a) a nucleic acid sequence which encodes residues 1 to 421 of the PR0216 polypeptide shown in Figure 4 (SEQ ID NO 7) or (b) a nucleic acid sequence which encodes another specifically derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID NO 7) PR0216 polynucle
• PRO302 polynucleotide variants do not encompass the native PRO302 nucleotide sequence
• PRO230, PR0216 and PRO302 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in
• Percent (%) nucleic acid sequence identity with respect to the PRO230.
• PR0216 and PRO302 polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage ot nucleotides in a candidate sequence that are identical with the nucleotides in a PRO230.
• PR0216 or PRO302 polypeptide-encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity Alignment for pu ⁇ oses 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
• the ALIGN-2 sequence comparison computer program was authored by Genentech. Inc , and the source code shown in Figures 8A-Q has been filed with user documentation in the U S Copyright Office, Washington D C .
• ALIGN-2 program is publicly available through Genentech. Inc . South San Francisco, California or mav be compiled from the source code provided in Figures 8A- Q
• the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX
• % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows
• % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al . Nucleic Acids Res . 25 3389-3402 (1997))
• NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih.gov
• % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows
• a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide- encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide- encoding nucleic acid and the comparison nucleic acid molecule of interest (; e , the sequence against which the
• PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide- encoding nucleic acid molecule of interest
• the nucleic acid sequence A is the comparison nucleic acid molecule of interest
• the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest
• PRO230, PR0216 and PRO302 variant polynucleotides are nucleic acid molecules that encode an active PRO230, PR0216 or PRO302 polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO230, PR0216 or PRO302 polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 7) or Figure 6 (SEQ
• PRO230, PR0216 and PRO302 variant polypeptides may be those that are encoded by a PRO230, PR0216 or PRO302 variant polynucleotide
• ammo acid residues in the context of the ammo acid sequence identity comparisons performed as described above, includes ammo 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 ammo acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 1 below) of the amino acid residue of interest
• the % value of positives ot a given amino acid sequence A to with, or against a given am o 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 ammo acid sequence B) is calculated as follows
• isolated when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide. and mav include enzvmes.
• the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO230, PR0216 or PRO302 natural environment will not be present Ordinarily, however, isolated polypeptide will be prepared by at least one purification step
• An "isolated" nucleic acid molecule encoding a PRO230, PR0216 or PRO302 polypeptide or an "isolated” nucleic acid molecule encoding an ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the PRO230-.
• PR0216- or PRO302-encodmg nucleic acid or the natural source of the ant ⁇ -PRO230-, ant ⁇ -PR0216- or ant ⁇ -PRO302-encod ⁇ ng nucleic acid Preferably, the isolated nucleic acid is free of association with all components with which it is naturally associated
• An isolated PRO230-, PR0216- or PRO302-encod ⁇ ng nucleic acid molecule or an isolated ant ⁇ -PRO230-, ant ⁇ -PR0216- or ant ⁇ -PRO302-encod ⁇ ng nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated nucleic acid molecules therefore are distinguished from the PRO230-.
• ant ⁇ -PR0216 or ant ⁇ -PRO302 antibody includes PRO230-, PR0216- or PRO302-nucle ⁇ c acid molecules or ant ⁇ -PRO230-.
• control sequences refers to DNA sequences necessary for the expression ot an operably linked coding sequence in a particular host organism
• Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
• Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence
• DNA for a presequence or secretory leader is operably linked to DNA for a PRO230, PR0216 or PRO302 polypeptide if it is expressed as a preprotein that participates in the secretion of the — polypeptide
• a promoter or enhancer is operably linked to a coding sequence if it affect
• “St ⁇ ngencv” of hybridization reactions is readily determinable by one of ordinary skill in the art. and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hyb ⁇ dizable sequence, the higher the relative temperature that can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so For additional details and explanation of stringency of hybridization reactions, see, Ausubel et al , Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995)
• Stringent conditions or “high-stringency conditions”, as defined herein, may be identified by those that ( 1 ) employ low ionic strength and high temperature for washing, for example, 0 015 M sodium chlo ⁇ de/0 0015 M sodium c ⁇ trate/0 1% sodium dodecyl sulfate at 50°C.
• 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 niM sodium chloride 75 mM sodium citrate at 42°C, or (3) employ 50% formamide.
• 5 x SSC (0 75 M NaCl. 0 075 M sodium citrate), 50 mM sodium phosphate (pH 6 8), 0 1% sodium pyrophosphate, 5 x Denhardt ' s solution, sonicated salmon sperm DNA (50 ⁇ g/ml).
• Moderately-stringent conditions may be identified as described by Sambrook et al Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Press, 1989). and include the use of washing solution and hybridization conditions (e g , temperature, ionic strength, and % SDS) less stringent than those described ab ⁇ ve
• moderately stringent conditions is overnight incubation at 37 C 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
• 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.
• 20 mg/ml denatured sheared salmon sperm DNA followed by washing the filters in 1 x SSC at about 37-50°C
• the skilled a ⁇ isan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like
• epitope-tagged refers to a chimeric polypeptide comprising a PRO230, PR0216 or PRO302 polypeptide fused to a "tag polypeptide"
• the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused
• the tag polypeptide preferably also is fairly unique so that the antibody does— not substantially cross-react with other epitopes
• Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)
• PR0216 or PRO302 variants refers to form(s) of PRO230, PR0216 or PRO302 proteins that retain the biologic and/or lmmunologic activities of a native or naturally- occurring PRO230, PR0216 or PRO302 polypeptide
• Bioactivity in the context of a molecule that antagonizes a PRO230 PR0216 or PRO302 polypeptide that can be identified by the screening assays disclosed herein is used to refer to the ability of such molecules to bind or complex with the PRO230, PR0216 or PRO302 polypeptide identified herein, or otherwise interfere with the interaction of the PRO230, PR0216 or PRO302 polypeptides with other cellular proteins or otherwise inhibits the transcription or translation of the PRO230, PR0216 or PRO302 polypeptide
• Particularly preferred biological activity includes cardiac hypertrophy, activity that acts on systemic disorders that affect vessels, such as diabetes mel tus. as well as diseases of the arteries, capillaries, veins, and/or lymphatics, and cancer
• antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes one or more of the biological activities of a native PRO230, PR0216 or PRO302 polypeptide disclosed herein, for example, if applicable, its mitogenic or angiogenic activity Antagonists of a PRO230.
• PR0216 or PRO302 polypeptide may act bv interfering with the binding of a PRO230.
• PR0216 or PRO302 polypeptide to a cellular receptor, by incapacitating or killing cells that have been activated by a PRO230, PR0216 or PRO302 polypeptide, or by interfering with vascular endothelial cell activation after binding of a PRO230, PRO216 or PRO302 polypeptide to a cellular receptor All such points of intervention by a PRO230, PR0216 or PRO302 polypeptide antagonist shall be considered equivalent for pu ⁇ oses of this invention
• the antagonists inhibit the mitogenic, angiogenic, or other biological activity of PRO230, PR0216 or PRO302 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, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular gla
• Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibodv fragments, fragments, or amino acid sequence variants of native PRO230 PR0216 or PRO302 polypeptides. peptides. small organic molecules, etc
• PRO230, PR0216 or PRO302 polypeptide receptor refers to a cellular receptor for __ a PRO230, PR0216 or PRO302 polypeptide. ordinarily a cell-surface receptor found on vascular endothelial cells, as well as variants thereof that retain the ability to bind a PRO230.
• PR0216 or PRO302 polypeptide "Antibodies” (Abs) and “immunoglobuhns” (Igs) are glycoproteins having the same structural characteristics
• immunoglobuhns 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 immunoglobuhns” 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 lntrachain 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 of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains
• variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody to and for its particular antigen
• 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.
• Antibody fragments comprise a portion ot an intact antibody, preferably the antigen-binding or variable region of the intact ant ⁇ bod ⁇ Examples of antibody fragments include Fab. Fab', F(ab'),, and Fv fragments, diabodies. linear antibodies (Zapata et al .
• Fv is the minimum antibodv 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
• variable domains each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer
• the six CDRs confer antigen-binding specificity to the antibody
• 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 of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine res ⁇ due(s) of the constant domains bear a free thiol group F(ab') : antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them Other chemical coupling
• the "light chains” of antibodies (immunoglobuhns) 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
• immunoglobuhns can be assigned to different classes There are five major classes ot immunoglobuhns IgA. IgD, IgE IgG, and IgM and several of these may be further divided into subclasses (isotypes), e g , IgG 1 , IgG2, lgG3, IgG4.
• the heavy-chain constant domains that correspond to the different classes of immunoglobuhns are called ⁇ , ⁇ , e ⁇ , and ⁇ , respectively.
• the subunit structures and three-dimensional configurations of different classes of immunoglobuhns are well known.
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies. / e .
• the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts
• Monoclonal antibodies are highly specific, being directed against a single antigenic site
• each monoclonal antibody is directed against a single determinant on the antigen
• the monoclonal antibodies are advantageous in that they are synthesized by the hyb ⁇ doma culture, uncontaminated by other immunoglobuhns
• the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population ot antibodies, and is not to be construed as requiring production of the antibody by any particular method
• the monoclonal antibodies to be used in accordance with the present invention mav be made by the hyb ⁇ doma method first described by Kohler et al Nature.
• the "monoclonal antibodies” may also be isolated from phage antibodv libraries using the techniques described in Clackson et al Nature. 352
• the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobuhns) in which a— portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, w hile the remainder of the cha ⁇ n(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 immunoglobuhns in which a— portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, w hile the remainder of the cha ⁇ n(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
• humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobuhns. 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 are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity
• donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity
• Fv FR residues of the human immunoglobulin are replaced by corresponding non-human residues
• humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences
• the humanized antibody includes a PRIMATIZEDTM antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest
• Single-chain Fv or “sFv” antibody fragments comprise the 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[ domains that enables the sFv to form the desired structure for antigen binding
• d bodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (Verne) connected to a light-chain variable domain (V L ) in the same polypeptide chain (Vêt - V L )
• V flavour 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.
• 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 ofN-terminal or internal amino acid sequence by use of a spinning cup sequenator.
• label when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody
• the label may be detectable by itself (e g , radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable
• 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), poiyacrylamides, polystyrene, polyvinyl alcohol and si cones
• the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column)
• This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275, 149
• a "liposome” is a small vesicle composed of various types of lipids.
• phospholipids and/or surfactant that is useful for delivery of a drug (such as the PRO230. PR0216 or PRO302 poly peptide or antibodies thereto disclosed herein) to a mammal
• a drug such as the PRO230. PR0216 or PRO302 poly peptide or antibodies thereto disclosed herein
• the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes
• the term “lmmunoadhesin” designates antibody-like molecules that combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains.
• the immunoadhesins comprise a fusion ot an am o acid sequence with the desired binding specificity that is other than the antigen recognition and binding site of an antibody (; e .
• the adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence comp ⁇ sing at least the binding site of a receptor or a ligand
• the immunoglobulin constant domain sequence in the lmmunoadhesin may be obtained from any immunoglobulin, such as IgG-1. IgG-
• IgG-3, or IgG-4 subtypes IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM II Compositions and Methods of the Invention
• PRO230 PR0216 and PRO302 polypeptides can be prepared.
• PRO230 PR0216 and PRO302 variants can be prepared by introducing appropriate nucleotide changes into the PRO230, PR0216 or PRO302 DNA, and/or by synthesis of the desired PRO230, PR0216 or PRO302 polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO230 PR0216 or PRO302, such __ as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
• Variations in the native full-length sequence PRO230, PR0216 or PRO302 or in various domains of the PRO230, PR0216 or PRO302 described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth for instance, in U S Patent No 5,364,934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO230. PR0216 or PRO302 that results in a change in the amino acid sequence of the PRO230 PR0216 or PRO302 as compared with the native sequence PRO230.
• the variation is bv substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO230 PR0216 or PRO302
• Guidance in determining which am o acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO230.
• Amino acid substitutions can be the result of replacing one amino acid with another am o acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, ; e , conservative amino acid replacements
• Insertions or deletions may optionally be in the range of about 1 to 5 amino acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence
• Substantial modifications in function or immunological identity of the PRO230 PR0216 or PRO302 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hvdrophobicitv 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 conservative substitution sites or more preferably, into the remaining (non-conserved) sites
• the variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis Site-directed mutagenesis [Carter et al , Nucl Acids Res , 13 4331 (1986). Zoller et al . Nucl Acids Res . JO 6487 ( 1987)], cassette mutagenesis [Wells et al , Gene. 34 315 ( 1985)], restriction selection mutagenesis [Wells et al , Philos Trans R Soc London SerA, 317 415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO230, PR0216 or PRO302 variant
• Scanning amino acid analysis can also be employed to identify one or more am o acids along a contiguous sequence
• preferred scanning amino acids are relatively small, neutral ammo acids
• amino acids include alanine. glycine, serine. and cysteine
• Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells. Science. 244 1081 -1085 ( 1989)] Alanine is also typically preferred because it is the most common amino acid Further, it is frequently found in both buried and exposed
• One type of covalent modification includes reacting targeted amino acid residues of a PRO230.
• PR0216 or PRO302 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 PRO230, PR0216 or PRO302 De ⁇ vatization with bifunctional agents is useful, for instance, for crosslinking PRO230, PR0216 or PRO302 to a water-insoluble support matrix or surface for use in the method for purifying ant ⁇ -PRO230.
• crosslinking agents include, e g , 1 , 1 -b ⁇ s(d ⁇ azoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccm ⁇ m ⁇ de esters, forexample,estersw ⁇ th4-az ⁇ dosal ⁇ cyhcac ⁇ d.
• homobifunctional imidoesters including disuccimmidyl esters such as 3,3'-d ⁇ th ⁇ ob ⁇ s(succ ⁇ n ⁇ m ⁇ dylprop ⁇ onate), bifunctional maleimides such as b ⁇ s-N-male ⁇ m ⁇ do- 1 8-octane and agents such as methyl-3-[(p-az ⁇ dophenyl)d ⁇ th ⁇ o]prop ⁇ o ⁇ m ⁇ date
• Other modifications include deamidation of glutaminyl and asparagmyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of prohne and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methy lation of the cc-amino groups of lysine, arginine, and histid e side chains [T E Creighton, Proteins Structure and Molecular Properties.
• Another type of covalent modification of the PRO230, PR0216 or PRO302 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended for pu ⁇ oses herein to mean deleting one or more carbohydrate moieties found in native sequence PRO230, PR0216 or PRO302 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO230.
• PR0216 or PRO302 the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present
• PR0216 or PRO302 polypeptide may be accomplished by altering the amino acid sequence The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO230.
• PR0216 or PRO302 (for O-linked glycosylation sites)
• PR0216 or PRO302 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO230, PR0216 or PRO302 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids
• PR0216 or PRO302 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e g , in WO 87/05330 published 1 1 September 1987 and in Ap n and W ⁇ ston CRC C ⁇ t Rev Biochem , pp 259-306 ( 1981 ) Removal ot carbohydrate moieties present on the PRO230 PR0216 or PRO302 polypeptide mav be accomplished chemically or enzvmaticalK or by mutational substitution of codonsencoding for amino acid residues that serve as targets for glycosylation Chemical deglycosylation techniques are known in the art and described, for instance, bv Hakimudd et al Arch Biochem Biophvs .
• Enzvmatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al , Meth Enzymol 138 350 ( 1987)
• Another type of covalent modification of PRO230 PR0216 or PRO302 comprises linking the PRO230, — PR0216 or PRO302 polypeptide to one of a variety of nonprotemaceous polymers, e g , polyethylene glycol (PEG), polypropylene glycol. or polyoxyalkylenes in the manner set forth in U S Patent Nos 4 640.835 4 496.689, 4,301 , 144, 4,670,417, 4,791 , 192 or 4, 179.337
• PRO230. PR0216 or PRO302 of the present invention may also be modified in a way to form a chimeric molecule comprising PRO230 PR0216 or PRO302 fused to another, heterologous polypeptide or amino acid sequence
• such a chimeric molecule comprises a fusion of the PRO230.
• PR0216 or PRO302 with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO230, PR0216 or PRO302 The presence of such epitope-tagged forms of the PRO230.
• PR0216 or PRO302 can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the PRO230, PR0216 or PRO302 to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag
• tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidine
• tag polypeptides include the Flag-peptide [Hopp et al , BioTechnology. 6 1204- 1210 ( 1988)], the KT3 epitope peptide
• the chimeric molecule may comprise a fusion of the PRO230, PR0216 or PRO302 with an immunoglobulin or a particular region of an immunoglobulin
• an immunoglobulin or a particular region of an immunoglobulin For a bivalent form of the chimeric molecule (also referred to as an 'lmmunoadhesin"), such a fusion could be to the Fc region of an IgG molecule
• the lg fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO230.
• PR0216 or PRO302 polypeptide in place of at least one variable region within an lg molecule
• the immunoglobulin fusion includes the hinge. CH2 and CH3 or the hinge, CHI, CH2 and CH3 regions of an IgG 1 molecule For the production of immunoglobulin fusions see also. -US
• Patent No 5,428.130 issued June 27. 1995 C Preparation of the PRO230 PRQ216 and PRO302 polypeptides
• the present invention provides newlv identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO230 PR0216 or PRO302 In particular. cDNAs encoding PRO230 PR0216 or PRO302 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed However, for sake of simplicity, in the present specification the protein encoded by DNA33223-1 136, DNA33087-1 158 or __ DNA40370-1217, as well as all further native homologues and variants included in the foregoing definition of PRO230, PR0216 or PRO302. will be referred to as "PRO230 PR0216 or PRO302 ', respectively, regardless of their origin or mode of preparation
• PRO230, PR0216 or PRO302 polypeptides by culturing cells transformed or transfected with a vector containing nucleic acid encoding PRO230, PR0216 or PRO302 polypeptides It is, of course, contemplated that alternative methods that are well known in the art may be employed to prepare PRO230.
• PR0216 or PRO302 For instance, the PRO230, PR0216 or PRO302 polypeptide sequence or portions thereof mav be produced by direct peptide synthesis using solid-phase techniques See, e g , Stewart et al .
• DNA encoding PRO230, PR0216 or PRO302 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the mRNA encoding PRO230, PR0216 or PRO302 and to express it at a detectable level Accordingly, DNAs encoding human PRO230, PR0216 or PRO302 can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in the Examples The gene encoding PRO230,
• PR0216 or PRO302 polypeptide may also be obtained from a genomic library or by oligonucleotide synthesis
• Probes such as antibodies to the PRO230, PR0216 or PRO302 polypeptide or oligonucleotides of at least about 20-80 bases
• Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , supra
• An alternative means to isolate the gene encoding PRO230, PR0216 or PRO302 is to use PCR methodology Sambrook et al , supra. Dieffenbach et al .
• oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized
• the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened
• Methods ot labeling are well known in the art. and include the use ot radiolabels like ' P-labeled ATP biotinylation, or enzvme labeling Hybridization conditions, including moderate stringency and high stringency are provided in Sambrook el al . supra
• Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases
• Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as __ ALIGN. DNAstar. and INHERIT, which employ various algorithms to measure homology
• Nucleic acid having protein coding sequence may be obtained bv screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al , supra to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA
• Host cells are transfected or transformed with expression or cloning vectors described herein tor PRO230
• the culture conditions such as media, temperature, pH, and the like, can be selected by the skilled artisan without undue experimentation
• principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology A Practical Approach. M Butler, ed (IRL Press, 1991) and Sambrook et al , supra
• polybrene or polyornithine may also be used for various techniques for transforming mammalian cells, see, Keown et al , Methods in Enzymology , 185 527-537 ( 1990) and Mansour et l . Nature, 336 348-352 (1988)
• Suitable host cells for cloning or expressing the DNA in the vectors herein include prokarvote, yeast, or higher eukaryote cells
• Suitable prokaryotes include, but are not limited to. eubacte ⁇ a. such as Gram-negative or Gram- positive organisms for example Enterobacte ⁇ aceae such as E colt
• Various E coli strains are pubhclv available, such as £ coli K12 strain MM294 (ATCC 31.446).
• E coli X I 776 ATCC 3 1.537
• E coli strain W31 10 ATCC 27.325
• K5 772 ATCC 53.635
• Other suitable prokarvotic host cells include Enterobacte ⁇ aceae such as Escherichia.
• Strain W31 10 is one particularly preferred host — or parent host because it is a common host strain for recombinant DNA product fermentations
• the host cell secretes minimal amounts of proteolytic enzymes
• 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 1A2, which has the complete genotype tonA .
• E coli W31 10 strain 9E4 which has the complete genotype tonA ptri.
• E co// W31 10 strain 27C7 (ATCC 55.244) which has the complete genotype to 4 ptr3 phoA El 5 (argF-lac) 169 degP ompT karf
• E coli W31 10 strain 37D6 which has the complete genotype tonA ptri phoA E15 (argF-lac)169 degP ompT rbs7 vG kari
• E coli W31 10 stram 40B4. which is strain 37D6 with a non- kanamycin resistant degP deletion mutation, and an E coli strain having mutant periplasmic protease disclosed in
• eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding PRO230, PR0216 or PRO302 Saccharomyces cerevisiae is a commonly used lowereukaryotichostmicroorganism Others include Schizosaccharomvces pombe (Beach and Nurse, Nature,
• K lactis MW98-8C, CBS683. CBS4574, Louvencourt et al . J Bacteriol . 737 [19831).
• K frastlis ATCC 12,424)
• K bitlgaricus ATCC 16 045)
• K wickeramu ATCC 24.178
• A: waltu ATCC 56.500
• K drosophilarum ATCC 36.906 Van den Berg et al . Bio/Technology.
• Pichia pastor is (EP 183.070. Sreek ⁇ shna et al . J Basic Microbiol . 28 265-278 [ 1988]), Candida Trichoderma reesta (EP 244,234), Neurospora crassa (Case et al , Proc Nati Acad Sci USA 76 5259-5263 [ 1979]), Schwanmomvces such as Schwanniomvces occidentals (EP394 538pubhshed31 October 1990), and filamentous fungi such as, e g Neurospora Penictlltum.
• Methylotropic yeasts are suitable herein and include, but are not limited to yeast capable of growth on methanol selected from the genera consisting of Hansemda.
• Candida kloeckera Pichia Saccharomyces Torulopsis and Rhodotorula A list of specific species that are exemplary of this class of yeasts may be found in C Anthony, The Biochemistry of Methylotrophs. 269 ( 1982)
• Suitable host cells for the expression of nucleic acid encoding glycosylated PRO230 are derived from multicellular organisms
• invertebrate cells include insect cells such as Drosophila S2 and Spodoptera SP9 as well as plant cells
• useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkev kidney CV 1 line transformed by SV40 (COS-7, ATCC CRL 1651 ), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al J Gen Virol 36 59 ( 1977)), Chinese hamster ovarv cells/-DHFR (CHO. Urlaub and Chasm, Proc Nati Acad Sci USA, 77 4216 ( 1980)) mouse sertoli cells (TM4. Mather. Biol Reprod .23 243-
• the nucleic acid (e , cDN A or genomic DNA) encoding PRO230, PR0216 or PRO302 may be inserted into a rephcable vector for cloning (amplification of the DNA) or for expression
• a rephcable vector for cloning (amplification of the DNA) or for expression
• the vector may, for example, be in the form of a plasmid. cosmid. viral particle or phage
• the appropriate nucleic acid sequence may be inserted into the vector a variety ot procedures in general, 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.
• a signal sequence if the sequence is to be secreted, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence
• Construction of suitable vectors containing one or more of these components employs standard hgation techniques that are known to the skilled artisan The PRO230.
• PR0216 or PRO302 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide
• the signal sequence may be a component of the vector, or it may be a part of the DNA encoding PRO230, PR0216 or PRO302 that is inserted into the vector
• the signal sequence mav be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase. penicil nase.
• the signal sequence may be, e g , the yeast invertase leader, alpha factor leader( ⁇ nclud ⁇ ng 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 va ⁇ etv 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 clorung vectors in mammalian cells
• Selection genes encode proteins that (a) confer resistance to antibiotics or other toxins e g . ampicilhn neomvcin. methotrexate. or tetracvchne. (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e . the gene encoding D-alanine racemase for Bacilli
• selectable markers for mammalian cells are those that enable the identification of cells competent to take up the nucleic acid encoding PRO230 PR0216 or PRO302. such as DHFR or thvmidine kinase
• DHFR 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 1 gene present in the yeast plasmid YRp7 Stinchcomb et al Nature. 282 39 (1979). Kmgsman et al . Gene. 7 141 ( 1979). Tschemper et al , Gene, 10 157 (1980)
• the trp ⁇ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example. ATCC No
• Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding PRO230, PR0216 or PRO302 to direct mR
• a synthesis Promoters recognized by a variety of potential host cells are well known Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems (Chang et al . Nature 275 615 ( 1978), Goeddel et al . Nature. 281 544 ( 1979)), alkaline phosphatase. a tryptophan (t ⁇ ) promoter system (Goeddel, Nucleic Acids Res . 8 4057 (1980).
• Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S D ) sequence operably linked to the DNA encoding PRO230, PR0216 or PRO302
• S D Shine-Dalgarno
• suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase (Hitzeman et al . J Biol Chem . 255 2073 (1980)) or other glycolytic enzymes (Hess et al , J Adv Enzyme Reg .
• enolase such as 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
• PRO230, PR0216 or PRO302 nucleic acid transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2.21 1 ,504 published 5 July 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 , the actin promoter or an immunoglobulin promoter, and by heat-shock promoters, provided such promoters are compatible with the host cell systems
• viruses such as polyoma virus, fowlpox virus (UK 2.21 1 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus avian sarcoma virus cytome
• 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, ⁇ -fetoprotem.
• an enhancer from a eukaryotic cell virus examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers
• the enhancer may be spliced into the vector at a position 5' or 3' to the sequence coding for PRO230, PR0216 or PRO302. but is preferably located at a site 5' from the promoter __
• Expression vectors used in eukaryotic host cells will also contain sequences necessary tor the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO230, PR0216 or PRO302
• Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc Nati Acad. Sci USA.
• antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes
• the antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected
• Gene expression alternatively, mav be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product
• Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal.
• the antibodies may be prepared against a native-sequence PRO230, PR0216 or PRO302 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to DNA encoding PRO230, PR0216 or
• PRO302 and encoding a specific antibody epitope
• PRO230, PR0216 or PRO302 polypeptides may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (e g
• TRITON-XTM 100 or by enzymatic cleavage
• Cells employed in expression of nucleic acid encoding the PRO230, PR0216 or PRO302 polypeptide can be disrupted by various physical or chemical means, such as treeze-thaw cvchng. sonication. mechanical disruption, or cell-lysing agents
• PRO230, PR0216 or PRO302 polypeptide may be desired to purify the PRO230, PR0216 or PRO302 polypeptide from recombinant cell proteins or polypeptides
• the following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC. chromatography on silica or on a cation- exchange resinsuch as DEAE.chromatofocusmg,SDS-PAGE.
• PRQ216 or PRO302 polypeptides l Assays for Cardiovascular Endothelial and Angiogenic Activity
• 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 endothehn antagonist activity include a rat heart ventricle binding assay where the polypeptide is tested for its ability to inhibit lodinized endothehn- 1 binding in a receptor assay, an endothehn receptor binding assay testing for intact cell binding of radiolabeled endothehn- 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 endothelin-stimulatedarachidonic acid release in cultured vascular smooth muscles, in vitro (isolated vessel) studies using endothe um 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
• 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 .
• An assay to screen for a test molecule relating to a PRO230, PR0216 or PRO302 polypeptide that binds an endothehn B, (ETB , ) receptor polypeptide and modulates signal transduction activity involves providing a host cell transformed with a DNA encoding endothehn B, receptor polypeptide, exposing the cells to the test candidate, and measuring endothehn B, receptor signal transduction activity , as described, e . 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 by 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, 1 90), pp 36-60
• This procedure permits the isolation ot adult ventricular mvocvtes and the long-term culture of these cells in the rod-shaped phenotype Phenvleph ⁇ ne and Prostaglandin F 2 ⁇ (PGF ⁇ J have been shown to induce a spreading response in these adult cells
• PGF ⁇ J Prostaglandin F 2 ⁇
• This pharmacological model tests the ability of the PRO230, PR0216 or PRO302 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 ⁇ ) It is known that rats with pathologic cardiac hypertrophy induced by myocardial infarction have chronically elevated levels of extractable PGF 2 ⁇ in their myocardium Lai et al . Am J Phvsiol (Heart Circ Physiol ).
• breast cancer, colon cancer, prostate cancer, lung cancer, etc include both non-recombinant and recombinant (transgenic) animals
• Non-recombinant animal models include, for example, rodent, e , 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 mtraperitoneal implantation, implantation underthe renal capsule, ororthopin implantation, e g colon cancer cells implanted in colonic tissue See, e g PCT publication No WO 97/33551.
• the cells introduced into such animals can be derived from known tumor/cancer cell lines, such as any of the above-listed tumor cell lines, and, for example, the B 104-1- 1 cell line (stable NIH-3T3 cell line transfected with the nett protooncogene). r ⁇ s-transfected NIH-3T3 cells Caco-2 (ATCC HTB-37). or a moderately weli- 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 new-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 et al Cancer Research. 54 4726-4728 (1994) and
• Tumors that arise in animals can be removed and cultured in vitro Cells from the in vitro cultures can then be passaged to animals Such tumors can serve as targets for further testing or drug screening Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest Such passaging techniques can be performed with any known tumor or cancer cell lines For example, Meth A CMS4. CMS5. CMS21.
• tumor cells are propagated m 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 10x10" to lOx l O 7 cells/ml The animals are then infected subcutaneously with 10 to 100 w 1 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 in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small-cell carcinoma of the lung (SCCL)
• 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 ofcells maintained in culture Zupi er ⁇ / Br J Cancer. 41 suppl 4 30 (1980)
• Evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells survive For further information about this tumor model see. Zacharski. Haemostasis. 16. 300-320 (1986)
• recombinant (transgenic) animal models can be engineered by introducing the coding portion of the PRO230, PR0216 or PRO302 genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals
• Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g , baboons, chimpanzees and monkeys
• Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (U S Patent No 4.873,191 ), retrovirus-mediated gene transfer into germ lines (e g , Van der Putten et al Proc Nati Acad Sci USA, 82 6148-615 (1985)), gene targeting in embryonic stem cells (Thompson et al CeH.
• transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals”).
• the transgene can be integrated either as a single transgene. or in concatamers, e g , head-to-head or head-to-tail tandems
• Selective introduction ot a transgene into a particular cell type is also possible by following, for example the technique of Lasko et al Proc Nati Acad Sci USA, 89 6232-636 ( 1992)
• the expression of the transgene in transgenic animals can be monitored by standard techniques For example.
• Southern blot analysis or PCR amplification can be used to verify the integration of the transgene
• the level of mR A expression can then be analyzed using techniques such as in situ hybridization.
• Northern blot analysis, PCR, or immunocytochemistry The animals are further examined for signs of tumor or cancer development
• knock-out animals can be constructed that have a defective or altered gene encoding a_ PRO230, PR0216 or PRO302 polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the PRO230, PR0216 or PRO302 polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal For example.
• cDNA encoding a particular polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the PRO230, PR0216 or PRO302 polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal
• PRO230, PR0216 or PRO302 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques
• a portion of the genomic DNA encoding a particular PRO230, PR0216 or PRO302 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration
• a selectable marker that can be used to monitor integration
• the vector is introduced into an embryonic stem cell line ( g , by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected See, e g , Li et al , Cell, 69 915 ( 1992)
• the selected cells are then
• chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock-out" animal
• Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA
• Knockout animals can be characterized, for instance, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to absence of the PRO230,
• mammary adenocarcinoma in dogs and __ cats is a preferred model as its appearance and behavior are very similar to those in humans
• the use of this model is limited by the rare occurrence of this type of tumoi in animals
• Other in vitro and in vivo cardiovascular, endothelial, and angiogenic tests known in the art are also suitable herein
• gene amplification and/or gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc 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
• Gene expression in various tissues may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product
• immunological methods such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product
• Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal.
• the antibodies may be prepared against a native-sequence PRO230, PR0216 or PRO302 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO230, PR0216 or PRO302 DNA and encoding a specific antibody epitope
• a native-sequence PRO230, PR0216 or PRO302 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO230, PR0216 or PRO302 DNA and encoding a specific antibody epitope
• ant ⁇ -PRO230 ant ⁇ -PR0216 or ant ⁇ -PRO302 antibodies to inhibit the effect of the PRO230, PR0216 or PRO302 polypeptides on endothelial cells or other cells used in the cardiovascular, endothelial. and angiogenic assays is tested
• Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow
• Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assavs and immunoprecipitation assavs Zola Monoclonal Antibodies A Manual of Techniques (CRC Press. Inc 1987), pp 147-158
• the antibodies preferably are lnsolubihzed before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and _ analyte that remain unbound
• Sandwich assays involve the use of two antibodies, each capable of binding to a different lmmunogenic 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.
• 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
• iv Cell-Based Tumor Assavs Cell-based assays and animal models for cardiovascular, endothelial, and angiogenic disorders, such as tumors can be used to verify the findings of a cardiovascular, endothelial, and angiogenic assay herein, and further to understand the relationship between the genes identified herein and the development and pathogenesis of undesirable cardiovascular, endothelial. and angiogenic cell growth
• angiogenic cell growth, e tumor cells
• cells or cells lines that have been identified as being stimulated or inhibited by the PRO230, PR0216 or PRO302 polypeptide herein
• Such cells include, for example, those set forth in the Examples below
• suitable tumor cells include, for example, stable tumor cells lines such as the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and r ⁇ s-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumo ⁇ genic growth
• stable tumor cells lines such as the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and r ⁇ s-transfected NIH-3T3 cells, which can be transfected with the desired gene and monitored for tumo ⁇ genic growth
• Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumo ⁇ genic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC) Cells transfected with the coding sequences of the genes identified herein
• PRO230, PR0216 or PRO302 polypeptide herein and polypeptidvl 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 PRO230, PR0216 or PRO302 polypeptide is required, ; e , the site of synthesis of the PRO230, PR0216 or PRO302 polypeptide. if known, and the site (e g , wound) where biological activity of PRO230.
• PR0216 or PRO302 polypeptide is needed
• the patient's cells are removed the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or. for example encapsulated within porous membranes that are implanted into the patient (see. e . U S Pat Nos 4 892.538 and
• nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or transferred in vivo in the cells of the intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction. cell fusion. DEAE-dextran, the calcium phosphate precipitation method, etc Transduction involves the association of a replication-defective, recombinant viral (preferably retroviral) particle with a cellular receptor, followed by introduction of the nucleic acids contained by the particle into the cell A commonly used vector for ex vivo delivery of the gene is a retrovirus
• the currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral vectors (such as adenovirus, lentivirus. He ⁇ es simplex I virus, or adeno-associated virus (AAV)) and hpid-based systems (useful lipids for hpid-mediated transfer of the gene are, for example, DOTM A DOPE, and DC-Choi. see. e g , Tonkmson et al , Cancer Investigation.
• viral or non-viral vectors such as adenovirus, lentivirus. He ⁇ es simplex I virus, or adeno-associated virus (AAV)
• hpid-based systems useful lipids for hpid-mediated transfer of the gene are, for example, DOTM A DOPE, and DC-Choi. see. e g , Tonkmson et al , Cancer Investigation.
• the most preferred vectors for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses
• a viral vector such as a retroviral vector includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger
• a viral vector such as a retroviral vector includes a nucleic acid molecule that, when transcribed in the presence of a gene encoding PRO230, PR0216 or PRO302 polypeptide.
• Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof and positive and negative strand primer binding sites appropriate to the virus used (if these are not already present in the viral vector)
• LTRs long terminal repeats
• such vector typically includes a signal sequence for secretion of the PRO230, PR0216 or PRO302 polypeptide from a host cell in which it is placed
• the signal sequence for this pu ⁇ ose is a mammalian signal sequence, most preferably the native signal sequence for the PRO230, PR0216 or PRO302 polypeptide
• the vector construct may also include a signal that directs polyadenylation.
• such vectors will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof
• Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dend ⁇ mers
• it is desirable to provide the nucleic acid source with an agent that targets the target cells such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc
• proteins that bind to a cell-surface membrane protein associated— with endocytosis may be used for targeting and/or to facilitate uptake, e , capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo intemahzation in cycling, and proteins that target mtracellular localization and enhance intracellular half-life
• This invention is also related to the use of the gene encoding the PRO230, PR0216 or PRO302 polypeptide as a diagnostic Detection of a mutated form of the PRO230, PR0216 or PRO302 polypeptide will allow a diagnosis of a cardiovascular, endothelial, and angiogenic disease or a susceptibilityto a cardiovascular, endothelial, and angiogenic disease, such as a tumor, since mutations in the PRO230, PR0216 or PRO302 polypeptide may cause tumors
• Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva tissue biopsy, and autopsy material
• the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al . Nature.
• RNA or cDNA may also be used for the same pu ⁇ ose
• PCR primers complementary to the nucleic acid encoding the PRO230, PR0216 or PRO302 polypeptide can be used to identify and analyze PRO230, PR0216 or PRO302 polypeptide mutations
• deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype
• Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA encoding the PRO230.
• PR0216 or PRO302 polypeptide or alternatively, radiolabeled antisense DNA sequences encoding the PRO230, PR0216 or PRO302 polypeptide Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures Genetic testing based on DNA sequence differences may be achieved by detection of alteration- in electrophoretic mobility of DNA fragments in gels with or without denaturing agents Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis DNA fragments of different sequences may be distinguished on denaturing formamidine gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures See. e g . Mvers et al , Science. 230 1242 ( 1985)
• Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method, for example, Cotton et al , Proc 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 of restriction enzymes, e g , restriction fragment length polymo ⁇ hisms (RFLP), and Southern blotting of genomic DNA
• nucleic acid encoding the PRO230 PR0216 or PRO302 polypeptide may be linked to vascular disease or neovascula ⁇ zation associated with tumor formation If the PRO230, PR0216 or PRO302 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 PRO230, PR0216 or PRO302 polypeptide is present in serum Accordingly, an anti- PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 polypeptide antibody could be used to diagnose vascular disease or neovascula ⁇ zation associated with tumor formation, since an altered level of this PRO230, PR0216 or PRO302 polypeptide may be indicative of such disorders
• a competition assay may be employed wherein antibodies specific to the PRO230, PR0216 or PRO302 polypeptide are attached to a solid support and the labeled PRO230.
• PR0216 or PRO302 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 PRO230, PR0216 or PRO302 polypeptide in the sample
• sequences of the present invention are also valuable for chromosome identification
• the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome Moreover, there is a current need for identifying particular sites on the chromosome Few chromosome marking reagents based on actual sequence data (repeat polymo ⁇ hisms) are presently available for marking chromosomal location
• the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease
• sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA Computer analysis for the 3'- untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process These primers are then usedfor PCR screening of somatic cell hybrids containing individual human chromosomes Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner Other mapping strategies that can similarly be used to map to its chromosome include m situ hybridization prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome- specific cDNA
• 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 Fluorescence in situ hybridization
• This technique can be used with cDNA as short as 500 or 600 bases, however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection FISH requires use of the clones from which the gene encoding the PR0230, PR0216 or PRO302 polypeptide was derived, and the longer the better For example, 2,000 bp is good, 4,000 bp is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time
• Verma e/ ⁇ / Human Chromosomes a Manual of Basic Techniques (Pergamon Press, New York.
• a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes (This assumes 1 megabase mapping resolution and one gene per 20 kb)
• This invention encompasses methods of screening compounds to identify those that mimic the PRO230 PR0216 or PRO302 polypeptide (agonists) or prevent the effect of the PRO230, PR0216 or PRO302 polypeptide (antagonists)
• Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO230, PR0216 or PRO302 polypeptide encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins
• Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates
• the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art
• the interaction is binding and the complex formed can be isolated or detected in the reaction mixture
• the PRO230 PR0216 or PRO302 polypeptide encoded bv the gene identified herein or the drug candidate is immobilized on a solid phase, e , on a microtiter plate by covalent or non-covalent attachments
• Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the PRO230 PR0216 or PRO302 polypeptide and drying
• an immobilized antibody eg , a _ monoclonal antibody, specific for the PRO230 PR0216 or PRO302 polypeptide to be immobilized can be used to anchor it to a solid surface
• the assay is performed bv adding the non-immobi zed 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, g bv washing, and complexes
• the candidate compound interacts with but does not bind to a particular PRO230 PR0216 or PRO302 polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions
• assays include traditional approaches, such as, e g , cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns
• protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London).340 245-246 ( 1989), Chien et al , Proc Nati Acad Sci USA, 88 9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc Nati Acad Sci USA.
• yeast GAL4 Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain
• yeast expression system described in the foregoing publications (generally referred to as the ' two-hvb ⁇ d svstem") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA- bindmg domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain
• the expression of a GAL l-/ ⁇ 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
• MATCHMAKERTM for identifying protein-protein lnteractionsbetweentwo specific proteins using the two-hybrid technique is commercial
• a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products
• a 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 ot the test compound and its reaction partner
• PRO230, PR0216 or PRO302 polypeptide has the ability to stimulate the proliferation of endothelial __ cells in the presence of the co-mitogen ConA, then one example of a screening method takes advantage of this ability Specifically, in the proliferation assay, human umbilical vein endothelial cells are obtained and cultured in 96-well flat-bottomed culture plates (Costar.
• the assay described above is performed, however, in this assay the PRO230, PR0216 or PRO302 polypeptide is added along with the compound to be screened and the ability of the compound to inhibit ' (H)thym ⁇ d ⁇ ne inco ⁇ oration in the presence of the PRO230, PR0216 or PRO302 polypeptide indicates that the compound is an antagonist to the PRO230, PR0216 or PRO302 polypeptide
• antagonists may be detected by combining the PRO230, PR0216 or PRO302 polypeptide and a potential antagonist with membrane- bound PRO230, PR0216 or PRO302 polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay
• the PRO230, PR0216 or PRO302 polypeptide can be labeled, such as by radioactivity, such that the number of PRO230.
• PR0216 or PRO302 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 .
• RNA is prepared from a cell responsive to the PRO230 PR0216 or PRO302 polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the PRO230, PR0216 or PRO302 polypeptide Transfected cells that are grown on glass slides are exposed to the labeled PRO230, PR0216 or PRO302 polypeptide The PRO230.
• PR0216 or PRO302 polypeptide can be labeled by a variety of means including lodination or inclusion of a recognition site for a site- specific protein kinase Following fixation and incubation, the slides are subjected to autoradiographic analysis Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re- screening process, eventually yielding a single clone that encodes the putative receptor
• labeled PRO230 As an alternative approach for receptor identification, labeled PRO230.
• PR0216 or PRO302 polypeptide can be photoaffinity- nked with cell membrane or extract preparations that express the receptor molecule Cross-linked material is resolved bv PAGE and exposed to X-rav 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 ot degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor
• mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO230 PR0216 or PRO302 polypeptide in the presence of the candidate compound The ability of the compound to enhance or block this interaction could then be measured —
• compositions useful in the treatment of cardiovascular, endothelial, and angiogenic disorders include, without limitation, antibodies small organic and inorganic molecules, peptides. phosphopeptides antisense and ribozyme molecules, t ⁇ ple-hehx molecules etc that inhibit the expression and/or activity of the target gene product
• potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with a PRO230 PR0216 or PRO302 polypeptide. and in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments single-chain antibodies anti-idiotvpic 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 PRO230, PR0216 or PRO302 polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO230, PR0216 or PRO302 polypeptide
• Another potential PRO230, PR0216 or PRO302 polypeptide antagonist or agonist is an antisense RNA or DNA construct prepared using antisense technology, where, e , 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 PRO230 PR0216 or PRO302 polypeptides herein is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length
• a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see, Lee et al , Nucl Acids Res , 6 3073 ( 1979
• RNA oligonucleotide hybridizes to the mRNA m vivo and blocks translation of the mRNA molecule into the PRO230 PR0216 or PRO302 polypeptide (antisense - Okano, Neurochem , 56 560
• 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 PRO230, PR0216 or PRO302 polypeptide
• antisense DNA 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 m 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 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 PRO230, PR0216 or PRO302 polypeptide.
• Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g Rossi, Current Biology, 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18 1997) Nucleic acid molecules in 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 triple-
• the PRO230. PR0216 or PRO302 polypeptides, or agonists or antagonists thereto, that have activity in the cardiovascular, angiogenic, and endothelial assays described herein, and/or whose gene product has been found to be localized to the cardiovascular system, are likely to have therapeutic uses in a variety of cardiovascular, endothelial, and angiogenic disorders, including systemic disorders that affect vessels, such as diabetes mel tus
• 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 ofcells 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 m vitro to enhance human marrow erythroid and granulocytic progenitor cell growth)
• the PRO230, PR0216 or PRO302 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 PRO230 PR0216 or PRO302 polypeptide or antagonist would be beneficial to lschemic tissues and to collateral coronary development in the heart subsequent to coronarv stenosis Antagonists are used to inhibit the action ot such polypeptides, for example, to limit the production of excess connective tissue during wound healing or pulmonary fibros
• the treatment for cardiac hypertrophy can be performed at any of its various stages, which may result from a variety of diverse pathologic conditions, including myocardial infarction, hypertension, hypertrophic cardiomyopathy, and valvular regurgitation
• the treatment extends to all stages of the progression ofcardiac hypertrophy, with or without structural damage of the heart muscle, regardless of the underlying cardiac disorder
• vascular tumors such as haemangioma, tumor angiogenesis, neovascula ⁇ zation in the retina, choroid, or cornea, associated with diabetic retinopathy or premature infant retinopathy or macular degeneration and prohferative vitreoretinopathy, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation, psoriasis, endomet ⁇ osis associated with neovascula ⁇ zation, restenosis subsequent to balloon angioplasty, scar tissue ove ⁇ roduction, for example
• vascular tumors such as haemangioma, tumor angiogenesis, neovascula ⁇ zation in the retina, choroid, or cornea, associated with diabetic retinopathy or premature infant retinopathy or macular degeneration and prohferative vitreoretinopathy, rheumatoid arthritis, Crohn's disease, atherosclerosis, ovarian hyperstimulation,
• angiogenesis it would be used itself (or an agonist thereof) for indications where angiogenesis is desired such as peripheral vascular disease, hypertension, inflammatory vascu tides, 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
• the molecule inhibits angiogenesis.
• an antagonist thereof would be used for treatment of those conditions where angiogenesis is desired
• Atherosclerosis is a disease characterized by accumulation of plaques of intimal thickening in arteries, due to accumulation of lipids, proliferation ot 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 vascu tides include giant cell arte ⁇ tis, Takayasu's arte ⁇ tis, polyarte ⁇ tis nodosa (including the__ microangiopathic form), Kawasaki's disease, microscopic polyangntis, Wegener's granuiomatosis, and a variety of infectious-related vascular disorders (including Henoch-Schonlein prupura) Altered endothelial cell function has been shown to be important in these diseases
• Reynaud's disease and Reynaud's phenomenon are characterized by intermittent abnormal impairment of the circulation through the extremities on exposure to cold Altered endothelial cell function has been shown to be important in this disease
• Aneurysms are saccular or fusiform dilatations of the arterial or venous tree that are associated with altered endothelial cell and/or vascular smooth muscle cells
• Arterial restenosis (restenosis of the arterial wall) may occur following angioplasty as a result of alteration in the function and proliferation of endothelial and vascular smooth muscle cells
• Thrombophlebitis and lymphangitis are inflammatory disorders of veins and lymphatics, respectively, that may result from, and/or in, altered endothelial cell function
• lymphedema is a condition involving impaired lymphatic vessels resulting from endothelial cell function
• lymphangiomas are benign tumors of the lymphatic system that are congenital, often cystic, malformations of the lymphatics that usually occur in newborns Cystic tumors tend to grow into the adjacent tissue Cystic tumors usually occur in the cervical and axillary region They can also occur in the soft tissue of the extremities
• the main symptoms are dilated, sometimes reticular, structured lymphatics and lymphocysts surrounded by connective tissue Lymphangiomas are assumed to be caused by improperly connected embryonic lymphatics or their deficiency The result is impaired local lymph drainage Gnener et al , Lymphology, 4 140-144 (1971)
• tumor angiogenesis involves vascularization of a tumor to enable it to growth and/or metastasize This process is dependent on the growth of new blood vessels
• neoplasms and related conditions that involve tumor angiogenesis include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endomet ⁇ al carcinoma, endomet ⁇ al hype ⁇ lasia, endomet ⁇ osis, fibrosarcomas, cho ⁇ ocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma,
• ghoblastoma Schwannoma, o godendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma, abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
• AMD Age-related macular degeneration
• AMD Age-related macular degeneration
• the exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment. Because choroidal neovascularization is associated with a dramatic worsening in prognosis, the PRO230, PR0216 or PRO302 polypeptides or antagonist thereto is expected to be useful in reducing the severity__ of AMD.
• PRO230, PR0216 or PRO302 polypeptides herein or their antagonists are also a targeted use for the PRO230, PR0216 or PRO302 polypeptides herein or their antagonists. Formation and regression of new blood vessels is essential for tissue healing and repair. This category includes bone, cartilage, tendon, ligament, and/or nerve tissue growth or regeneration, as well as wound healing and tissue repair and replacement, and in the treatment of burns, incisions, and ulcers.
• a PRO230, PR0216 or PRO302 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 PRO230, PR0216 or
• PRO302 polypeptide or antagonist thereof may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma-induced, or oncologic, resection-induced craniofacial defects, and also is useful in cosmetic plastic surgery.
• PRO230, PR0216 or PRO302 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 PRO230, PR0216 or PRO302 polypeptide or antagonist thereto may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, or endothehum), muscle (smooth, skeletal, or cardiac), and vascular (including vascular endothehum) tissue, or for promoting the growth ofcells comprising such tissues.
• organs including, for example, pancreas, liver, intestine, kidney, skin, or endothehum
• muscle smooth, skeletal, or cardiac
• vascular including vascular endothehum
• a PRO230, PR0216 or PRO302 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 PRO230, PR0216 or PRO302 polypeptide or antagonist thereto may be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells, or for inhibiting the growth of tissues described above.
• a PRO230, PR0216 or PRO302 polypeptide or antagonist thereto may also be used in the treatment of periodontal diseases and in other tooth-repair processes. Such agents may provide an environment to attract bone- forming cells, stimulate growth of bone-forming cells, or induce differentiation of progenitors of bone-forr ng cells.
• a PRO230, PR0216 or PRO302 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 PRO230, PR0216 or PRO302 polypeptide herein or antagonist thereto is tendon/ligament formation
• a protein that induces tendon/hgament- ke 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
• the PRO230, PR0216 or PRO302 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, ortrauma to neural cells or nerve tissue More specifically, a PRO230, PR0216 or PRO302 polypeptide or its antagonist may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateralscleros ⁇ s,and Shy-Dragersyndrome Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma, and cerebrovascular diseases such as stroke Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a PRO230, PR0216 or PRO302 polypeptide herein
• Endothelial cell dysfunction may be important in both the initiation of, and in regulation of the sequelae of events that occur following ischemia-reperfusion injury
• Rheumatoid arthritis is a further indication
• Blood vessel growth and targeting of inflammatory cells through the vasculature is an important component in the pathogenesis of rheumatoid and sero-negative forms of arthritis
• PRO230, PR0216 or PRO302 polypeptide or its antagonist may also be administered prophylactically to patients with cardiac hypertrophy, to prevent the progression of the condition, and avoid sudden death, including death of asymptomatic patients Such preventative therapy is particularly warranted in the case of patients diagnosed with massive left ventricular cardiac hypertrophy (a maximal wall thickness of 35 mm or more-in adults, or a comparable value in children), or in instances when the hemodynamic burden on the heart is particularly strong PRO230.
• PR0216 or PRO302 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 prohferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, thyroid hype ⁇ lasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, pe ⁇ cardial effusion (such as that associated w ⁇ th__ pericarditis), and pleural effusion
• the PRO230. PR0216 or PRO302 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 PRO230, PR0216 or PRO302 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,
• Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidantsincludmg ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben. catechol, resorcinol.
• polypeptides such as serum albumin, gelatin, or immunoglobuhns
• hydrophilic polymers such as polyvinylpyrrohdone
• amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccha ⁇ des, disaccha ⁇ des, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA.
• sugars such as sucrose, mannitol. trehalose or sorbitol. salt-forming counter-ions such as sodium, metal complexes (e g , Zn- protein complexes), and/or non-ionic surfactants such as TWEEN TM, PLURONICS TM or polyethylene glycol (PEG)
• 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 glyce ⁇ de mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate. disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium t ⁇ sihcate, polyvinyl pyrrohdone.
• depot forms are suitably used Such forms include, for example, microcapsules, nano-capsules, liposomes piasters, inhalation forms, nose sprays, subhngual tablets, and sustained-release preparations
• the PRO230, PR0216 or PRO302 polypeptides or agonists or antagonists will typically be formulated in such vehicles at a concentration of about 0 1 mg/ml to 100 mg/ml
• Another formulation comprises inco ⁇ orating a PRO230, PR0216 or PRO302 polypeptide or antagonist thereof into formed articles
• Such articles can be used in modulating endothelial cell growth and angiogenesis
• tumor invasion and metastasis may be modulated with these articles
• PRO230, PR0216 or PRO302 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
• PRO230, PR0216 or PRO302 polypeptide ordinarily will be stored in lyophi zed form or in solution if administered systemically If in lyophihzed form.
• PRO230, PR0216 or PRO302 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 PRO230, PR0216 or PRO302 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) PRO230, PR0216 or PRO302 polypeptide or agonist or antagonist thereto, b) a buffer capable of maintaining the pH in a range of maximum stability of the polypeptide or other molecule in solution, preferably about 4-8, c) a detergent/surfactant primarily to stabilize the polypeptide or molecule against agitation-induced aggregation, d) an isotonifier, e) a preservative selected from the group of phenol, benzyl alcohol and a
• the detergent employed is non-ionic, it may, for example, be polysorbates (e , 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 isotonicity of a liquid composition of the PRO230, PR0216 or
• PRO302 polypeptide or antagonist thereto includes polyhyd ⁇ c sugar alcohols, preferably t ⁇ hyd ⁇ c or higher sugar alcohols, such as glycerin, eryth ⁇ tol, arabitol, xyhtol, sorbitol, and mannitol These sugar alcohols can be used alone or in combination Alternatively, sodium chloride or other appropriate inorganic salts may be used to render the solutions isotonic
• the buffer may, for example, be an acetate, citrate, succinate. or phosphate buffer depending on the pH desired
• the pH of one type of liquid formulation of this invention is buffered in the range of about 4 to 8, preferably about physiological pH
• the preservatives phenol, benzyl alcohol and benzethonium halides. e g , chloride, are known antimicrobial agents that may be employed
• Therapeutic PRO230, PR0216 or PRO302 polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle
• the formulations are preferably administered as repeated intravenous (I v ),__ subcutaneous (s c ), or intramuscular (I m ) injections, or as aerosol formulations suitable for intranasal or mtrapulmonary delivery (for intrapulmonary delivery see, e g , EP 257,956)
• PRO230, PR0216 or PRO302 polypeptide can also be administered in the form of sustained-released preparations Suitable examples of sustained-release preparations include sem ⁇ ermeable matrices of solid hydrophobic polymers containing the protein, which matrices are
• non-degradable ethylene-vinyl acetate Langer et al , supra
• degradable lactic acid-glyco c acid copolymers such as the Lupron DepotTM (injectable microspheres composed of lactic acid-glycohc acid copolymer and leupro de acetate), and poly-D-(-)-3-hydroxybuty ⁇ c acid (EP 133,988)
• polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days
• certain hydrogels release proteins for shorter time periods
• When encapsulated proteins remain in the body for a long time they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised for protein stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be lntermolecular S-S bond formation through
• Sustained-release PRO230, PR0216 or PRO302 polypeptide compositions also lnclude posomailyentrapped PRO230, PR0216 or PRO302 polypeptides Liposomes containing the PRO230.
• PR0216 or PRO302 polypeptide are prepared by methods known per se DE 3.218.121, Epstein et al . Proc Nati Acad Sci USA, 82 3688-3692
• the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy
• the therapeutically effective dose of PRO230, PR0216 or PRO302 polypeptide or antagonist thereto will.
• the pathological condition to be treated including prevention
• the method of administration the type of compound being used for treatment, any co-therapy involved, the patient's age, weight, general medical condition, medical history, etc , and its determination is well with the skill of a practicing physician Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the maximal therapeutic effect
• the PRO230, PR0216 or PRO302 polypeptide has a narrow host range, for the treatment of human patients formulations comprising human PRO230, PR0216 or PRO302 polypeptide, more preferably native-sequence human PRO230, PR0216 or PRO302 polypeptide, are preferred
• the clinician will administer PRO230, PR0216 or PRO302 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 cardio
• the effective dose generally is within the range of from about 0 001 to about 1 0 mg/kg, more preferably about 0 01-1 0 mg/kg, most preferably about 0 01-0 1 mg/kg
• a molecule based on the PRO230, PR0216 or PRO302 polypeptide is preferably administered at about 5 mg to 1 g, preferably about 10 to 100 mg, per kg body weight, 1 to 3 times daily
• endotoxin contamination should be kept minimally at a safe level, for example, less than 0 5 ng/mg protein
• the formulations preferably meet sterility, pyrogenicity, general safety, and purity as required by FDA Office and Biologies standards
• the dosage regimen of a pharmaceutical composition containing PRO230, PR0216 or PRO302 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, histomo ⁇ homet ⁇ c determinations, and tetracyclme labeling
• the route of PRO230, PR0216 or PRO302 polypeptide or antagonist or agonist administration is in accord with known methods, e , by injection or infusion by intravenous, intramuscular, lntracerebral, mtraperitoneal, intracerobrospinal, subcutaneous, intraocular, lntraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes, or by sustained-release systems as noted below
• the PRO230, PR0216 or PRO302 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 mtraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors If a peptide or small molecule is employed as an antagonist or agonist, it is preferably administered orally or non-orally in the form of a liquid or solid to mammals.
• Examples of pharmacologically acceptable salts of molecules that form salts and are useful hereunder include alkali metal salts (e.g., sodium salt, potassium salt), alkaline earth metal salts (e.g., calcium salt, magnesium salt). ammonium salts, organic base salts (e.g. , pyridine salt, triethylamine salt), inorganic acid salts (e.g., hydrochloride, sulfate, nitrate), and salts of organic acid (e.g., acetate, oxalate, p-toluenesulfonate).
• alkali metal salts e.g., sodium salt, potassium salt
• alkaline earth metal salts e.g., calcium salt, magnesium salt
• ammonium salts e.g., organic base salts (e.g. , pyridine salt, triethylamine salt)
• inorganic acid salts e.g., hydrochloride, sulfate,
• the therapeutic method includes administering the composition topically, systemically, or locally as an implant or device.
• the therapeutic composition for use is in a pyrogen-free, physiologically acceptable form.
• the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage, ortissue damage.
• Topical administration may be suitable for wound healing and tissue repair.
• the composition would include a matrix capable of delivering the protein- containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and preferably capable of being resorbed into the body.
• Such matrices may be formed of materials presently in use for other implanted medical applications.
• compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid, and polyanhydrides.
• Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.
• Further matrices are comprised of pure proteins or extracellular matrix components.
• Other potential matrices are nonbiodegradabie and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.
• Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.
• the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
• One specific embodiment is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.
• a sequestering agent such as carboxymethyl cellulose or autologous blood clot
• cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,hydoxyethylcelluloseJ ⁇ ydroxypropylcellulose.
• hydroxypropylmethylcellulose and carboxymethylcellulose, one preferred being cationic salts of carboxymethylcellulose (CMC).
• CMC carboxymethylcellulose
• Other preferred sequestering agents include hyaluronic acid, sodium alginate. poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and poly(vinyl alcohol).
• the amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1 - 10 wt%, based on total formulation weight, which represents the amount necessary to prevent deso ⁇ tion of the polypeptide (or its antagonist) from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide (or its antagonist) the opportunity to assist the osteogenic activity of the progenitor cells
• the effectiveness of the PRO230. PR0216 or PRO302 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 pu ⁇ oses, either in the same composition or as separate compositions
• PRO230, PR0216 or PRO302 polypeptide therapy can be combined with the administration of inhibitors of known cardiac myocyte hypertrophy factors, e , inhibitors of ⁇ -adrenergic agonists such as phenylephrine, endothehn- 1 inhibitors such as BOSENTANTM and MOXONODINTM, inhibitors to CT-1 (US Pat No 5,679,545), inhibitors to LIF, ACE inhibitors, des-aspartate- angiotensm I inhibitors (U S Pat No 5,773,415), and angiotensin II inhibitors
• PRO230, PR0216 or PRO302 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 , qumap ⁇ l, captop ⁇ l, enalap ⁇ l, ramip ⁇ l, benazep ⁇ l, fosinop ⁇ l, or lisinop ⁇ l, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlo ⁇ henamide, acetazolamide, or
• ⁇ -adrenergic-blocking drugs e g , propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol
• verapamil difedipine
• diltiazem Treatment of hypertrophy associated with high blood pressure may require the use of antihypertensive drug therapy, using calcium channel blockers, e g , diltiazem, nifedipine, verapamil, or nicardipine, ⁇ -adrenergic blocking agents, diuretics, e g , chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthi
• PRO230, PR0216 or PRO302 polypeptides or their antagonists may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question These agents include various growth factors such as EGF, PDGF, TGF- ⁇ or TGF- ⁇ , IGF, FGF, and CTGF
• agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question include various growth factors such as EGF, PDGF, TGF- ⁇ or TGF- ⁇ , IGF, FGF, and CTGF
• PRO230, PR0216 or PRO302 polypeptides or their antagonists used to treat cancer may be combined with cytotoxic, chemotherapeutic. or growth-inhibitory agents as identified above
• the PRO230, PR0216 or PRO302 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 PRO230, PR0216 or PRO302 polypeptide or antagonist thereof will be at the physician s or veterinarian's discretion Dosage administration and adjustment is done to achieve maximal management of the conditions to be treated For example, for treating hypertension, these amounts ideally take into account use of diuretics or digitalis, and conditions such as hyper- or hypotension, renal impairment, etc
• the dose will additionally depend on such factors as the type of the therapeutic agent to be used and the specific patient being treated Typically, the amount employed will be the same dose as that used, if the given therapeutic agent is administered without PRO230, PR0216 or PR0302 polypeptide
• An article of manufacture such as a kit containing PRO230, PR0216 or PRO302 polypeptide or antagonists thereof useful for the diagnosis or treatment of the disorders described above comprises at least a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes
• the containers may be formed from a variety of materials such as glass or plastic
• the container holds a composition that is effective for diagnosing or treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle)
• the active agent in the composition is the PRO230, PR0216 or PRO302 polypeptide or an agonist or antagonist thereto
• the label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice
• the article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer'
• Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments that may inhibit the production or the gene product of the genes identified herein and/or reduce the activity of the gene products
• polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or mtraperitoneal injections
• the immunizing agent may include the PRO230, PR0216 or PRO302 polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be lmmunogenic in the mammal being immunized Examples of such lmmunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobuhn, and soybean trypsin inhibitor Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A or synthetic trehalose dicorynomycolate) The imm
• the ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibodies may, alternatively, be monoclonal antibodies
• Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature. 256 495 (1975)
• a hybridoma method a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro
• the immunizing agent will typically include the PRO230, PR0216 or PRO302 polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired
• PBLs peripheral blood lymphocytes
• spleen cells or lymph node cells are used if non-human mammalian sources are desired
• the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell Goding, Monoclonal Antibodies Principles and Practice (New York Academic Press, 1986), pp 59- 103
• Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin Usually, rat or mouse myeloma cell lines are employed
• the hybridoma cells may be cultured in a suitable culture
• the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the PRO230, PR0216 or PRO302 polypeptide
• the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA)
• the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem , 07 220 ( 1980)
• the clones may be subcloned bv limiting dilution procedures and grown by standard methods Goding supra Suitable culture media for this pu ⁇ ose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium
• 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 ( g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies)
• the hybridoma cells of the invention serve as a preferred source of such DNA
• the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein to obtain the synthesis of monoclonal antibodies in the recombinant host cells
• the DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U S Patent No 4,816,567, Morrison et
• the antibodies may be monovalent antibodies
• Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain
• the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosslinking
• the relevant cysteme residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking
• the ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibodies may further comprise humanized antibodies or human antibodies
• Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobuhns, 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 immunoglobuhns (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity
• donor antibody such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity
• Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues
• Humanized antibodies may also comprise residues that are found
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody preferably also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al. , Nature, 321 : 522-525 ( 1986); Riechmann et al. , Nature, 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).
• Fc immunoglobulin constant region
• a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import” residues, which are typically taken from an “import” variable domain.
• Humanization can be essentially performed following the method of Winter and co-workers (Jones et at. Nature. 321 : 522-525 ( 1986); Riechmann et al. , Nature, 332: 323-327 ( 1988); Verhoeyen et al., Science, 239: 1534- 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol.. 227: 381 (1991); Marks et al.. J. Mol. Biol., 222: 581 (1991). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al.. Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol., 147(1): 86-95 (1991). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g.
• mice in which the endogenous immunoglobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
• This approach is described, for example, in U.S. Patent Nos. 5,545,807: 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661 ,016, and in the following scientific publications: Marks et al. , Bio/Technology, K): 779-783 ( 1992); Lonberg etal.. Nature, 368: 856-859( 1994); Morrison, Nature, 368: 812-813 1994): Fishwild et al.. Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, F4: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol.. 13: 65-93 (1995).
• Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
• one of the binding specificities is for the PRO230, PR0216 or PRO302 polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
• bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/hght-chain pairs, where the two heavy chains have different specificities Milstein and Cuello, Nature, 305 537-539 (1983) Because of the random assortment of immunoglobulin heavy and light chains, these hyb ⁇ domas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure The purification of the correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993 , and in Traunecker et al , EMBO J , 10 3655-3659 (1991) Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant-domain sequences The fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part of the hinge,
• Heteroconjugate antibodies are composed of two covalently joined antibodies Such antibodies have, for example, been proposed to target immune-system cells to unwanted cells (U S Patent No 4,676,980), and for treatment of HIV infection WO 91/00360, WO 92/200373, EP 03089 It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents For example, lmmunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond Examples of suitable reagents for this pu ⁇ ose include lminofhiolate 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 intemahzation capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See, Caron et al , J Exp Med , 176 1 191-1 195 (1992) and
• 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 Design, 3 219-230 (1989)
• the invention also pertains to lmmunoconjugates 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 (t 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)
• toxin e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof
• t e a radioactive isotope
• Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxm A chain (from Pseudomonas aeruginosa), ⁇ cin A chain, ab ⁇ n A chain, modeccin A chain, alpha-sarcin, Aleurttes fordu proteins, dianthin proteins Phytolaca amencana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotm, sapaona ⁇ a officinalis inhibitor, gelonin, mitogelhn, restrictocin, phenomycm, enomycin, and the tricothecenes
• a variety of radionuclides are available for the production of radioconjugated antibodies Examples include 21 B ⁇ , lj, I, 13l In, 90 Y, and
• Carbon- 14-labeled 1 - ⁇ soth ⁇ ocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid is an exemplary chelatmg agent for conjugation of radionucleotide to the antibody See, W094/ 1 1026
• 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 , avidin) that is conjugated to a cytotoxic agent (e g , a radionucleotide)
• a "receptor” such as streptavidin
• a ligand e , 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 et ⁇ l Proc Nati Acad Sci USA, 82 3688 (1985), Hwang et ⁇ l , 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 lipid composition comprising phosphatidylcho ne, cholesterol, and PEG-de ⁇ vatized phosphatidylethanolamine (PEG-
• 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 ⁇ l J Biol Chem , 257 286-288 (1982) via a disulfide-interchange reaction
• a chemotherapeutic agent such as Doxorubicin
• is optionally contained withm the liposome See, Gabizon et ⁇ l , J National Cancer Inst , 81(19) 1484 (1989) lx Pharmaceutical Compositions of Antibodies
• Antibodies specifically binding a PRO230, PR0216 or PRO302 polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders as noted above and below in the form of pharmaceutical compositions If the PRO230, PR0216 or PRO302 polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred However, pofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred For example, based upon the variable- region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence Such peptides can be synthesized chemically and/or produced by recombinant DNA technology See, e g, Marasco et al , Proc 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 other
• the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent
• cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent
• Such molecules are suitably present in combination in amounts that are effective for the pu ⁇ ose intended
• the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by mterfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-( ethylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra
• the formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes
• sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g films, or microcapsules
• 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 DEPOT TM (injectable microspheres composed of lactic acid-glycohc acid copolymer and leupro de acetate) and poly-D-(-)-3-hydroxybuty ⁇ c acid
• antibodies to a PRO230, PR0216 or PRO302 polypeptide may be used to treat various cardiovascular, endothelial, and angiogenic conditions as noted above.
• the antibodies are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous administration of the antibody is preferred.
• Other therapeutic regimens may be combined with the administration of the antibodies of the instant invention as noted above. For example, if the antibodies are to treat cancer, the patient to be treated with such antibodies may also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient.
• Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, Ed., M.C. Perry (Williams &
• the chemotherapeutic agent may precede, or follow administration of the antibody, or may be given simultaneously therewith.
• the antibody may be combined with an anti-estrogen compound such as tamoxifen or EVISTATM or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
• an anti-estrogen compound such as tamoxifen or EVISTATM or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
• an anti-estrogen compound such as tamoxifen or EVISTATM or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
• an anti-estrogen compound such as tamoxifen or EVISTATM or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules.
• the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances.
• two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to the patient.
• the antibodies herein are co- administered with a growth-inhibitory agent.
• the growth-inhibitory agent may be administered first, followed by an antibody of the present invention.
• simultaneous administration or administration of the antibody of the present invention first is also contemplated. Suitable dosages for the growth-inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth-inhibitory agent and the antibody herein.
• vascularization of tumors is attacked in combination therapy.
• the anti-PRO230, anti- PR0216 or anti-PRO302 polypeptide and another antibody are administered to tumor-bearing patients at therapeutically effective doses as determined, for example, by observing necrosis of the tumor or its metastatic foci, if any. This therapy is continued until such time as no further beneficial effect is observed or clinical examination shows no trace of the tumor or any metastatic foci.
• TNF is administered, alone or in combination with an auxiliary agent such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin, anti-heregulin antibody, D-factor, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), or agents that promote microvascular coagulation in tumors, such as anti-protein C antibody, anti-protein S antibody, or C4b binding protein (see. WO 91/01753, published 21 February 1991 ), or heat or radiation. Since the auxiliary agents will vary in their effectiveness, it is desirable to compare their impact on the tumor by matrix screening in conventional fashion.
• an auxiliary agent such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin, anti-heregulin antibody, D-factor, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating
• anti-PRO230, anti-PR0216 or anti-PRO302 polypeptide antibody and TNF is repeated until the desired clinical effect is achieved.
• the anti- — PRO230, anti-PR0216 or anti-PRO302 polypeptide antibody is administered together with TNF and, optionally, auxiliary agent(s).
• the therapeutic agents described herein are administered to the isolated tumor or organ.
• a FGF or PDGF antagonist such as an anti-FGF or an anti-PDGF neutralizing antibody, is administered to the patient in conjunction with the anti-PRO230, anti-PR0216 or anti-PRO302 polypeptide antibody.
• Treatment with anti-PRO230, anti-PR0216 or anti-PRO302 polypeptide antibodies preferably may be suspended during periods of wound healing or desirable neovascularization.
• 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 pu ⁇ oses, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
• the antibody is suitably administered to the patient at one time or over a series of treatments.
• g/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
• a typical daily or weekly dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
• the treatment is repeated or sustained until a desired suppression of disorder symptoms occurs.
• other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging.
• An article of manufacture containing a container with the antibody and a label is also provided. Such articles are described above, wherein the active agent is an anti-PRO230, anti-PR0216 or anti-PRO302 antibody.
• the same proteins along with PRO230, PR0216 or PRO302 polypeptides find additional use in the diagnosis and prognosis of tumors.
• antibodies directed against the PRO230, PR0216 or PRO302 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 PRO230, PR0216 or PRO302 polypeptide.
• the antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. Such binding assays are performed essentially as described above.
• In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this pu ⁇ ose, 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 hereinabove and in the following textbooks: Sambrook et al, supra; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N.Y., 1989); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.: N.Y., 1990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide
• the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases.
• the EST databases included public EST databases (e.g., GenBank) and a proprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA).
• the search was performed using the computer program BLAST or BLAST2 [Altschul et al. , Methods in Enzymology. 266:460-480 ( 1996)] as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap” (Phil Green, University of Washington, Seattle, Washington).
• a consensus DNA sequence was assembled relative to other EST sequences using phrap as described above.
• DNA30857 An EST proprietary to Genentech was employed in the consensus assembly and is herein designated DNA20088.
• the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above.
• oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO230.
• Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length.
• the probe sequences are typically 40-55 bp in length.
• additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp.
• DNA from the libraries was screened by PCR amplification, as per Ausubel et al. , Current Protocols in Molecular Biology, supra, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
• hybridization probe was constructed from the consensus DNA30857 sequence which had the following nucleotide sequence: hybridization probe:
• RNA for construction of the cDNA libraries was isolated from human fetal lung 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 cDN A was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al. , Science.253: 1278- 1280 ( 1991 )) in the unique Xhol and Notl sites.
• a suitable cloning vector such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al. , Science.253: 1278- 1280 ( 1991 )
• the predicted polypeptide precursor is 164 amino acids long [Figure 2; (SEQ ID NO:2)] and has a calculated molecular weight of approximately 18,359 daltons and an estimated pi of about 7.45.
• Analysis of the full-length PRO230 sequence shown in Figure 2 (SEQ ID NO 2) evidences the presence of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above
• Analysis of the full-length PRO230 sequence ( Figure 2, SEQ ID NO 2) evidences the following a signal peptide from about amino acid 1 to about amino acid 21 , an N-hnked glycosylation site from about amino acid 78 to about ammo acid 82, casein kinase II phosphorylation sites from about amino acid 80 to about amino acid 84, from about am o acid 1 17 to about ammo acid 121 , from about amino acid 126 to about am o acid 130 and from about amino acid 137 to about amino acid 141 , N-my ⁇ stoylation
• EXAMPLE 2 Isolation of cDNA Clones Encoding Human PRQ216 (an osteomodulin/fibromoduhn homolog)
• the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases
• the EST databases included public EST databases (e g , GenBank) and a proprietary EST database (LIFESEQ®, Incyte
• DNA28754 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA28754 In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
• oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0216
• Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length The probe sequences are typically 40-55 bp in length
• additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp
• DNA from the libraries was screened by PCR amplification, as per Ausubel etal , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs PCR primers (forward and reverse
• RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue
• the cDN A libraries used to isolate the cDN A clones were constructed bv standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
• the cDN A was primed with ohgo 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
• DNA330 DNA sequence for construction of the clones isolated as DNA330
• the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 268-270 and a stop signal at nucleotide positions 1531-1533 ( Figure 3, SEQ ID NO 6)
• the predicted polypeptide precursor is 421 am o acids long [Figure 4, (SEQ ID NO 7)] and has a calulated molecular weight of approximately 49,492 daltons and an estimated pi of about 5 51
• Analysis of the full-length PR0216 sequence shown in Figure 4 evidences the presence of important polypeptide domains as shown in Figure 4, wherein the locations given for those important polypeptide domains are approximate as described above
• Analysis of the full-length PR0216 sequence ( Figure 4, SEQ ID NO 7) evidences the following N-hnked glycosylation sites from about amino acid 1 13 to about amino acid 1 17, from about amino acid 121 to about amino acid 125, from about amino acid 187 to about amino acid 191, from about amino acid 242 to about amino acid 246 and from about am
• DNA35953 This consensus sequence is herein designated DNA35953
• the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
• oligonucleotides were synthesized 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PRO302
• Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length The probe sequences are typically 40-55 bp in length
• additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5 kbp
• DNA from the libraries was screened by PCR amplification, as per Ausubel et al ,
• PCR primers (forward and reverse) were synthesized forward PCR primer 1 5'-GTCCGCAAGGATGCCTACATGTTC-3' (SEQ ID NO 13) forward PCR primer 2
• RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228)
• the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
• the cDNA was primed with ohgo dT containing a Notl site, linked with blunt to Sail hemik ased 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 ofpRK5D 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
• the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 34-36 and a stop signal at nucleotide positions 1390-1392 (Figure 5, SEQ ID NO 1 1)
• the predicted polypeptide precursor is 452 amino acids long [Figure 6, (SEQ ID NO 12)] and has a calculated molecular weight of approximately 50,831 daltons and an estimated pi of about 5 74
• Analysis of the full-length PRO302 sequence shown in Figure 6 evidences the presence of important polypeptide domains as shown in Figure 6, wherein the locations given for those important polypeptide domains are approximate as described above
• Analysis of the full-length PRO302 sequence ( Figure 6, SEQ ID NO 12) evidences the following a signal peptide from about amino acid 1 to about amino acid 25, N-hnked glycosylation sites from about amino acid 64 to about amino acid 68, from about am o acid 126 to about ammo acid 130 and from about amino acid 362 to about ammo acid 366
• casein kinase II phosphorylation sites from about ammo acid 204 to about ammo acid 208, from about amino acid 220 to about amino acid 224, from about amino acid 280 to about ammo acid 284, from about amino acid 284 to about amino acid 288, from about amino acid 351 to about amino acid 355 and from about ammo acid 449 to about amino acid 453, and N-my ⁇ stoylation sites from about amino acid 22 to about amino acid 28, from about amino acid 76 to about amino acid 82, from about amino acid 79 to about amino acid 85, from about ammo acid 80 to about amino acid 86, from about amino acid 1 19 to about amino acid 125, from about amino acid 169 to about amino acid 175, from about amino acid 187 to about ammo acid 193, from about amino acid 195 to about amino acid 201 , from about amino acid 331 to about amino acid 337, from about amino acid 332 to about amino acid 338 and from about amino acid 360 to about amino acid 366 Clone DNA40370-1217 has been
• PRO302 ammo acid sequence and vitellogenic carboxvpeptidase EXAMPLE 4 Stimulation of Endothelial Tube Formation This assay follows the assay described in Davis and Cama ⁇ llo, Experimental Cell Research, 224 39-51 (1996), or one modified from it as follows Protocol Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) (passage number less than 8 from primary) are mixed with type I rat tail collagen, final concentration 2 6 mg/ml at a density of 6 x 10 5 cells/ml and plated at 50 l per well on a 96-well plate The gel is allowed to solidify for 1 hr at 37°C, then 50 ⁇ l per well of ⁇ ⁇ Ml 99 culture media supplemented with 1% FBS and a PRO230 polypeptide sample (at dilutions of 1%, 0 1%, and
• This assay will identify factors that facilitate cell survival in a 3-d ⁇ mens ⁇ onal matrix in the presence of exogenous growth factors (VEGF, bFGF without PMA)
• This assay is designed to identify factors that are involved in stimulating pinocytosis, ion pumping, permeability, and junction formation 3 Tube Formation Assay
• This assay is to identify factors that stimulate endothelial tube formation in a 3-d ⁇ mens ⁇ onal matrix This assay will identify factors that stimulate endothelial cells to differentiate into a tube-like structure in a 3-d ⁇ mens ⁇ onal matrix in the presence of exogenous growth factors (VEGF, bFGF)
• VEGF exogenous growth factors
• the amounts of the TM Lysis Buffer and Probes needed for the tests were calculated based on information provided by the manufacturer
• the approp ⁇ ate amounts of thawed Probes were added to the TM Lysis Buffer
• the Capture Hybridization Buffer was warmed to room temperature
• the bDNA strips were set up in the metal strip holders, and 100 ⁇ l of Capture Hybridization Buffer was added to each b-DNA well needed, followed by incubation for at least 30 minutes
• the test plates with the cells were removed from the incubator, and the media was gently removed using the vacuum manifold 100 ⁇ l of Lysis Hybridization Buffer with Probes were quickly pipetted into each well of the microtiter plates
• the plates were then incubated at 55 °C for 15 minutes Upon removal from the incubator, the plates were placed on the vortex mixer with the microtiter adapter head and vortexed on the #2 setting for one minute 80 ⁇ l of the lysate was removed and added to the b
• This assay is designed to determine whether PRO302 polypeptide shows the ability to induce vascular permeability
• Test samples containing the PRO302 polypeptide or a physiological buffer without the test polypeptide are injected into skin on the back of the test animals with 100 ⁇ l per injection site intradermally There were approximately 16-24 injection sites per animal
• One ml of Evans blue dye (1% in PBS) is then injected intracardially Skin vascular permeability responses to the compounds (/ e , blemishes at the injection sites of injection) are visually scored by measuring the diameter (in mm) of blue-colored leaks from the site of injection at 1 and 6 hours post administration of the test materials
• the mm diameter of blueness at the site of injection is observed and recorded as well as the severity of the vascular leakage Blemishes of at least 5 mm in diameter are considered positive for the assay when testing purified proteins, being indicative of the ability to induce vascular
• 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 1 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 M EDTA pH 8 0) was added, and the mixture was pipetted onto DE81 paper The remaining solution was loaded in a MICROCON-50TM ultrafiltration unit, and spun using program 10 (6 minutes) The filtration unit was inverted over a second tube and spun using program 2 (3 minutes) After the final recovery spin, a total of 100 ⁇ l TE was added, then 1 ⁇ l of the final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR IITM The probe was run on a TBE/urea gel A total of 1-3 ⁇ l of the probe or 5 ⁇ l of RNA Mrk III was added to
• 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 2 0)
• the sections were dehydrated in 70%, 95%, and 100% ethanol,
• 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) for human embryo tissue, or 8 x proteinase K ( 100 ⁇ l in 250 ml Rnase buffer, 37°C, 30 minutes) for formalin tissues Subsequent rinsing in 0 5 x SSC and dehydration were performed as described above
• the slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper
• the tissue was covered with 50 ⁇ l of hybridization buffer (3 75 g dextran sulfate + 6 ml SQ H 2 0), vortexed, and heated in the microwave for 2 minutes with the cap loosened After cooling on ice, 18 75 ml formamide, 3 75 ml 20 x SSC. and 9 ml SQ H 2 0 were added, and the tissue was vortexed well and incubated at 42 ⁇ C for 1-4 hours
• Sections showed an intense signal associated with arterial and venous vessels in the fetus.
• the signal appeared to be confined to smooth-muscle/pericyte cells.
• the signal was also seen in capillary vessels and in glomeruli. It was not clear whether endothelial cells were expressing this mRNA. Expression was also observed in epithelial cells in the fetal lens. Strong expression was also seen in cells within placental trophoblastic villi; these cells lie between the trophoblast and the fibroblast-like cells that express HGF-uncertain histogenesis. In the adult, there was no evidence of expression and the wall of the aorta and most vessels appeared to be negative. However, expression was seen over vascular channels in the normal prostate and in the epithelium lining the gallbladder. Insurers expression was seen in the vessels of the soft-tissue sarcoma and a renal cell carcinoma.
• PRO230 is a molecule that shows relatively specific vascular expression in the fetus as well as in some adult organs. Expression was also observed in the fetal lens and the adult gallbladder.
• vascular expression was observed in fetal blocks, similar to that observed above. Expression was on vascular smooth muscle, rather than endothehum. Expression was also seen in smooth muscle of the developing oesophagus, so this molecule is not vascular specific. Expression was examined in four lung and four breast carcinomas. Substantial expression was seen in vascular smooth muscle of at least three out of four lung cancers and two out of four breast cancers. In addition, in one breast carcinoma, expression was observed in peritumoral stromal cells of uncertain histogenesis (possibly myofibroblasts). No endothelial cell expression was observed in this study. (2) DNA33087 (PRO216)
• Sections showed strong specific expression in osteoblasts at all sites of enchondral and periosteal new bone formation. Additional sites of expression included the developing pulmonary arterial and aortic trunks. Otherwise, all fetal tissues tested negative. The fetal tissues examined included: placenta, umbilical cord, brain, spinal cord, eye, optic nerve, trachea, lung, heart, thymus, liver, spleen, esophagus, small intestine, pancreas, adrenal, thyroid, body wall, and lower limb. All of the adult tissues were negative. The adult tissues examined included: liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung and skin.
• PR0216 has a probable role in control of bone matrix deposition and/or osteoblast growth. All adult tissues in the multiblock were positive for beta-actin.
• EXAMPLE 8 Use of PRO230, PRQ216 or PRO302 as a Hybridization Probe The following method describes use of a nucleotide sequence encoding PRO230, PR0216 or PRO302 as a hybridization probe DNA comprising the coding sequence of full-length or mature PRO230, PR0216 or PRO302 (as shown in
• Figures 1 A- 1 B, 3 and 5, respectively, SEQ ID NOS 1 , 6 and 1 1 , respectively) or a fragment thereof is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO230, PR0216 or PRO302) in human tissue cDNA libraries or human tissue genomic libraries
• Hybridization and washing of filters containing either library DNAs is performed under the following high- stringency conditions
• Hybridization of radiolabeled probe derived from the gene encoding a PRO230, PR0216 or PRO302 polypeptide to the filters is performed in a solution of 50% formamide, 5x SSC, 0 1% SDS, 0 1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6 8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20 hours
• Washing of the filters is performed in an aqueous solution of 0 I x SSC and 0 1% SDS at 42°C
• DNAs having a desired sequence identity with the DNA encoding full-length native sequence can then be identified using standard techniques known in the art
• EXAMPLE 9 Expression of Nucleic Acid Encoding PRO230. PRQ216 or PRO302 in E coli This Example illustrates preparation of an unglycosylated form of PRO230, PR0216 or PRO302 by recombinant expression in E coli
• the DNA sequence encoding PRO230, PR0216 or PRO302 (SEQ ID NOS 1, 6 and 1 1, respectively) is initially amplified using selected PCR primers
• the primers should contain restriction enzyme sites that correspond to the restriction enzyme sites on the selected expression vector
• a variety of expression vectors may be employed
• An example of a suitable vector is pBR322 (derived from E coli, see Bolivar et al , Gene, 2 95 (1977)), which contains genes for ampicilhn and tetracychne resistance
• the vector is digested with restriction enzyme and dephosphorylated
• the PCR-amphfied sequences are then hgated into the vector
• the vector will preferably include sequences that encode an antibiotic-resistance gene, a t ⁇ promoter, a poly-His leader (including the first six STII codons, poly-His sequence, and enterokinase cleavage site), the region encoding PRO230, PR0216 or PRO302, lambda transcription
• Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics
• the overnight culture may subsequently be used to inoculate a larger-scale culture
• the cells are then grown to a desired optical density, during which the expression promoter is turned on
• the cells After culturing the cells for several more hours, the cells can be harvested by cent ⁇ fugation
• the cell pellet obtained by the centrifiigation can be solubihzed using various agents known in the art, and the solubilized PRO230, PR0216 or PRO302 polypeptide can then be purified using a metal-chelating column under conditions that allow tight binding of the polypeptide
• the vector, pRK5 (see, EP 307,247, published March 15, 1989), is employed as the expression vector
• the PRO230, PR0216 or PRO302 DNA is hgated into pRK5 with selected restriction enzymes to allow insertion of the DNA encoding PRO230, PR0216 or PRO302 using gation methods such as described in Sambrook et al , supra
• the resulting vector is called pRK5-(DNA encoding PRO230, PR0216 or PRO302)
• the selected host cells are 293 cells Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics About 10 ⁇ g DNA of pRK5-(DNA encoding PRO230, PR0216 or
• PRO302 is mixed with about 1 ⁇ g DNA encoding the VA RNA gene (Thimmappaya et al , Cell, 3 . 543 (1982)) and dissolved in 500 ⁇ l of 1 mM T ⁇ s-HCl, 0 1 mM EDTA, 0 227 M CaCl, To this mixture is added, dropwise,
• the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 35 S-cyste ⁇ ne and 200 ⁇ Ci/ml 35 S-meth ⁇ omne After a
• the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel
• the processed gel may be dried and exposed to film for a selected period of time to reveal the presence of the
• PRO230, PR0216 or PRO302 polypeptide The cultures containing transfected cells may undergo further incubation (in serum-free medium) and the medium is tested in selected bioassays
• the gene encoding PRO230, PR0216 or PRO302 may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Nati Acad Sci . 12 7575 ( 1981 ) 293 cells are grown to maximal density in a spinner flask and 700 ⁇ g pRK5-(DNA encoding PRO230, PR0216 or PRO302) is added The cells are first concentrated from the spinner flask by cent ⁇ fugation and washed with PBS The DNA-dextran precipitate is incubated on the cell pellet for four hours The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 ⁇ g/ml bovine insulin, and 0 1 ⁇ g/ml bovine transfer ⁇ n After about four days, the conditioned media is centrifuged and filtered to remove cells and debris The sample containing the expressed
• PRO230 and PR0216 were stably expressed in CHO cells by the above described method Epitope-tagged gene encoding the PRO230, PR0216 or PRO302 polypeptide may also be expressed in host
• the gene encoding PRO230. PR0216 or PRO302 may be subcloned out of the pRK5 vector
• the subclone insert can undergo PCR amplification to fuse in frame with a selected epitope tag such as a poly-His tag into a baculovirus expression vector
• the gene insert encoding the polv-H ⁇ s-tagged-PRO230, -PR0216 or - PRO302 can then be subcloned into a SV40- driven vector containing a selection marker such as DHFR for selection of stable clones
• the CHO cells can be transfected (as described above) with the SV40-d ⁇ ven vector Labeling may be performed, as described above, to verify expression
• the culture medium containing the expressed gene encoding the poly-H ⁇ s-tagged-PRO230, -PR0216 or -PRO302 can then be concentrated and purified by any selected method, such as by Nr + -chelate affinity chromatography
• yeast expression vectors are constructed for intracellular production or secretion of PRO230 PR0216 or PRO302 from the ADH2'GAPDH promoter DNA encoding PRO230 PR0216 or PRO302 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the gene encoding PRO230, PR0216 or PRO302
• DNA encoding PRO230, PR0216 or PRO302 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO230, PR0216 or PRO302 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of the gene encoding
• yeast cells such as yeast strain AB 1 10
• yeast cells can then be transformed with the expression plasmids described above and cultured in selected fermentation media
• the transformed yeast supernatants can be analyzed by precipitation with 10% t ⁇ chloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain
• Recombinant PRO230, PR0216 or PRO302 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by cent ⁇ fugation and then concentrating the medium using selected cartridge filters The concentrate containing PRO230, PR0216 or PRO302 may further be purified using selected column- chromatography resins
• the sequence coding for PRO230, PR0216 or PRO302 is fused upstream of an epitope tag contained within ⁇ ⁇ a baculovirus expression vector
• epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG)
• plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding PRO230, PR0216 or PRO302 or the desired portion of the coding sequence of PRO230, PR0216 or PRO302 [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may inco ⁇ orate flanking (selected) restriction enzyme sites The product is then digested with those selected
• Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA
• Expressed poly-H ⁇ stagged-PRO230, -PRO216or-PRO302canthen be purified, for example, byNi "-chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al , Nature, 362 175- 179 ( 1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes, pH 7 9, 12 5 mM MgCl 2 , 0 1 mM EDTA, 10% glycerol, 0 l% NP-40, 0 4 M KC1), and sonicated twice for 20 seconds on ice The sonicates are cleared by centrifiigation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7 8) and filtered through a 0 45 ⁇ m filter A Ni
• purification of the IgG-tagged (or Fc tagged)-PRO230, -PR0216 or -PRO302 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography PRO230 and PR0216 were expressed in baculovirus infected Sf9 insect cells While expression was actually performed in a 0 5-2 L scale, it can be readily scaled up for larger (e g , 8 L) preparations
• the proteins were expressed as an IgG construct (lmmunoadhesin), in which the protein extracellular region was fused to an IgGl constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms Following PCR amplification, the respective coding sequences were subcloned into a baculovirus expression vector (pb PH IgG for IgG fusions and pb PH His c for poly-His tagged proteins), and the vector and Baculogold® baculovirus DNA (
• the first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0 1 Cells were incubated for 3 days at 28 °C The supernatant was harvested and filtered Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed
• the conditioned medium from the transfected cells (0 5 to 3 L) was harvested by cent ⁇ fugation to remove the cells and filtered through 0 22 micron filters
• the protein construct were purified using a Ni 2* -NTA column (Qiagen) Before purification, imidazole was added to the conditioned media to a concentration of 5 mM The conditioned media were pumped onto a 6 ml Ni ,+ -NTA column equilibrated in
• a modified baculovirus procedure mav be used inco ⁇ orating h ⁇ gh-5 cells
• the DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector
• epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG)
• a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIE 1 - 1 (Novagen)
• the pIE 1 - 1 and pIE 1 -2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus lei promoter in stably-transformed insect cells
• the plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lei -mediated gene expression in uninfected insect cells as well as
• H ⁇ gh-5 cells are grown to a confluency of 50% under the conditions of, 27 °C, no C02, NO pen/strep
• 30 ⁇ g of pIE based vector containing the sequence was mixed with 1 ml Ex-Cell medium (Media Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401-78P (note this media is light sensitive)), and in a separate tube, 100 ⁇ l of CellFectin (CellFECTIN (GibcoBRL # 10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium
• the two solutions were combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex-Cell media was added to the 2 ml of DNA/CellFECTIN mix and this is layered on h ⁇ gh-5 cells that have been washed once with Ex-Cell media The plate is then incubated in darkness for 1 hour at room temperature
• Immunogens that may be employed include purified PRO230, PR0216 or PRO302 fusion proteins containing PRO230, PR0216 or PRO302, and cells expressing the gene encoding PRO230, PR0216 or PRO302 on the cell surface Selection of the immunogen can be made by the skilled artisan without undue experimentation
• mice such as Balb/c are immunized with the PRO230, PR0216 or PRO302 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1 to 100 micrograms Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research,
• mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional immunization injections Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 antibodies
• the animals "positive" for antibodies can be injected with a final intravenous injection of PRO230, PR0216 or PRO302
• the mice are sacrificed and the spleen cells are harvested
• the spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU 1, available from ATCC, No CRL 1597
• the fusions generate hybridoma cells that can then be plated in 96-well tissue culture plates containing HAT medium to inhibit proliferation of non- fused cells, myeloma hybrids, and spleen cell hybrids
• the hybridoma cells will be screened in an ELISA for reactivity against PRO230, PR0216 or PRO302
• hybridoma cells secreting the desired monoclonal antibodies aga ⁇ nstPRO230, PR0216 or PRO302 are withm the skill in the art
• the positive hybridoma cells can be injected lntrape ⁇ toneally into syngeneic Balb/c mice to produce ascites containing the ant ⁇ -PRO230, ant ⁇ -PR0216 or ant ⁇ -PRO302 monoclonal antibodies
• the hybridoma cells can be grown m tissue-culture flasks or roller bottles Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium-sulfate precipitation, followed by gel-exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed

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