WO2001028586A1 - Traitement de la fibrose - Google Patents

Traitement de la fibrose Download PDF

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Publication number
WO2001028586A1
WO2001028586A1 PCT/US2000/029270 US0029270W WO0128586A1 WO 2001028586 A1 WO2001028586 A1 WO 2001028586A1 US 0029270 W US0029270 W US 0029270W WO 0128586 A1 WO0128586 A1 WO 0128586A1
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zvegβ
antagonist
cells
fibrosis
mammal
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PCT/US2000/029270
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English (en)
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Debra G. Gilbertson
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Zymogenetics, Inc.
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Priority to AU80317/00A priority Critical patent/AU8031700A/en
Publication of WO2001028586A1 publication Critical patent/WO2001028586A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • Fibrosis is the abnormal accumulation of fibrous tissue that can occur as a part of the wound-healing process in damaged tissue. Such tissue damage may result from physical injury, inflammation, infection, exposure to toxins, and other causes. Examples of fibrosis include dermal scar formation, keloids, liver fibrosis, lung fibrosis (e.g., silicosis, asbestosis), kidney fibrosis (including diabetic nephropathy), and glomerulosclerosi .
  • Liver (hepatic) fibrosis occurs as a part of the wound- healing response to chronic liver injury. Fibrosis occurs as a complication of haemochromatosis, Wilson's disease, alcoholism, schistosomiasis, viral hepatitis, bile duct obstruction, exposure to toxins, and matabolic disorders. This formation of scar tissue is believed to represent an attempt by the body to encapsulate the injured tissue. Liver fibrosis is characterized by the accumulation of extracellular matrix that can be distinguished qualitatively from that in normal liver. Left unchecked, hepatic fibrosis progresses to cirrhosis (defined by the presence of encapsulated nodules), liver failure, and death.
  • the present invention provides materials and methods for reducing cell proliferation or extracellular matrix production, treating fibrosis, and reducing stellate cell activation in a mammal.
  • a method of treating fibrosis in a mammal comprising administering to the mammal a composition comprising a therapeutically effective amount of a zvegf3 antagonist in combination with a pharmaceutically acceptable delivery vehicle, wherein the zveg ⁇ antagonist is selected from the group consisting of anti-zvegf3 antibodies, mitogenically inactive receptor-binding zveg ⁇ variant polypeptides, and inhibitory polynucleotides.
  • the fibrosis is liver fibrosis or kidney fibrosis.
  • a method of reducing stellate cell activation in a mammal comprising administering to the mammal a composition comprising a zvegf3 antagonist in combination with a pharmaceutically acceptable delivery vehicle, wherein the zvegf3 antagonist is selected from the group consisting of anti-zvegf3 antibodies, mitogenically inactive receptor-binding zveg ⁇ variant polypeptides, and inhibitory polynucleotides, in an amount sufficient to reduce stellate cell activation.
  • the stellate cells are liver stellate cells.
  • compositions for use within the above methods.
  • the compositions comprise a zvegf3 antagonist in combination with a pharmaceutically acceptable delivery vehicle, wherein the zvegf3 antagonist is selected from the group consisting of anti-zveg ⁇ antibodies, mitogenically inactive receptor-binding zveg ⁇ variant polypeptides, and inhibitory polynucleotides.
  • Fig. 1 is a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO:2. The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated in the figure by lower case letters.
  • Fig. 2 is an alignment of human (SEQ ID NO:2) and mouse (SEQ ID NO:2) and mouse (SEQ ID NO:2)
  • a “zveg ⁇ antagonist” is a compound that reduces the receptor-mediated biological activity (e.g., mitogenic activity) of zveg ⁇ on a target cell. Antagonists may exert their action by competing with zveg ⁇ for binding sites on a cell-surface receptor, by binding to zveg ⁇ and preventing it from binding to a cell-surface receptor, by otherwise interfering with receptor function, by reducing production of zveg ⁇ , or by other means.
  • an “inhibitory polynucleotide” is a DNA or RNA molecule that reduces or prevents expression (transcription or translation) of a second (target) polynucleotide.
  • Inhibitory polynucleotides include antisense polynucleotides, ribozymes, and external guide sequences.
  • the term “inhibitory polynucleotide” further includes DNA and RNA molecules that encode the actual inhibitory species, such as DNA molecules that encode ribozymes.
  • the terms “treat” and “treatment” are used broadly to denote therapeutic and prophylactic interventions that favorably alter a pathological state. Treatments include procedures that moderate or reverse the progression of, reduce the severity of, prevent, or cure a disease.
  • Zveg ⁇ is a protein that is structurally related to platelet- derived growth factor (PDGF) and the vascular endothelial growth factors (VEGF). This protein has also been designated “VEGF-R” (WIPO Publication WO 99/37671) and, more recently, "PDGF-C” (WO 00/18212). Zveg ⁇ /PDGF-C is a multi-domain protein with significant homology to the PDGF/VEGF family of growth factors. Representative amino acid sequences of human and mouse zveg ⁇ are shown in SEQ ID NO: 2 and SEQ ID NO:4, respectively. DNAs encoding these polypeptides are shown in SEQ ID NOS:l and 3, respectively.
  • zveg ⁇ protein is used herein to denote proteins comprising the growth factor domain of a zveg ⁇ polypeptide (e.g., residues 235-345 of human zveg ⁇ (SEQ ID NO:2) or mouse zveg ⁇ (SEQ ID NO:4)), wherein said protein is mitogenic for cells expressing cell-surface PDGF ⁇ -receptor subunit.
  • Zveg ⁇ has been found to bind to the ⁇ and ⁇ isoforms of PDGF receptor.
  • Zveg ⁇ proteins include homodimers and heterodimers as disclosed below.
  • zveg ⁇ proteins can be prepared in a variety of forms, including glycosylated or non- glycosylated, pegylated or non-pegylated, with or without an initial methionine residues, and as fusion proteins as disclosed in more detail below.
  • Fig. 2 An alignment of mouse and human zveg ⁇ polypeptide sequences is shown in Fig. 2. Analysis of the amino acid sequence shown in SEQ ID NO:2 indicates that residues 1 to 14 form a secretory peptide.
  • the CUB domain extends from residue 46 to residue 163.
  • a propeptide-like sequence extends from residue 164 to residue 234, and includes two potential cleavage sites at its carboxyl terminus, a dibasic site at residues 231-232 and a target site for furin or a furin-like protease at residues 231-234.
  • the growth factor domain extends from residue 235 to residue 345. Those skilled in the art will recognize that domain boundaries are somewhat imprecise and can be expected to vary by up to ⁇ 5 residues from the specified positions.
  • interdomain region may be truncated at its amino terminus by a like amount. See Table 1. Corresponding domains in mouse and other non-human zveg ⁇ s can be determined by those of ordinary skill in the art from sequence alignments.
  • Zveg ⁇ proteins can be prepared as fusion proteins comprising amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, an affinity tag, or a targetting polypeptide.
  • a zveg ⁇ protein can be prepared as a fusion with an affinity tag to facilitate purification.
  • any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include, for example, a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol.
  • Zveg ⁇ can also be fused to a targetting peptide, such as an antibody (including polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like) or other peptidic moiety that binds to a target tissue.
  • a targetting peptide such as an antibody (including polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like) or other peptidic moiety that binds to a target tissue.
  • Variations can be made in the zveg ⁇ amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4 to provide mitogenically inactive, receptor-binding polypeptides that act as zveg ⁇ antagonists.
  • mitogenically inactive means that the protein does not show statistically significant activity in a standard mitogenesis assay as compared to a wild-type zveg ⁇ control.
  • Such variations include amino acid substitutions, deletions, and insertions.
  • residues Arg260-Trp271 of human zveg ⁇ form a loop that define the ability of the protein to bind to PDGF- ⁇ receptors, although binding is also permitted to alpha receptors. It is thus predicted that binding to either receptor subunit can be blocked or enhanced by mutations in this region.
  • residues Leu311-His321 of SEQ ID NO:2 are predicted to form a loop (loop3) that may be mutated to block receptor binding. Peptides that mimic this region of the molecule may act as antagonists.
  • the effects of amino acid sequence changes can be predicted by computer modeling (using, e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA) or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nature 369:455, 1994), and can be assessed using art-recognized mutagenesis procedures in combination with activity assays.
  • Representative mutagenesis procedures include, for example, site-directed mutagenesis and alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991).
  • Mutagenesis can be combined with high volume or high-throughput screening methods to detect biological activity of zveg ⁇ variant polypeptides, in particular biological activity in modulating cell proliferation. For example, mitogenesis assays that measure dye incorporation or 3 H- thymidine incorporation can be carried out on large numbers of samples. Competition assays can be employed to confirm antagonist activity.
  • Zveg ⁇ proteins including full-length polypeptides, fragments, and fusion proteins, can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells (including cultured cells of multicellular organisms).
  • a DNA sequence encoding a zveg ⁇ polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. See, WO 00/34474.
  • Zveg ⁇ proteins can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3- methylproline, 2,4-methanoproline, c/.s-4-hydroxyproline, tr ⁇ ras-4-hydroxyproline, N- methylglycine, //o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert- leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4- fluorophenylalanine.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine).
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
  • Zveg ⁇ polypeptides or fragments thereof can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Oxford, 1989.
  • Zveg ⁇ proteins are purified by conventional protein purification methods, typically by a combination of chromatographic techniques.
  • Proteins comprising a polyhistidine affinity tag are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988.
  • the growth factor domain itself binds to nickel resin at pH 7.0-8.0 and 25 mM Na phosphate, 0.25 M NaCl.
  • Bound protein can be eluted with a descending pH gradient down to pH 5.0 or an imidazole gradient.
  • Recombinant zveg ⁇ growth factor domain protein can be purified using a combination of chromatography on a strong cation exchanger followed by a tandem column array comprising a strong anion exchanger followed by an immobilized metal affinity column in series. It has also been found that zveg ⁇ binds to various dye matrices (e.g., BLUE1, BLUE 2, ORANGE 1, ORANGE 3, and RED3 from Lexton Scientific, Signal Hill, CA) in PBS at pH 6-8, from which the bound protein can be eluted in 1-2 M NaCl in 20 mM boric acid buffer at pH 8.8.
  • various dye matrices e.g., BLUE1, BLUE 2, ORANGE 1, ORANGE 3, and RED3 from Lexton Scientific, Signal Hill, CA
  • Protein eluted from RED3 may be passed over RED2 (Lexton Scientific) to remove remaining contaminants.
  • Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
  • zveg ⁇ dimers appear to resemble the previously described PDGF isoforms in being potent mitogens of hepatic stellate cells and appearing to play a role in liver fibrosis.
  • transgenic mice thus provide a model for testing zveg ⁇ antagonists as well as other antifibrotic agents.
  • altered zveg ⁇ expression may initiate or exacerbate a variety of fibrotic conditions.
  • inhibiting the action of zveg ⁇ using a zveg ⁇ antagonist will limit the progress of such conditions. While not wishing to be bound by theory, it is believed that the pro-fibrotic effects of zveg ⁇ are due at least in part to the induction of TGF- ⁇ production.
  • Zveg ⁇ antagonists include, without limitation, anti-zveg ⁇ antibodies (including neutralizing antibodies), inhibitory polynucleotides (including antisense polynucleotides, ribozymes, and external guide sequences), and other peptidic and non- peptidic agents, including small molecule inhibitors and mitogenically inactive receptor-binding zveg ⁇ polypeptides.
  • Such antagonists can be use to block the mitogenic effects of zveg ⁇ and thereby reduce, inhibit, prevent, or otherwise treat fibrosis, including, without limitation, scar formation, keloids, scleroderma, liver fibrosis, lung fibrosis, kidney fibrosis, pancreatic fibrosis, myelofibrosis, post-surgical fibrotic adhesions, fibroproliferative disorders of the vasculature, fibroproliferative disorders of the prostate, fibroproliferative disorders of bone, fibromatosis, fibroma, fibrosarcoma, and the like.
  • Cirrhosis a diffuse process characterized by fibrosis and a conversion of normal architecture into structurally abnormal nodules, can come about for a variety reasons including alcohol abuse, post necrotic cirrhosis (usually due to chronic active hepatitis), biliary cirrhosis, pigment cirrhosis, cryptogenic cirrhosis, Wilson's disease, and alpha- 1 -an titrypsin deficiency.
  • a zveg ⁇ antagonist to suppress the activation of stellate cells, which have been implicated in the production of extracellular matrix in fibrotic liver (Li and Friedman, ibid.).
  • the glomerulus is a major target of many types of renal injury, including immunologic (e.g., immune-complex- or T-cell- mediated), hemodynamic (systemic or renal hypertension), metabolic (e.g., diabetes), "atherosclerotic” (accumulation of lipids in the glomerulus), infiltrative (e.g., amyloid), and toxic (e.g., snake venom) (Johnson, Kidney Int. 45:1769-1782, 1994).
  • immunologic e.g., immune-complex- or T-cell- mediated
  • hemodynamic systemic or renal hypertension
  • metabolic e.g., diabetes
  • "atherosclerotic” accumulation of lipids in the glomerulus
  • infiltrative e.g., amyloid
  • toxic e.g., snake venom
  • Fibrotic disorders of the lung include, for example, silicosis, asbestosis, idiopathic pulmonary fibrosis, bronchiolitis obliterans-organizing pneumonia, pulmonary fibrosis associated with high-dose chemotherapy, idiopathic pulmonary fibrosis, and pulmonary hypertension. These diseases are characterized by cell proliferation and increased production of extracellular matrix components, such as collagens, elastin, fibronectin, and tenascin-C.
  • Pancreatic fibrosis occurs in chronic pancreatitis. This condition is characterized by duct calcification and fibrosis of the pancreatic parenchyma. Like liver cirrhosis, chronic pancreatitis is associated with alcohol abuse. See, Fogar et al., J. Medicine 29:277-287, 1998.
  • Fibroproliferative disorders of bone are characterized by aberrant and ectopic bone formation, commonly seen as active proliferation of the major cell types participating in bone formation as well as elaboration by those cells of a complex bone matrix. Exemplary of such bone disorders is the fibrosis that occurs with prostate tumor metastases to the axial skeleton.
  • prostate carcinoma cells can interact reciprocally with osteoblasts to produce enhanced tumor growth and osteoblastic action when they are deposited in bone (Zhau et al., Cancer 88:2995-3001, 2000; Ritchie et al., Endocrinology 138:1145-1150. 1997).
  • Fibroproliferative responses of the bone originating in the skeleton per se include ostepetrosis and hyperstosis.
  • a defect in osteoblast differentiation and function is thought to be a major cause in osteopetrosis, an inherited disorder characterized by bone sclerosis due to reduced bone resorption, marrow cavities fail to develop, resulting in extramedullary hematopoiesis and severe hematologic abnormalities associated with optic atrophy, deafness, and mental retardation (Lajeunesse et al., J. Clin Invest. 98:1835-1842, 1996).
  • osteoarthritis bone changes are known to occur, and bone collagen metabolism is increased within osteoarthritic femoral heads. The greatest changes occur within the subchondral zone, supporting a greater proportion of osteoid in the diseased tissue (Mansell and Bailey, J. Clin. Invest. 101:1596-1603, 1998).
  • zveg ⁇ has been found to be produced by prostate cells and to stimulate an osteoblast cell line.
  • Fibroproliferative disorders of the vasculature include, for example, transplant vasculopathy, which is a major cause of chronic rejection of heart transplantation.
  • Transplant vasculopathy is characterized by accelerated atherosclerotic plaque formation with diffuse occlusion of the coronary arteries, which is a "classic" fibroproliferative disease. See, Miller et al., Circulation 101:1598-1605, 2000).
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody).
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics.
  • biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • Monoclonal antibodies can also be produced in mice that have been genetically altered to produce antibodies that have a human structure.
  • polyclonal antibodies can be generated by inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zveg ⁇ polypeptide or a fragment thereof.
  • Immunogenic polypeptides will comprise an epitope-bearing portion of a zveg ⁇ polypeptide (e.g., as shown in SEQ ID NO:2) or receptor.
  • An "epitope" is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 8J_: 3998-4002, 1984.
  • Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length.
  • Immunogenic, epitope-bearing polypeptides contain a sequence of at least six, often at least nine, more often from 15 to about 30 contiguous amino acid residues of a zveg ⁇ protein. Polypeptides comprising a larger portion of a zveg ⁇ protein, i.e. from 30 to 50 residues up to the entire sequence are included.
  • the amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided.
  • Such regions include residues 43-48, 96-101, 97-102, 260-265, and 330-335 of SEQ ID NO:2.
  • the latter peptide can be prepared with an additional N-terminal Cys residue to facilitate coupling.
  • the immunogenicity of a polypeptide immunogen may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a zveg ⁇ polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid tetan
  • Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to a polypeptide immunogen, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled polypeptide).
  • Techniques for creating and screening such random peptide display libraries are known in the art (e.g., Ladner et al., US Patent No. 5,223,409; Ladner et al., US Patent No. 4,946,778; Ladner et al., US Patent No. 5,403,484 and Ladner et al., US Patent No.
  • Random peptide display libraries can be screened using the zveg ⁇ sequences disclosed herein to identify proteins which bind to zveg ⁇ .
  • Antibodies are determined to be specifically binding if they bind to their intended target (e.g., zveg ⁇ protein or receptor) with an affinity at least 10-fold greater than the binding affinity to control (e.g., non-zveg ⁇ or non-receptor) polypeptide or protein.
  • a "non-zveg ⁇ polypeptide” includes the related molecules VEGF, VEGF-B, VEGF-C, VEGF-D, PIGF, PDGF-A, and PDGF-B, but excludes zveg ⁇ polypeptides from non-human species.
  • antibodies specific for human zveg ⁇ may also bind to zveg ⁇ from other species.
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 5 660-672, 1949). Methods for screening and isolating specific antibodies are well known in the art. See, for example, Paul (ed.), Fundamental Immunology, Raven Press,. 1993; Getzoff et al., Adv. in Immunol. 43: 1- 98, 1988; Goding (ed.), Monoclonal Antibodies: Principles and Practice, Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2:67-101, 1984.
  • Binding affinity can also be determined using a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ), wherein protein is immobilized onto the surface of a receptor chip. See, Karlsson, /. Immunol. Methods 145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-563, 1993. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • assays known to those skilled in the art can be utilized to detect antibodies that specifically bind to zveg ⁇ proteins or receptors. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assays, inhibition or competition assays, and sandwich assays.
  • Zveg ⁇ antagonists further include antisense polynucleotides, which can be used to inhibit zveg ⁇ gene transcription and thereby inhibit cell activation and/or proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a zveg ⁇ - encoding polynucleotide e.g., a polynucleotide as set forth in SEQ ID NO:l
  • Antisense polynucleotides can be targetted to specific tissues using a gene therapy approach with specific vectors and/or promoters, such as viral delivery systems as disclosed in more detail below.
  • Ribozymes can also be used as zveg ⁇ antagonists within the present invention.
  • Ribozymes are RNA molecules that contain a catalytic center and a target RNA binding portion.
  • the term includes RNA enzymes, self-splicing RNAs, self- cleaving RNAs, and nucleic acid molecules that perform these catalytic functions.
  • a ribozyme selectively binds to a target RNA molecule through complementary base pairing, bringing the catalytic center into close proximity with the target sequence. The ribozyme then cleaves the target RNA and is released, after which it is able to bind and cleave additional molecules.
  • Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a mRNA molecule (see, for example, Draper and Macejak, U.S. Patent No. 5,496,698, McSwiggen, U.S. Patent No. 5,525,468, Chowrira and McSwiggen, U.S. Patent No. 5,631,359, and Robertson and Goldberg, U.S. Patent No. 5,225,337).
  • An expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a ribozyme.
  • An external guide sequence generally comprises a ten- to fifteen-nucleotide sequence complementary to zveg ⁇ mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine.
  • the external guide sequence transcripts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5 '-side of the base-paired region.
  • the growth factor domain of zveg ⁇ has been found to be the active (PDGF receptor-binding) species of the molecule. Proteolytic processing to remove the active (PDGF receptor-binding) species of the molecule.
  • zveg ⁇ antagonists are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • pharmaceutical formulations will include a zveg ⁇ antagonist in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, or the like.
  • a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% dextrose in water, or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995.
  • a “therapeutically effective amount” of a composition is that amount that produces a statistically significant effect, such as a statistically significant reduction in disease progression or a statistically significant improvement in organ function.
  • the exact dose will be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
  • the therapeutic formulations will generally be administered over the period required to achieve a beneficial effect, commonly up to several months and, in treatment of chronic conditions, for a year or more. Dosing is daily or intermittently over the period of treatment, intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed.
  • a zveg ⁇ antagonist can be delivered by aerosolization according to methods known in the art. See, for example, Wang et al., US Patent No. 5,011,678; Gonda et al., US Patent No. 5,743,250; and Lloyd et al., US Patent No. 5,960,792.
  • Antibodies are preferably administered parenterally, such as by bolus injection or infusion (intravenous, intramuscular, intraperitoneal or subcutaneous) over the course of treatment.
  • Antibodies are generally administered in an amount sufficient to provide a minimum circulating level of antibody throughout the treatment period of between approximately 20 ⁇ g and 1 mg/kg body weight.
  • Chimeric and humanized antibodies are expected to have circulatory half-lives of up to four and up to 14-21 days, respectively. In many cases it will be preferable to. administer daily doses during a hospital stay, followed by less frequent bolus injections during a period of outpatient treatment.
  • Antibodies can also be delivered by slow-release delivery systems, pumps, and other known delivery systems for continuous infusion. Dosing regimens may be varied to provide the desired circulating levels of a particular antibody based on its pharmacokinetics. Thus, doses will be calculated so that the desired circulating level of therapeutic agent is maintained. Daily doses referred to above may be administered as larger, less frequent bolus administrations to provide the recited dose averaged over the term of administration .
  • zveg ⁇ antagonists Those skilled in the art will recognize that the same principles will guide the use of other zveg ⁇ antagonists.
  • the dosing regimen for a given antagonist will be determined by a number of factors including potency, pharmacokinetics, and the physicochemical nature of the antagonist.
  • non-peptidic zveg ⁇ antagonists may be administered enterally.
  • Therapeutic polynucleotides can be delivered to patients or test animals by way of viral delivery systems.
  • viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997.
  • the adenovirus system offers several advantages.
  • Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body. Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinj ection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963- 967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
  • Activity of zveg ⁇ antagonists can be measured in vitro using assays (including cell-based assays) designed to measure zveg ⁇ activity. Antagonists will reduce the effects of zveg ⁇ within the assay.
  • Ligand-receptor binding can be assayed by a variety of methods well known in the art, including receptor competition assays (Bowen-Pope and Ross, Methods Enzymol. 109:69-100, 1985) and through the use of soluble receptors, including receptors produced as IgG fusion proteins (U.S. Patent No. 5,750,375).
  • Receptor binding assays can be performed on cell lines that contain known cell-surface receptors for evaluation.
  • Suitable mitogenesis assays measure incorporation of 3 H-thymidine into (1) 20% confluent cultures to look for the ability of zveg ⁇ proteins to further stimulate proliferating cells, and (2) quiescent cells held at confluence for 48 hours to look for the ability of zveg ⁇ proteins to overcome contact- induced growth inhibition.
  • Suitable dye incorporation assays include measurement of the incorporation of the dye Alamar blue (Raz et al., ibid.) into target cells. See also, Gospodarowicz et al., J. Cell. Biol. 70:395-405, 1976; Ewton and Florini, Endocrinol. 106:577-583, 1980; and Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 86:7311- 7315, 1989.
  • zveg ⁇ antagonists can be studied in non- human animals by administration of exogenous compounds, by expression of zveg ⁇ antisense polynucleotides, and by suppression of endogenous zveg ⁇ expression through knock-out techniques.
  • Viral delivery systems can be employed.
  • Zveg ⁇ antagonists can be administered or expressed individually, in combination with other zveg ⁇ antagonists, or in combination other compounds, including other growth factor antagonists.
  • Test animals are monitored for changes in such parameters as clinical signs, body weight, blood cell counts, clinical chemistry, histopathology, and the like.
  • bleomycin is a known causative agent of pulmonary fibrosis in humans and can induce interstitial lung disease in mice, including an increase in the number of fibroblasts, enhanced collagen deposition, and dysregulated matrix remodeling.
  • C57B1/6 mice are administered bleomycin by osmotic minipump for 1 week. There follows a period of inflammation, with cutaneous toxicity beginning approximately 4-7 days after bleomycin administration and continuing for about a week, after which the mice appear to regain health. About 3-4 weeks after the finish of bleomycin delivery, the mice are sacrificed, and the lungs are examined histologically for signs of fibrosis.
  • Scoring is based on the extent of lung fibrotic lesions and their severity. Serum is assayed for lactic dehydrogenase, an intracellular enzyme that is released into the circulation upon general cell death or injury. Lung tissue is assayed for hydroxyproline as a measure of collagen deposition. The invention is further illustrated by the following non-limiting examples.
  • Example 1 A human salivary gland library was screened for a full-length clone of zveg ⁇ by PCR. This library was an arrayed library representing 9.6 X 10 5 clones made in the vector pZP5x.
  • the vector pZP5x is the same as vector pZP-9 (deposited with American Type Culture Collection, 10801 University Boulevard., Manassas, VA under Accession Number 98668), but contains a cytomegalovirus promoter instead of a metallothionein promoter between the Asp718 and BamHI sites.
  • the plasmid thus comprises a dihyrofolate reductase gene under control of the SV40 early promoter and SV40 polyadenylation site, and a cloning site to insert the gene of interest under control of the CMV promoter and the human growth hormone (hGH) gene polyadenylation site.
  • the working plate containing 80 pools of 12,000 colonies each was screened by PCR using oligonucleotide primers ZC19.045 (SEQ ID NO:6) and ZC19,047 (SEQ ID NO:7) with an annealing temperature of 60°C for 35 cycles. There were two strong positives, pools 58 (T-8 F1-F12) and 77 (T-7 H1-H12).
  • the corresponding pools in the transfer plate were then screened by PCR using the same conditions. Two positives were obtained at the transfer level. The positives were T-7 HI 1 and T-8 F10. 5' RACE reactions were done on the transfer plate pools, and the fragments were sequenced to check zveg ⁇ sequence and determine if a full-length clone was present.
  • oligonucleotide primers ZC12 00 (SEQ ID NO:8) and ZC19,045 (SEQ ID NO:6) were used at an annealing temperature of 61°C for 5 cycles, then 55°C for 30 cycles. Sequencing showed that the pool T-7 Hl l had a frameshift. Transfer plate 8 pool F10 sequence appeared to be correct, so this pool of DNA was used in filter lifts.
  • the filters were prewashed at 65°C in prewash buffer consisting of 0.25X SSC, 0.25% SDS, and lmM EDTA. The solution was changed a total of three times over a 45-minute period to remove cell debris. Filters were prehybridized for approximately 3 hours at 65°C in 25 ml of ExpressHybTM.
  • the probe was generated using an approximately 400-bp fragment produced by digestion of the first proprietary database clone with EcoRI and Bgi ⁇ and gel-purified using a spin column as disclosed above. The probe was radioactively labeled with P by random priming as disclosed above and purified using a push column. ExpressHybTM solution was used for the hybridizing solution for the filters. Hybridization took place overnight at 65°C.
  • a PCR panel was screened for mouse zveg ⁇ DNA.
  • the panel contained 8 cDNA samples from brain, bone marrow, 15-day embryo, testis, salivary gland, placenta, 15-day embryo (Clontech Laboratories), and 17-day embryo (Clontech Laboratories) libraries.
  • PCR mixtures contained oligonucleotide primers ZC21,222 (SEQ ID NO:9) and ZC21.224 (SEQ ID NO: 10).
  • the reaction was run at an annealing temperature of 66°C with an extension time of 2 minutes for a total of 35 cycles using Ex TaqTM DNA polymerase (PanVera, Madison, WI) plus antibody.
  • the mouse 15-day embryo library was screened for full-length zveg ⁇ DNA.
  • This library was an arrayed library representing 9.6 X 10 5 clones in the pCMV*SPORT 2 vector (Life Technologies, Gaithersburg, MD).
  • the working plate containing 80 pools of 12,000 colonies each, was screened by PCR using oligonucleotide primers ZC21.223 (SEQ ID NO:l l) and ZC21.224 (SEQ ID NO:10) with an annealing temperature of 66°C for 35 cycles. Eighteen positives were obtained. Fragments from four pools (A2, A 10, B2, and C4)were sequenced; all were confirmed to encode zveg ⁇ . Additional rounds of screening using the same reaction conditions and pools from the working and source plates identified one positive pool (5D).
  • a probe was generated by PCR using oligonucleotide primers ZC21.223 (SEQ ID NO:l l) and ZC21.224 (SEQ ID NO: 10), and a mouse 15-day embryo template at an annealing temperature of 66°C for 35 cycles.
  • the PCR fragment was gel purified using a spin column containing a silica gel membrane (QIAquickTM Gel Extraction Kit; Qiagen, Inc., Valencia, CA).
  • the DNA was radioactively labeled with 32 P using a commercially available kit (RediprimeTM II random-prime labeling system; Amersham Corp., Arlington Heights, IL) according to the manufacturer's specifications.
  • the probe was purified using a commercially available push column (NucTrap® column; Stratagene, La Jolla, CA; see U.S. Patent No. 5,336,412).
  • the filters were prewashed at 65°C in prewash buffer consisting of 0.25X SSC, 0.25% SDS and lmM EDTA. The solution was changed a total of three times over a 45-minute period to remove cell debris. Filters were prehybridized overnight at 65°C in 25 ml of a hybridization solution (ExpressHybTM Hybridization Solution; Clontech Laboratories, Inc., Palo Alto, CA), then hybridized overnight at 65° C in the same solution.
  • a hybridization solution ExpressHybTM Hybridization Solution
  • Filters were rinsed twice at 65°C in pre-wash buffer (0.25X SSC, 0.25% SDS, and lmM EDTA), then washed twice in pre-wash buffer at 65°C. Filters were exposed to film for 2 days at -80°C. There were 10 positives on the filters. 3 clones were picked from the positive areas, streaked out, and 15 individual colonies from these three positives were screened by PCR using primers ZC21,223 (SEQ ID NO: 11) and ZC21,334 (SEQ ID NO: 12) at an annealing temp of 66°C. Two positives were recovered and sequenced. Both sequences were found to be the same and encoded full-length mouse zveg ⁇ (SEQ ID NO:4).
  • the amino acid sequence is highly conserved between mouse and human zveg ⁇ s, with an overall amino acid sequence identity of 87%.
  • the secretory peptide, CUB domain, inter-domain, and growth factor domain have 82%, 92%, 79% and 94% amino acid identity, respectively.
  • a mammalian cell expression vector for the growth factor domain of zveg ⁇ was constructed by joining the zveg ⁇ fragment to a sequence encoding an optimized t-PA secretory signal sequence (U.S. Patent No. 5,641,655) in the linearized pZMPl l vector downstream of the CMV promoter.
  • the plasmid pZMPl l is a mammalian expression vector containing an expression cassette having the CMV immediate early promoter, a consensus intron from the variable region of mouse immunoglobulin heavy chain locus, Kozak sequences, multiple restriction sites for insertion of coding sequences, a stop codon, and a human growth hormone terminator.
  • the plasmid also contains an IRES element from poliovirus, the extracellular domain of CD8 truncated at the C-terminal end of the transmembrane domain, an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene, the SV40 terminator, and the URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae.
  • the resulting vector was designated pZMPll/zv3GF-otPA.
  • BHK 570 cells were transfected with pZMPl l/zv3GF-otPA by liposome-mediated transfection (using (LipofectamineTM; Life Technologies) and cultured according to conventional procedures.
  • the zveg ⁇ growth factor domain eluted at 25 to 50 mS conductivity during the evolving gradient. Reducing SDS-PAGE revealed a distinct band at 20 kD, which was confirmed as zveg ⁇ by Western blotting.
  • This material was pooled and prepared for loading to a tandem column array comprising a strong anion exchange resin (Poros® HQ 50; PerSeptive Biosystems) followed by an immobilized metal (nickel) affinity column in series. The system of columns was equilibrated in 20 mM MOPS buffer at pH 7.0. The veg ⁇ pool was in-line diluted at 1:10 (V:V) with the MOPS equilibration buffer while loading.
  • transgenic animals expressing zveg ⁇ genes requires adult, fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl, 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2fl, 2-4 months, (Taconic Farms)).
  • the donors are acclimated for 1 week, then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma, St. Louis, MO) IP., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin (hCG (Sigma)) IP. to induce superovulation. Donors are mated with studs subsequent to hormone injections.
  • Pregnant Mare's Serum gonadotrophin Sigma, St. Louis, MO
  • hCG human Chorionic Gonadotropin
  • Ovulation generally occurs within 13 hours of hCG injection. Copulation is confirmed by the presence of a vaginal plug the morning following mating.
  • the oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma Chemical Co.). Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (Table 2; all reagents available from Sigma Chemical Co.) that has been incubated with 5% CO2,
  • Zveg ⁇ cDNA is inserted into the expression vector pHB12-8.
  • Vector pHB12-8 was derived from p2999B4 (Palmiter et al., Mol. Cell Biol. 13:5266-5275, 1993) by insertion of a rat insulin ⁇ intron (ca. 200 bp) and polylinker (Fse I/Pme I/Asc I) into the Nru I site.
  • the vector comprises a mouse metallothionein (MT-1) promoter (ca. 750 bp) and human growth hormone (hGH) untranslated region and polyadenylation signal (ca. 650 bp) flanked by 10 kb of MT-1 5' flanking sequence and
  • the cDNA is inserted between the insulin II and hGH sequences.
  • Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO2-equilibrated mineral oil.
  • the DNA is drawn into an injection needle (pulled from a 0.75mm ID, 1mm OD borosilicate glass capillary) and injected into individual eggs. Each egg is penetrated with the injection needle into one or both of the haploid pronuclei.
  • Picoliters of DNA are injected into the pronuclei, and the injection needle is withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transferred into an organ tissue-culture dish with pregassed W640 medium for storage overnight in a 37°C/5% CO2 incubator.
  • 2-cell embryos are transferred into pseudopregnant recipients.
  • the recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds.
  • Recipients are anesthetized and shaved on the dorsal left side and transferred to a surgical microscope.
  • a small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen.
  • the reproductive organs are exteriorized onto a small surgical drape.
  • the fat pad is stretched out over the surgical drape, and a baby serrefine (Roboz, Rockville, MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in.
  • a baby serrefine Robot, Rockville, MD
  • primers ZC17,156 SEQ ID NO:15
  • ZC17,157 SEQ ID NO:16
  • DNA from animals positive for the transgene will generate two bands, a 368-base-pair band corresponding to the hGH 3' UTR fragment and a band of variable size corresponding to the cDNA insert.
  • a partial hepatectomy is performed.
  • a surgical prep is made of the upper abdomen directly below the xiphoid process.
  • a small 1.5-2 cm incision is made below the sternum, and the left lateral lobe of the liver is exteriorized.
  • a tie is made around the lower lobe securing it outside the body cavity.
  • An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid, Wayne, N.J.) is placed proximal to the first tie.
  • a distal cut is made from the Dexon tie, and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish.
  • the excised liver section is transferred to a 14-ml polypropylene round bottom tube, snap frozen in liquid nitrogen, and stored on dry ice.
  • the surgical site is closed with suture and wound clips, and the animal's cage is placed on a 37°C heating pad for 24 hours post-operatively.
  • the animal is checked daily post- operatively, and the wound clips are removed 7-10 days after surgery.
  • Analysis of the mRNA expression level of each transgene is done using an RNA solution hybridization assay or real-time PCR on an ABI Prism 7700 (PE Applied Biosystems, Inc., Foster City, CA) following the manufacturer's instructions.
  • An adenovirus vector was prepared using a liver-specific albumin gene enhancer and basal promoter (designated "AEO promoter").
  • the resulting PmeVAscI fragment was subcloned into the polylinker of pKFO114, a basal keratinocyte-restricted transgenic vector comprising the human keratin 14 (K14) promoter (an approximately 2.3 Kb fragment amplified from human genomic DNA [obtained from Clontech Laboratories, Inc.] based on the sequence of Staggers et al., "Sequence of the promoter for the epidermal keratin gene, K14", GenBank accession #U 11076, 1994), followed by a heterologous intron (a 294- bp BstXUPstl fragment from pIRESlhyg (Clontech Laboratories, Inc.; see, Huang and Gorman, Nucleic Acids Res.
  • K14 human keratin 14
  • heterologous intron a 294- bp BstXUPstl fragment from pIRESlhyg (Clontech Laboratories, Inc
  • the transgene insert was separated from the plasmid backbone by /Votl digestion and agarose gel purification, and fertilized ova from matings of B6C3FlTac mice or inbred FVB/NTac mice were microinjected and implanted into pseudopregnant females essentially as described by Malik et al., Molec. Cell. Biol. 15:2349-2358, 1995.
  • Transgenic founders were identified by PCR on genomic tail DNA using primers specific for the human growth hormone poly A signal (ZC17,252, SEQ ID NO:14; and ZC17,251, SEQ ID NO:13) to amplify a 368-bp diagnostic product.
  • Transgenic lines were initiated by breeding founders with C57BL/6Tac or FVB/NTac mice.
  • telomere sequence For construction of adenovirus vectors, the protein coding region of human zveg ⁇ was amplified by PCR using primers that added Pmel and Ascl restriction sites at the 5' and 3' termini respectively.
  • PCR primers ZC20,180 (SEQ ID NO: 17) and ZC20,181 (SEQ ID NO: 18) were used with a full-length zveg ⁇ cDNA template in a PCR reaction as follows: incubation at 95°C for 5 minutes; followed by 15 cycles at 95°C for 1 min., 61°C for 1 min., and 72°C for 1.5 min.; followed by 72°C for 7 min.; followed by a 4°C soak.
  • the reaction product was loaded onto a 1.2 % low- melting-temperature agarose gel in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA).
  • the zveg ⁇ PCR product was excised from the gel and purified using a commercially available kit comprising a silica gel mambrane spin column (QIAquickTM PCR Purification Kit and gel cleanup kit; Qiagen, Inc.) as per kit instructions.
  • the zveg ⁇ product was then digested with Pmel and Ascl, phenol/chloroform extracted, EtOH precipitated, and rehydrated in 20 ml TE (Tris/EDTA pH 8).
  • the 1038 bp zveg ⁇ fragment was then ligated into the Pmel-Ascl sites of the transgenic vector pTG12-8 (also known as pHB12-8; see Example 4) and transformed into E. coli DH10BTM competent cells by electroporation.
  • Clones containing zveg ⁇ were identified by plasmid DNA miniprep followed by digestion with Pmel and Ascl. A positive clone was sequenced to insure that there were no deletions or other anomalies in the construct. The sequence of zveg ⁇ cDNA was confirmed. DNA was prepared using a commercially available kit (Maxi Kit,
  • the 1038bp zveg ⁇ cDNA was released from the pTG12-8 vector using Pmel and Ascl enzymes.
  • the cDNA was isolated on a 1% low melting temperature agarose gel and was excised from the gel. The gel slice was melted at 70°C, and the DNA was extracted twice with an equal volume of Tris-buffered phenol and precipitated with EtOH. The DNA was resuspended in 10 ⁇ l H 2 O.
  • the zveg ⁇ cDNA was cloned into the EcoRV-AscI sites of a modified pAdTrack-CMV (He, T-C. et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998).
  • This construct contains the green fluorescent protein (GFP) marker gene.
  • the CMV promoter driving GFP expression was replaced with the SV40 promoter, and the SV40 polyadenylation signal was replaced with the human growth hormone polyadenylation signal.
  • the native polylinker was replaced with Fsel, EcoRV, and Ascl sites.
  • This modified form of pAdTrack-CMV was named pZyTrack.
  • Ligation was performed using a commercially available DNA ligation and screening kit (Fast-LinkTM kit; Epicentre Technologies, Madison, WI). Clones containing zveg ⁇ were identified by digestion of mini prep DNA with Fsel and Ascl. In order to linearize the plasmid, approximately 5 ⁇ g of the resulting pZyTrack zveg ⁇ plasmid was digested with Pmel. Approximately 1 ⁇ g of the linearized plasmid was cotransformed with 200 ng of supercoiled pAdEasy (He et al., ibid.) into E. coli BJ5183 cells (He et al., ibid.).
  • the co-transformation was done using a Bio-Rad Gene Pulser at 2.5 kV, 200 ohms and 25 ⁇ Fa.
  • the entire co-transformation mixture was plated on 4 LB plates containing 25 ⁇ g/ml kanamycin. The smallest colonies were picked and expanded in LB/kanamycin, and recombinant adenovirus DNA was identified by standard DNA miniprep procedures. Digestion of the recombinant adenovirus DNA with Fsel and Ascl confirmed the presence of the zveg ⁇ insert.
  • the recombinant adenovirus miniprep DNA was transformed into E. coli DH10BTM competent cells, and DNA was prepared using a Maxi Kit (Qiagen, Inc.) according to kit instructions.
  • recombinant adenoviral DNA was digested with Pad enzyme (New England Biolabs) for 3 hours at 37°C in a reaction volume of 100 ⁇ l containing 20-30U of Pad.
  • the digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol.
  • the DNA pellet was resuspended in lO ⁇ l distilled water.
  • a T25 flask of QBI-293A cells Quantantum Biotechnologies, Inc. Montreal, Qc. Canada, inoculated the day before and grown to 60-70% confluence, were transfected with the Pad digested DNA.
  • the Pad-digested DNA was diluted up to a total volume of 50 ⁇ l with sterile HBS (150mM NaCl, 20mM HEPES).
  • HBS 150mM NaCl, 20mM HEPES
  • 20 ⁇ l of lmg/ml N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethyl-ammonium salts (DOTAP) (Boehringer Mannheim, Indianapolis, IN) was diluted to a total volume of 100 ⁇ l with HBS.
  • the DNA was added to the DOTAP, mixed gently by pipeting up and down, and left at room temperature for 15 minutes.
  • NP-40 detergent was added to a final concentration of 0.5% to the bottles of crude lysate in order to lyse all cells.
  • Bottles were placed on a rotating platform for 10 minutes agitating as fast as possible without the bottles falling over.
  • the debris was pelleted by centrifugation at 20,000 X G for 15 minutes.
  • the supernatant was transferred to 250-ml polycarbonate centrifuge bottles, and 0.5 volume of 20% PEG8000/2.5 M NaCl solution was added.
  • the bottles were shaken overnight on ice.
  • the bottles were centrifuged at 20,000 X G for 15 minutes and, the supernatant was discarded into a bleach solution.
  • the white, virus/PEG precipitate from 2 bottles was resuspended in 2.5 ml PBS.
  • the resulting virus solution was placed in 2-ml microcentrifuge tubes and centrifuged at 14,000 X G in the microcentrifuge for 10 minutes to remove any additional cell debris.
  • the supernatant from the 2-ml microcentrifuge tubes was transferred into a 15-ml polypropylene snapcap tube and adjusted to a density of 1.34 g/ml with CsCl.
  • the volume of the virus solution was estimated, and 0.55 g/ml of CsCl was added.
  • the CsCl was dissolved, and 1 ml of this solution weighed 1.34 g.
  • the solution was transferred to 3.2-ml, polycarbonate, thick-walled centrifuge tubes and spun at 348,000 X G for 3-4 hours at 25°C.
  • the virus formed a white band. Using wide-bore pipette tips, the virus band was collected.
  • the virus recovered from the gradient had a large amount of CsCl which had to be removed before it was used on cells.
  • Commercially available ion-exchange columns (PD-10 columns prepacked with Sephadex® G-25M; Pharmacia Biotech, Piscataway, NJ) were used to desalt the virus preparation.
  • the column was equilibrated with 20 ml of PBS. The virus was loaded and allowed to run into the column. 5 ml of PBS was added to the column, and fractions of 8-10 drops were collected.
  • glycerol was added to the purified virus to a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80°C.
  • TCID 5 o formulation used was as per Quantum Biotechnologies, Inc., above.
  • the titer (T) was determined from a plate where virus was diluted from 10 "2 to 10 " , and read 5 days after the infection. At each dilution a ratio (R) of positive wells for CPE per the total number of wells was determined.
  • R ratio of positive wells for CPE per the total number of wells was determined.
  • livers were pale and very enlarged, with enlarged vessels at the tips of the lobes.
  • the livers also showed sinusoidal cell proliferation. Changes were also seen in hepatocytes (hypertrophy, degeneration, and necrosis) and were most likely nonspecific effects of adenovirus infection. Splenic change consisted of increased extramedulary hematopoiesis, which was correlated with enlarged splenic size.
  • Polyclonal anti-peptide antibodies were prepared by immunizing two female New Zealand white rabbits with the peptides huzveg ⁇ -1 (residues 80-104 of SEQ ID NO:2), huzveg ⁇ -2 (residues 299-314 of SEQ ID NO:2), huzveg ⁇ -3 (residues 299-326 of SEQ ID NO:2 with an N-terminal cys residue), or huzveg ⁇ -4 (residues 195-225 of SEQ ID NO:2 with a C-terminal cys residue).
  • the peptides were synthesized using an Applied Biosystems Model 431 A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's instructions.
  • the peptides huzveg ⁇ -1, huzveg ⁇ -3, and huzveg ⁇ -4 were then conjugated to the carrier protein maleimide-activated keyhole limpet hemocyanin (KLH) through cysteine residues (Pierce Chemical Co., Rockford, IL).
  • KLH keyhole limpet hemocyanin
  • the peptide huzvefg3-2 was conjugated to the carrier protein KLH using gluteraldehyde.
  • the rabbits were each given an initial intraperitoneal (IP) injection of 200 ⁇ g of conjugated peptide in Complete Freund's Adjuvant (Pierce Chemical Co.) followed by booster IP injections of 100 ⁇ g conjugated peptide in incomplete Freund's Adjuvant every three weeks. Seven to ten days after the administration of the third booster injection, the animals were bled and the serum was collected. The rabbits were then boosted and bled every three weeks.
  • IP intraperitoneal
  • the huzveg ⁇ peptide-specific antibodies were affinity purified from the rabbit serum using an CNBr-Sepharose® 4B peptide column (Pharmacia Biotech) that was prepared using 10 mg of the respective peptides per gram CNBr-Sepharose®, followed by dialysis in PBS overnight.
  • Peptide specific-huzveg ⁇ antibodies were characterized by an ELISA titer check using 1 ⁇ g/ml of the appropriate peptide as an antibody target.
  • the huzveg ⁇ - 1 peptide-specific antibodies had a lower limit of detection (LLD) of 500 pg/ml by ELISA on the appropriate antibody target and recognize full-length recombinant protein (MBP-fusion) by ELISA.
  • the huzveg ⁇ -2 peptide-specific antibodies had an LLD of 1 ng/ml by ELISA.
  • the huzveg ⁇ -3 peptide- specific antibodies had an LLD of 50 pg/ml by ELISA and recognized recombinant protein by Western Blot analysis.
  • the huzveg ⁇ -4 peptide-specific antibodies had an LLD of 50 pg/ml by ELISA and recognized recombinant protein by Western Blot analysis.
  • Mouse hybridomas producing monoclonal antibodies (MAbs) specific for recombinant human zveg ⁇ growth factor domain (GFD) protein were generated using purified, untagged, recombinant human zveg ⁇ GFD produced in BHK cells (huzveg ⁇ -GFD-BHK).
  • Ten BALB/c mice were each injected IP on day 1 with 20 ⁇ g of huzveg ⁇ -GFD-BHK mixed 1:1 (v/v) in complete Freund's adjuvant.
  • Each mouse was subsequently injected IP with 10 ⁇ g of huzveg ⁇ -GFD-BHK mixed 1:1 in incomplete Freund's adjuvant on days 15, 29, 41, 57, 71, 89 and 115.
  • the fusion mixture was plated into 24 96-well plates at an average density of 1.2 x 10 5 total cells/well in Iscove's modified Dulbecco's medium (IMDM; Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal clone I serum (HyClone Laboratories, Inc., Logan, UT), 10% hybridoma cloning supplement (BM Condimed® HI; Roche Diagnostics Corp., Indianapolis, IN), 2 mM L-glutamine (Life Technologies, Inc.), 100 U/mL penicillin G sodium (Life Technologies, Inc.), and 100 ⁇ g/mL streptomycin sulfate (Life Technologies, Inc.). Wells were fed on days 4 and 7 by aspiration and replacement of approximately three- fourths of the media contents in each well. This fusion was designated HH1.
  • IMDM Iscove's modified Dulbecco's medium
  • Anti-huzveg ⁇ mAbs of the IgG class were detected on days 9/10 post-fusion using a biotinylated huveg ⁇ -GFD capture ELISA.
  • Wells of plates (Immulon® II; Dynex Technologies, Chantilly, VA) were coated with 1 ⁇ g/mL of goat anti-mouse IgG (obtained from Kirkegaard & Perry Laboratories, Gaithersburg, MD) in 0.05 M carbonate/bicarbonate buffer (Sigma, St. Louis, MO), 50 ⁇ L/well.
  • Human zveg ⁇ - GFD protein was biotinylated with sulfo-NHS-LC-biotin (Pierce Chemical Company, Rockford, IL) according to the manufacturer's instructions for 45 minutes at RT. The reaction was stopped with 2M glycine. Biotinylated protein was diluted to 1 ⁇ g/mL in PTB buffer. The plates were washed four times with PBST, biotinylated huzveg ⁇ - GFD was added at 100 ⁇ L/well, and the plates were incubated at RT for 1 hour.
  • Optical density was read on an ELISA plate reader (Molecular Devices, Sunnyvale, CA) at wavelengths 450 nm (LI) and 650 nm (L2). Final OD measurement was determined by the formula L1-L2.
  • 78 master wells contained antibody that was capable of capturing biotinylated huzveg ⁇ -GFD. These master wells were expanded for hybridoma cryopreservation and additional supernatant generation. ELISA analysis of the expanded master well supematants demonstrated that 27 of the original 78 master wells retained antibody specific for huzveg ⁇ -GFD, with 20 of 27 possessing significant reactivity with the antigen.
  • Clones HH1-24, -40, -57 and -76 were all found to produce an Igd antibody.
  • Clones HH1-58 and -78 produced an IgG 2b antibody. All antibodies possessed a K light chain.
  • Recombinant zveg ⁇ was analyzed for mitogenic activity on rat stellate cells (Greenwel et al., Laboratory Invest. 65:644, 1991; Greenwel et al., Laboratory Invest. 69:210, 1993).
  • Stellate cells were plated at a density of 2,000 cells/well in 96- well culture plates and grown for approximately 72 hours in DMEM containing 10% fetal calf semm at 37°C. Cells were quiesced by incubating them for 20 hours in serum-free DMEM/Ham's F-12 medium containing insulin (5 ⁇ g/ml), transferrin (20 ⁇ g/ml), and selenium (16 pg/ml) (ITS).
  • Test samples consisted of either conditioned media (CM) from adenovirally-infected HaCaT human keratinocyte cells (Boukamp et al., J. Cell. Biol. 106:761-771, 1988) expressing full- length zveg ⁇ , purified growth factor domain expressed in BHK cells, or control media from cells infected with parental adenovirus (Zpar) containing an expression unit for green fluorescent protein.
  • CM conditioned media
  • Zpar parental adenovirus
  • RNA from mouse and rat liver were used as controls (20 ⁇ g each).
  • An approximately 680-bp DNA probe was generated by PCR using oligonucleotide primers ZC21,222 (SEQ ID NO:9) and ZC21.224 (SEQ ID NO: 10) and a mouse zveg ⁇ full-length cDNA clone as a template.
  • the DNA probe was purified by conventional procedures using a commercially available kit (QIAquickTM Gel Extraction Kit; Qiagen, Inc., Valencia, CA).
  • the probe was radioactively labeled with 32 P using a commercially available kit (RediprimeTM II DNA Labeling system; Amersham, Arlington Heights, IL) according to the manufacturer's specifications.
  • the probe was purified using a push column (NucTrap® column; Stratagene, La Jolla, CA; see U.S. Patent No. 5,336,412).
  • a commercially available hybridization solution (ExpressHybTM Hybridization Solution; Clontech Laboratories, Inc., Palo Alto, CA) was used for the blots. Hybridization took place overnight at 65°C.
  • the blots were then washed four times in 2X SCC and 0.1% SDS at room temperature, followed by two washes in 0.1X SSC and 0.1% SDS at 55° C.
  • One transcript size was detected at approximately 4 kb in Jakotay, Nelix, Paris and Tuvak cell lines. Signal intensity was highest for Jakotay, then Nelix and Tuvak.
  • Recombinant human zveg ⁇ GFD produced in BHK cells was assayed for growth factor activity using an osteoblast cell line containing a luciferase reporter gene constmct.
  • CCC4 cells derived from p53 knock-out mice
  • transfected with the reporter constmct were plated in 96-well plates (BioCoatTM; Becton Dickinson, Franklin Lakes, NJ) at 1 x 10 4 cells/well in 1% FBS alpha MEM (JRH Biosciences, Lexena, KS) containing L-Glutamine and sodium pyruvate (Life Technologies, Inc.).
  • the cells were cultured for 24 hours, the medium was removed, and the wells were washed twice with wash buffer (1% BSA in PBS), 100 ⁇ l/well at 37°C.
  • Zveg ⁇ , PDGF-AA, and PDGF-BB were diluted in assay medium (alpha MEM containing 1% BSA, 10 mM Hepes, L-Glutamate, and sodium pyruvate), and 100- ⁇ l samples were added to the wells.
  • the plates were incubated 4 hours at 37°C, then the wells were washed with wash buffer.
  • Lysis reagent Cell Culture Lysis Reagent; Promega Corporation, Madison, WI
  • Luciferase substrate (Luciferase Assay System; Promega Corporation) was diluted 1: 1 with assay medium and added to the wells. The plates were read on a luminometer. Results, shown in Table 4, indicted that all three growth factors exhibited mitogenic activity on the CCC4 cells. Table 4
  • CBC complete blood count
  • serum chemistry serum chemistry
  • slides were prepared for manual differential blood and marrow progenitor cell analysis.
  • femur, lung, heart, thymus, liver, kidney, spleen, pancreas, duodenum, and mesenteric lymph nodes were submitted for standard histology and assessment of BrdU incorporation.
  • the lining of the duodenum served as the control tissue for BrdU incorporation.
  • livers of the zveg ⁇ adenovims-treated mice tended to look more pale than animals treated with the parental vims. Proliferation of sinusoidal cells was observed in liver. Visual inspection suggested that these cells were stellate cells and/or fibroblasts. Spleen color was the same in both groups. Most of the animals that received the zveg ⁇ adenovims had paler femur shafts, with the marrow lighter in color.
  • BrdU labeling showed increased cell proliferation in kidney, mainly in the medulla and to a lesser extent in the cortex. Proliferating cells appeared to be interstitial cells, which may have included fibroblasts and/or mesangial cells.
  • TGF- ⁇ 1 levels were determined in these media using a commercially available ELISA kit (R&D Systems, St Paul, MN). Stimulation of stellate cells with 100 ng/ml zveg ⁇ GFD resulted in an approximately 5 -fold increase in the production of TGF- ⁇ compared to a BSA control.
  • Recombinant human zveg ⁇ GFD was analyzed for mitogenic activity on human mesangial cells (Clonetics, San Diego, CA).
  • Mesangial cells were plated at a density of 2,000 cells/well in 96-well culture plates and grown for approximately 72 hours in DMEM containing 10% fetal calf semm at 37°C. Cells were quiesced by incubating them for 20 hours in semm-free DMEM/Ham's F-12 medium containing insulin (5 ⁇ g/ml), transferrin (20 ⁇ g/ml), and selenium (16 pg/ml) (ITS). At the time of the assay, the medium was removed, and test samples were added to the wells in triplicate.
  • Test samples consisted of purified zveg ⁇ GFD at 3, 10, 30 and 100 ng/ml, and PDGF-AA, PDGF-AB and PDGF-BB at 30 ng/ml in a buffer containing 0.1% BSA. Samples were serially diluted into ITS medium and added to the test plate. A control buffer of 0.1% BSA was diluted identically to the highest concentration of zveg ⁇ protein and added to the plate. 10% fetal bovine semm (FBS) was used as a positive control. For measurement of [ 3 H]thymidine incorporation, 20 ⁇ l of a 50 ⁇ Cr/ml stock in DMEM was added directly to the cells, for a final activity of 1 ⁇ Ci/well.
  • FBS fetal bovine semm
  • PDGF-AB 30 ng/ml 1554 114
  • PDGF-BB 30 ng/ml 1852 464

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Abstract

L'invention concerne des substances et des techniques permettant de traiter la fibrose chez les mammifères. Ces techniques consistent à administrer à un mammifère une composition contenant une quantité efficace d'un point de vue thérapeutique d'un antagoniste de zvegf3 en combinaison avec un vecteur d'administration pharmaceutiquement acceptable. Parmi les antagonistes de zvegf3 figurent des anticorps anti-zvegf3, des polypeptides variants de zvegf3 de liaison à un récepteur et inactifs d'un point de vue mitogénique, et des polynucléotides inhibiteurs. Dans un mode de réalisation de l'invention, la fibrose concernée est une fibrose du foie.
PCT/US2000/029270 1999-10-21 2000-10-23 Traitement de la fibrose WO2001028586A1 (fr)

Priority Applications (1)

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AU80317/00A AU8031700A (en) 1999-10-21 2000-10-23 Method of treating fibrosis

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US16165399P 1999-10-21 1999-10-21
US60/161,653 1999-10-21
US16525599P 1999-11-12 1999-11-12
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US22222300P 2000-08-01 2000-08-01
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1335024A1 (fr) * 2000-10-19 2003-08-13 Kyowa Hakko Kogyo Co., Ltd. Anticorps inhibant l'activite vplf
WO2013160359A1 (fr) 2012-04-24 2013-10-31 Thrombogenics N.V. Anticorps anti-pdgf-c

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047677A2 (fr) * 1998-03-17 1999-09-23 Genentech, Inc. Polypeptides homologues de vegf et bmp1
WO2000034474A2 (fr) * 1998-12-07 2000-06-15 Zymogenetics, Inc. Zvegf3 homologue du facteur de croissance

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047677A2 (fr) * 1998-03-17 1999-09-23 Genentech, Inc. Polypeptides homologues de vegf et bmp1
WO2000034474A2 (fr) * 1998-12-07 2000-06-15 Zymogenetics, Inc. Zvegf3 homologue du facteur de croissance

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1335024A1 (fr) * 2000-10-19 2003-08-13 Kyowa Hakko Kogyo Co., Ltd. Anticorps inhibant l'activite vplf
EP1335024A4 (fr) * 2000-10-19 2004-09-01 Kyowa Hakko Kogyo Kk Anticorps inhibant l'activite vplf
WO2013160359A1 (fr) 2012-04-24 2013-10-31 Thrombogenics N.V. Anticorps anti-pdgf-c
US9884910B2 (en) 2012-04-24 2018-02-06 Thrombogenics Nv Anti-PDGF-C antibodies

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