[go: up one dir, main page]

US20100080810A1 - Methods of treating diseases with anti-vegf antibodies - Google Patents

Methods of treating diseases with anti-vegf antibodies Download PDF

Info

Publication number
US20100080810A1
US20100080810A1 US12/546,590 US54659009A US2010080810A1 US 20100080810 A1 US20100080810 A1 US 20100080810A1 US 54659009 A US54659009 A US 54659009A US 2010080810 A1 US2010080810 A1 US 2010080810A1
Authority
US
United States
Prior art keywords
hvegf
antibody
monoclonal antibodies
cells
monoclonal antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/546,590
Inventor
Kyung Jin Kim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Original Assignee
Genentech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genentech Inc filed Critical Genentech Inc
Priority to US12/546,590 priority Critical patent/US20100080810A1/en
Publication of US20100080810A1 publication Critical patent/US20100080810A1/en
Priority to US12/876,800 priority patent/US20100330096A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • This application relates to hybrid cell lines (lymphocyte hybridomas) for the production of monoclonal antibodies to human vascular endothelial growth factor (hVEGF), to such homogeneous monospecific antibodies, and to the use of such antibodies for diagnostic and therapeutic purposes.
  • hVEGF human vascular endothelial growth factor
  • the two major cellular components of the vasculature are the endothelial and smooth muscle cells.
  • the endothelial cells form the lining of the inner surface of all blood vessels, and constitute a non-thrombogenic interface between blood and tissue.
  • endothelial cells are an important component for the development of new capillaries and blood vessels.
  • endothelial cells proliferate during the neovascularization associated with tumor growth and a variety of diseases, including psoriasis, arthritis, and diabetic retinopathy.
  • FGF basic and acidic fibroblast growth factors
  • PD-ECGF platelet-derived endothelial cell growth factor
  • VEGF vascular endothelial growth factor
  • VEGF was first identified in media conditioned by bovine pituitary follicular or folliculostellate cells. Biochemical analyses indicate that bovine VEGF is a dimeric protein with an apparent molecular mass of approximately 45,000 Daltons, and with an apparent mitogenic specificity for vascular endothelial cells. DNA encoding bovine VEGF was isolated by screening a cDNA library prepared from such cells, using oligonucleotides based on the amino-terminal amino acid sequence of the protein as hybridization probes.
  • Human VEGF was obtained by first screening a cDNA library prepared from human cells, using bovine VEGF cDNA as a hybridization probe. One cDNA identified thereby encodes a 165-amino acid protein having greater than 95% homology to bovine VEGF, which protein is referred to as human VEGF (hVEGF). The mitogenic activity of human VEGF was confirmed by expressing the human VEGF cDNA in mammalian host cells. Media conditioned by cells transfected with the human VEGF cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not. Leung, et al, Science 246:1306 (1989).
  • the 121-amino acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 116 and 159 in hVEGF.
  • the 189-amino acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 116 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF).
  • hVPF human vascular permeability factor
  • VEGF vascular endothelial growth factor
  • the present invention is directed to monoclonal antibodies, and portions thereof, which are capable of specifically binding to hVEGF or hVEGF-related protein.
  • the invention also is directed to hybridoma cell lines that produce such monoclonal antibodies.
  • the monoclonal antibodies of the invention are useful as therapeutic agents, either by themselves or in conjunction with cytotoxic or other chemotherapeutic agents, to treat diseases that are characterized by excessive vascular endothelial cell proliferation.
  • the monoclonal antibodies of the invention also are useful in diagnostic and analytical assays for determining the presence of hVEGF or hVEGF related-protein in a test sample.
  • FIG. 1 shows the effect of anti-hVEGF monoclonal antibodies (A4.6.1 or B2.6.2) or an irrelevant anti-hepatocyte growth factor antibody (anti-HGF) on the binding of the anti-hVEGF monoclonal antibodies to hVEGF.
  • anti-HGF irrelevant anti-hepatocyte growth factor antibody
  • FIG. 2 shows the effect of anti-hVEGF monoclonal antibodies (A4.6.1 or B2.6.2) or an irrelevant anti-HGF antibody on the biological activity of hVEGF in cultures of bovine adrenal cortex capillary endothelial (ACE) cells.
  • ACE capillary endothelial
  • the present invention provides monoclonal antibodies, or portions thereof, which are capable of binding specifically to hVEGF or hVEGF-related protein.
  • hVEGF refers to the 165-amino acid human vascular endothelial growth factor
  • hVEGF-related protein refers to the 121- and 189-amino acid proteins, that are described by Leung, et. al., Science 246:1306 (1989).
  • recombinant used in reference to hVEGF or hVEGF-related protein refers to such proteins that are produced by recombinant DNA expression in a host cell.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the monoclonal antibodies included within the scope of the invention include hybrid and recombinant antibodies (e.g. “humanized” antibodies) regardless of species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab′) 2 , and Fv), so long as they are capable of binding specifically to hVEGF or hVEGF-related protein.
  • hybrid and recombinant antibodies e.g. “humanized” antibodies
  • antibody fragments e.g., Fab, F(ab′) 2 , and Fv
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from such a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975), or may be made by recombinant DNA methods. Cabilly, et al. U.S. Pat. No. 4,816,567.
  • a mouse or other appropriate host animal is immunized with hVEGF or hVEGF-related protein by subcutaneous, intraperitoneal, or intramuscular routes to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes maybe immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing ceils, and are sensitive to medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. Kozbor, J. Immunol. 133:3001 (1984). Brodeur, et. al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against hVEGF or hVEGF-related protein.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • the monoclonal antibodies of the invention are those that preferentially immunoprecipitate hVEGF or hVEGF-related protein that is present in a test sample, or that preferentially bind to hVEGF or hVEGF-related protein in a binding assay.
  • the monoclonal antibody will have an affinity for binding hVEGF or hVEGF-related protein of at least about 10 9 liters/mole, as determined, for example, by the Scatchard analysis of Munson & Pollard, Anal. Biochem 107:220 (1980).
  • the monoclonal antibody is a neutralizing antibody.
  • neutralizing antibody refers to a monoclonal antibody which is capable of specifically binding to hVEGF or hVEGF-related protein, and which is capable of substantially inhibiting or eliminating the mitogenic activity of one or more of such proteins with which it reacts.
  • a neutralizing antibody will inhibit such mitogenic activity at least about 50%, and preferably greater than 80%, as determined, for example, by an in vitro cell survival or proliferation assay, such as described in Example 2.
  • the neutralizing antibodies of the invention are especially useful in therapeutic applications, to prevent or treat unwanted endothelial cell proliferation or neovascularization.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods. Goding, Monoclonal - Antibodies: Principles and Practice , pp. 59-104 (Academic Press, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibody be reactive with fewer than all of the different proteins within the group of hVEGF and hVEGF-related proteins.
  • Such antibodies are readily identified by comparative ELISA assays or comparative immunoprecipitation of hVEGF and each of the hVEGF-related proteins.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma ceils 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, Morrison, et al., Proc. Nat. Acad. Sci. 81:6851 (1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptides include polypeptide toxins such as ricin, diphtheria toxin, or Pseudomonas exotoxin (to produce immunotoxins), viral sequences, cellular receptors such as a T-cell receptor, cytokines such as TNF, interferons, or interleukins, and other biologically or immunologically active polypeptides.
  • polypeptide toxins such as ricin, diphtheria toxin, or Pseudomonas exotoxin (to produce immunotoxins)
  • viral sequences such as a T-cell receptor
  • cytokines such as TNF, interferons, or interleukins
  • chimeric or “hybrid” antibodies are prepared that have the binding specificity of an anti-hVEGF or anti-hVEGF-related protein monoclonal antibody.
  • non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for hVEGF or hVEGF-related protein and another antigen-combining site having specificity for a different antigen.
  • Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond.
  • suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
  • the antibodies of the invention typically will be labeled with a detectable moiety.
  • the detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., 125 I, 32 P, 14 C, or 3 H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter, et al., Nature 144:945 (1962); David et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).
  • the antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques , pp. 147-158 (CRC Press, Inc., 1987).
  • ком ⁇ онентs rely on the ability of a labeled standard (which may be hVEGF, hVEGF-related protein, an immunologically reactive portion thereof) to compete with the test sample analyte (hVEGF or hVEGF-related protein) for binding with a limited amount of antibody.
  • a labeled standard which may be hVEGF, hVEGF-related protein, an immunologically reactive portion thereof
  • the amount of hVEGF or hVEGF-related protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected.
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays), or may be measured using an anti-immunoglobulin 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 antibodies of the invention also are useful for in vivo imaging, wherein an antibody labeled with a detectable moiety is administered to a host, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed.
  • This imaging technique is useful in the staging and treatment of neoplasms.
  • the antibody may be labeled with any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • the antibodies of the invention may be administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the antibody is also suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.
  • Such dosage forms encompass pharmaceutically acceptable carriers that are inherently nontoxic and nontherapeutic.
  • pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol.
  • Carriers for topical or gel-based forms of antibody include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols.
  • conventional depot forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, and sublingual tablets.
  • the antibody will typically be formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml.
  • the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibodies are administered for preventive or therapeutic purposes, 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.
  • 0.015 to 15 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.
  • the treatment is repeated until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful.
  • the effectiveness of the antibody in preventing or treating disease may be improved by administering the antibody serially or in combination with another agent that is effective for those purposes, such as another antibody directed against a different epitope or neutralizing a different protein than the first antibody, or one or more conventional therapeutic agents such as, for example, alkylating agents, folic acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil, purine nucleosides, amines, amino acids, triazol nucleosides, corticosteroids, calcium, retinoids, lipoxygenase and cyclooxygenase inhibitors, fumaric acid and its salts, analgesics, psychopharmaceuticals, local anesthetics, spasmolytics, and beta-blockers.
  • another agent that is effective for those purposes, such as another antibody directed against a different epitope or neutralizing a different protein than the first antibody
  • one or more conventional therapeutic agents such as, for
  • the antibodies of the invention also are useful as affinity purification agents.
  • the antibodies against hVEGF or hVEGF-related protein are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art.
  • the immobilized antibody then is contacted with a sample containing the hVEGF or hVEGF-related protein to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the hVEGF or hVEGF-related protein, which-is bound to the immobilized antibody.
  • the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the hVEGF or hVEGF-related protein from the antibody.
  • hVEGF conjugated to keyhole limpet hemocyanin (KLH) for immunization, recombinant hVEGF (165 amino acids), Leung, et al., Science 246:1306 (1989), was mixed with KLH at a 4:1 ratio in the presence of 0.05% glutaraldehyde and the mixture was incubated at room temperature for 3 hours with gentle stirring. The mixture then was dialyzed against phosphate buffered saline (PBS) at 4° C. overnight.
  • PBS phosphate buffered saline
  • mice were immunized four times every two weeks by intraperitoneal injections with 5 ⁇ g of hVEGF conjugated to 20 ⁇ g of KLH, and were boosted with the same dose of hVEGF conjugated to KLH four days prior to cell fusion.
  • Spleen cells from the immunized mice were fused with P3X63Ag8U. 1 myeloma cells, Yelton, et al., Curr. Top. Microbiol. Immunol. 81:1 (1978), using 35% polyethylene glycol (PEG) as described. Yarmush, et al., Proc. Nat. Acad. Sci. 77:2899 (1980). Hybridomas were selected in HAT medium,
  • the A4.6.1 and B2.6.2 hybridoma cell lines have been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 USA, and are maintained by the ATCC under accession numbers HB 10709 and HB 10710, respectively.
  • the binding specificities of the anti-hVEGF monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas were determined by ELISA.
  • the monoclonal antibodies were added to the wells of microtiter plates that previously had been coated with hVEGF, FGF, HGF, or epidermal growth factor (EGF). Bound antibody was detected with peroxidase conjugated goat anti-mouse IgG immunoglobulins. The results of those assays confirmed that the monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas bind to hVEGF, but not detectably to those other protein growth factors.
  • a competitive binding ELISA was used to determine whether the monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas bind to the same or different epitopes (sites) within hVEGF. Kim, et al., Infect. Immun. 57:944 (1989). Individual unlabeled anti-hVEGF monoclonal antibodies (A4.6.1 or B2.6.2) or irrelevant anti-HGF antibody (IgG1 isotype) were added to the wells of microtiter plates that previously had been coated with hVEGF. Biotinylated anti-hVEGF monoclonal antibodies (BIO-A4.6.1 or BIO-B2.6.2) were then added. The ratio of biotinylated antibody to unlabeled antibody was 1:1000.
  • Biotinylated antibodies Binding of the biotinylated antibodies was visualized by the addition of avidin-conjugated peroxidase, followed by o-phenylenediamine dihydrochloride and hydrogen peroxide.
  • the color reaction indicating the amount of biotinylated antibody bound, was determined by measuring the optical density (O.D) at 495 nm wavelength.
  • the isotypes of the anti-hVEGF monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas were determined by ELISA. Samples of culture medium (supernatant) in which each of the hybridomas was growing were added to the wells of microtiter plates that had previously been coated with hVEGF. The captured anti-hVEGF monoclonal antibodies were incubated with different isotype-specific alkaline phosphatase-conjugated goat anti-mouse immunoglobulins, and the binding of the conjugated antibodies to the anti-hVEGF monoclonal antibodies was determined by the addition of p-nitrophenyl phosphate. The color reaction was measured at 405 nm with an ELISA plate reader.
  • the isotype of the monoclonal antibodies produced by both the A4.6.1 and B2.6.2 hybridomas was determined to be IgG1.
  • the affinities of the anti-hVEGF monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas for hVEGF were determined by a competitive binding assays. A predetermined sub-optimal concentration of monoclonal antibody was added to samples containing 20,000-40,000 cpm 125 I-hVEGF (1-2 ng) and various known amounts of unlabeled hVEGF (1-1000 ng). After 1 hour at room temperature, 100 ⁇ l of goat anti-mouse Ig antisera (Pel-Freez, Rogers, Ark. USA) were added, and the mixtures were incubated another hour at room temperature.
  • Affinity constants were calculated from the data by Scatchard analysis.
  • the affinity of the anti-hVEGF monoclonal antibody produced by the A4.6.1 hybridoma was calculated to 1.2 ⁇ 10 9 liters/mole.
  • the affinity of the anti-hVEGF monoclonal antibody produced by the B2.6.2 hybridoma was calculated to be 2.5 ⁇ 10 9 liters/mole.
  • Bovine adrenal cortex capillary endothelial (ACE) cells Bovine adrenal cortex capillary endothelial (ACE) cells, Ferrara et al., Proc. Nat. Acad. Sci. 84:5773 (1987), were seeded at a density of 10 4 cells/ml in 12 multiwell plates, and 2.5 ng/ml hVEGF was added to each well in the presence or absence of various concentrations of the anti-hVEGF monoclonal antibodies produced by the A4.6.1 or B2.6.2 hybridomas, or an irrelevant anti-HGF monoclonal antibody. After culturing 5 days, the cells in each well were counted in a Coulter counter. As a control, ACE cells were cultured in the absence of added hVEGF.
  • both of the anti-hVEGF monoclonal antibodies inhibited the ability of the added hVEGF to support the growth or survival of the bovine ACE cells.
  • the monoclonal antibody produced by the A4.6.1 hybridoma completely inhibited the mitogenic activity of hVEGF, whereas the monoclonal antibody produced by the B2.6.2 hybridoma only partially inhibited the mitogenic activity of hVEGF.
  • Bovine ACE cells were seeded at a density of 2.5 ⁇ 10 4 cells/0.5 ml/well in 24 well microtiter plates in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% calf serum, 2 mM glutamine, and 1 ng/ml basic fibroblast growth factor. After culturing overnight, the cells were washed once in binding buffer (equal volumes of DMEM and FI2 medium plus 25 mM HEPES and 1% bovine serum albumin) at 4° C.
  • DMEM Dulbecco's Modified Eagle's Medium
  • the anti-hVEGF monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas inhibited the binding of hVEGF to the bovine ACE cells.
  • the anti-hVEGF monoclonal antibody produced by the A2.6.1 hybridoma had no apparent effect on the binding of hVEGF to the bovine ACE cells.
  • the monoclonal antibody produced by the A4.6.1 hybridoma inhibited the binding of hVEGF to a greater extent than the monoclonal antibody produced by the B2.6.2 hybridoma.
  • the monoclonal antibody produced by the A4.6.1 hybridoma completely inhibited the binding of hVEGF to the bovine ACE cells at a 1:250 molar ratio of hVEGF to antibody.
  • the antibody was assayed for its ability to immunoprecipate hVEGF-related, protein.
  • Human 293 cells were transfected with vectors comprising the nucleotide coding sequence of the 121- and 189-amino acid hVEGF-related proteins, as described. Leung, et al., Science 246:1306 (1989). Two days after transfection, the cells were transferred to medium lacking cysteine and methionine. The cells were incubated 30 minutes in that medium, then 100 ⁇ Ci/m 1 of each 35 S-methionine and 35 S-cysteine were added to the medium, and the cells were incubated another two hours. The labeling was chased by transferring the cells to serum free medium and incubating three hours.
  • the cell culture media were collected, and the cells were lysed by incubating for 30 minutes in lysis buffer (150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 8.0). Cell debris was removed from the lysates by centrifugation at 200 ⁇ G. for 30 minutes.
  • lysis buffer 150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 8.0.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention provides monoclonal antibodies, and portions thereof, which are capable of specifically binding to human vascular endothelial cell growth factor (hVEGF) or hVEGF-related protein. The invention also provides hybridoma cell lines that produce such monoclonal antibodies. The monoclonal antibodies of the invention are useful as therapeutic agents either by themselves or in conjunction with cytotoxic or other chemotherapeutic agents, to treat diseases that are characterized by excessive vascular endothelial cell proliferation. The monoclonal antibodies of the invention also are useful in diagnostic and analytical methods for determining the presence of hVEGF or hVEGF related-protein in a test sample.

Description

  • This is a continuation application filed under 37 CFR 1.53(b) of application Ser. No. 12/329,428, filed Dec. 5, 2008, which is a continuation of application Ser. No. 11/682,197 filed Mar. 5, 2007, now U.S. Pat. No. 7,482,005, which is a continuation of application Ser. No. 10/441,728, filed May 20, 2003, now U.S. Pat. No. 7,227,004 which is a continuation of application Ser. No. 08/950,863, filed Oct. 15, 1997, now U.S. Pat. No. 6,582,959, which is a continuation of Ser. No. 08/711,314, filed Sep. 3, 1996, now abandoned, which is a continuation of Ser. No. 08/558,042, filed Nov. 13, 1995, now abandoned, which is a continuation of Ser. No. 08/416,543, filed Mar. 29, 1995, now abandoned, which is a continuation of Ser. No. 08/185,291, filed Jan. 24, 1994, now abandoned, which is a continuation of Ser. No. 08/071,214, filed Jun. 2, 1993, now abandoned, which is a continuation of Ser. No. 07/677,215, filed Mar. 29, 1991, now abandoned, the contents of which are incorporated herein by reference.
    • FIELD OF THE INVENTION
  • This application relates to hybrid cell lines (lymphocyte hybridomas) for the production of monoclonal antibodies to human vascular endothelial growth factor (hVEGF), to such homogeneous monospecific antibodies, and to the use of such antibodies for diagnostic and therapeutic purposes.
    • BACKGROUND OF THE INVENTION
  • The two major cellular components of the vasculature are the endothelial and smooth muscle cells. The endothelial cells form the lining of the inner surface of all blood vessels, and constitute a non-thrombogenic interface between blood and tissue. In addition, endothelial cells are an important component for the development of new capillaries and blood vessels. Thus, endothelial cells proliferate during the neovascularization associated with tumor growth and a variety of diseases, including psoriasis, arthritis, and diabetic retinopathy.
  • Various naturally occurring polypeptides reportedly induce the proliferation of endothelial cells. Among these polypeptides are the basic and acidic fibroblast growth factors (FGF), Burgess and Maciag, Annual Rev. Biochem., 58:575 (1989), platelet-derived endothelial cell growth factor (PD-ECGF), Ishikawa, et. al., Nature, 338:557 (1989), and vascular endothelial growth factor (VEGF), Leung, et al., Science 246:1306 (1989); Ferrara & Henzel, Biochem. Biophys. Res, Commun. 161:851 (1989); Tischer, et al. Biochem. Biophys. Res. Commun. 165:1198 (1989); Ferrara, et. al, PCT Pat. Pub No WO 90/13649 (published Nov. 15, 1990); Ferrara, et al., U.S. patent application Ser. No. 07/360,229.
  • VEGF was first identified in media conditioned by bovine pituitary follicular or folliculostellate cells. Biochemical analyses indicate that bovine VEGF is a dimeric protein with an apparent molecular mass of approximately 45,000 Daltons, and with an apparent mitogenic specificity for vascular endothelial cells. DNA encoding bovine VEGF was isolated by screening a cDNA library prepared from such cells, using oligonucleotides based on the amino-terminal amino acid sequence of the protein as hybridization probes.
  • Human VEGF was obtained by first screening a cDNA library prepared from human cells, using bovine VEGF cDNA as a hybridization probe. One cDNA identified thereby encodes a 165-amino acid protein having greater than 95% homology to bovine VEGF, which protein is referred to as human VEGF (hVEGF). The mitogenic activity of human VEGF was confirmed by expressing the human VEGF cDNA in mammalian host cells. Media conditioned by cells transfected with the human VEGF cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not. Leung, et al, Science 246:1306 (1989).
  • Several additional cDNAs were identified in the human cDNA library that encode 121- and 189-amino acid hVEGF-related proteins. The 121-amino acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 116 and 159 in hVEGF. The 189-amino acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 116 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF). Keck, et. al., Science 246:1309 (1989); Connolly, et. al., J. Biol. Chem. 264:20017 (1989); Keck, et. al., EPO Pat. Pub. No. 0 370 989 (published May 30, 1990).
  • In view of the fundamental role of vascular endothelial cell growth in many diseases, it is desirable to have a means of regulating the mitogenic effect of VEGF. It is also desirable to have a means of assaying for the presence of VEGF in normal and pathological conditions, and especially cancer.
    • SUMMARY OF THE INVENTION
  • The present invention is directed to monoclonal antibodies, and portions thereof, which are capable of specifically binding to hVEGF or hVEGF-related protein. The invention also is directed to hybridoma cell lines that produce such monoclonal antibodies. The monoclonal antibodies of the invention are useful as therapeutic agents, either by themselves or in conjunction with cytotoxic or other chemotherapeutic agents, to treat diseases that are characterized by excessive vascular endothelial cell proliferation. The monoclonal antibodies of the invention also are useful in diagnostic and analytical assays for determining the presence of hVEGF or hVEGF related-protein in a test sample.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows the effect of anti-hVEGF monoclonal antibodies (A4.6.1 or B2.6.2) or an irrelevant anti-hepatocyte growth factor antibody (anti-HGF) on the binding of the anti-hVEGF monoclonal antibodies to hVEGF.
  • FIG. 2 shows the effect of anti-hVEGF monoclonal antibodies (A4.6.1 or B2.6.2) or an irrelevant anti-HGF antibody on the biological activity of hVEGF in cultures of bovine adrenal cortex capillary endothelial (ACE) cells.
  • FIG. 3 shows the effect of anti-hVEGF monoclonal antibodies (A4.6.1, B2.6.2, or A2.6.1) on the binding of hVEGF to bovine ACE cells.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides monoclonal antibodies, or portions thereof, which are capable of binding specifically to hVEGF or hVEGF-related protein. The term “hVEGF” refers to the 165-amino acid human vascular endothelial growth factor, and the term “hVEGF-related protein” refers to the 121- and 189-amino acid proteins, that are described by Leung, et. al., Science 246:1306 (1989). The term “recombinant” used in reference to hVEGF or hVEGF-related protein refers to such proteins that are produced by recombinant DNA expression in a host cell.
  • The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • The monoclonal antibodies included within the scope of the invention include hybrid and recombinant antibodies (e.g. “humanized” antibodies) regardless of species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab′)2, and Fv), so long as they are capable of binding specifically to hVEGF or hVEGF-related protein. Cabilly, et al., U.S. Pat. No. 4,816,567; Mage & Lamoyi, in Monoclonal Antibody Production Technique and Applications, pp. 79-97 (Marcel Dekker, Inc., New York, 1987).
  • Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from such a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975), or may be made by recombinant DNA methods. Cabilly, et al. U.S. Pat. No. 4,816,567.
  • In the hybridoma method, a mouse or other appropriate host animal is immunized with hVEGF or hVEGF-related protein by subcutaneous, intraperitoneal, or intramuscular routes to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes maybe immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, pp, 59-103 (Academic Press, 1986).
  • The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing ceils, and are sensitive to medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. Kozbor, J. Immunol. 133:3001 (1984). Brodeur, et. al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against hVEGF or hVEGF-related protein. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The monoclonal antibodies of the invention are those that preferentially immunoprecipitate hVEGF or hVEGF-related protein that is present in a test sample, or that preferentially bind to hVEGF or hVEGF-related protein in a binding assay.
  • In a preferred embodiment of the invention, the monoclonal antibody will have an affinity for binding hVEGF or hVEGF-related protein of at least about 109 liters/mole, as determined, for example, by the Scatchard analysis of Munson & Pollard, Anal. Biochem 107:220 (1980).
  • In another preferred embodiment of the invention, the monoclonal antibody is a neutralizing antibody. The term “neutralizing antibody” as used herein refers to a monoclonal antibody which is capable of specifically binding to hVEGF or hVEGF-related protein, and which is capable of substantially inhibiting or eliminating the mitogenic activity of one or more of such proteins with which it reacts. Typically a neutralizing antibody will inhibit such mitogenic activity at least about 50%, and preferably greater than 80%, as determined, for example, by an in vitro cell survival or proliferation assay, such as described in Example 2. The neutralizing antibodies of the invention are especially useful in therapeutic applications, to prevent or treat unwanted endothelial cell proliferation or neovascularization.
  • After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or neutralization activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. Goding, Monoclonal-Antibodies: Principles and Practice, pp. 59-104 (Academic Press, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • For some therapeutic and diagnostic applications, it is desirable that the monoclonal antibody be reactive with fewer than all of the different proteins within the group of hVEGF and hVEGF-related proteins. For example, it may be desirable to have a monoclonal antibody that is capable of specifically binding to hVEGF but not to hVEGF-related proteins. Such antibodies are readily identified by comparative ELISA assays or comparative immunoprecipitation of hVEGF and each of the hVEGF-related proteins.
  • The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma ceils of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese Hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison, et al., Proc. Nat. Acad. Sci. 81:6851 (1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides include polypeptide toxins such as ricin, diphtheria toxin, or Pseudomonas exotoxin (to produce immunotoxins), viral sequences, cellular receptors such as a T-cell receptor, cytokines such as TNF, interferons, or interleukins, and other biologically or immunologically active polypeptides. In that manner, “chimeric” or “hybrid” antibodies are prepared that have the binding specificity of an anti-hVEGF or anti-hVEGF-related protein monoclonal antibody.
  • Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for hVEGF or hVEGF-related protein and another antigen-combining site having specificity for a different antigen.
  • Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
  • For diagnostic applications the antibodies of the invention typically will be labeled with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., 125I, 32P, 14C, or 3H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter, et al., Nature 144:945 (1962); David et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).
  • The antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).
  • Competitive binding assays rely on the ability of a labeled standard (which may be hVEGF, hVEGF-related protein, an immunologically reactive portion thereof) to compete with the test sample analyte (hVEGF or hVEGF-related protein) for binding with a limited amount of antibody. The amount of hVEGF or hVEGF-related protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex. David & Greene, U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays), or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • The antibodies of the invention also are useful for in vivo imaging, wherein an antibody labeled with a detectable moiety is administered to a host, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. This imaging technique is useful in the staging and treatment of neoplasms. The antibody may be labeled with any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • For therapeutic applications, the antibodies of the invention may be administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibody is also suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.
  • Such dosage forms encompass pharmaceutically acceptable carriers that are inherently nontoxic and nontherapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol. Carriers for topical or gel-based forms of antibody include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols. For all administrations, conventional depot forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, and sublingual tablets. The antibody will typically be formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml.
  • For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibodies are administered for preventive or therapeutic purposes, 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.
  • Depending on the type and severity of the disease, about 0.015 to 15 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. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful.
  • According to another embodiment of the invention, the effectiveness of the antibody in preventing or treating disease may be improved by administering the antibody serially or in combination with another agent that is effective for those purposes, such as another antibody directed against a different epitope or neutralizing a different protein than the first antibody, or one or more conventional therapeutic agents such as, for example, alkylating agents, folic acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil, purine nucleosides, amines, amino acids, triazol nucleosides, corticosteroids, calcium, retinoids, lipoxygenase and cyclooxygenase inhibitors, fumaric acid and its salts, analgesics, psychopharmaceuticals, local anesthetics, spasmolytics, and beta-blockers. Such other agents may be present in the composition being administered or may be administered separately. Also, the antibody is suitably administered serially or in combination with radiological treatments, whether involving irradiation or administration of radioactive substances.
  • The antibodies of the invention also are useful as affinity purification agents. In this process, the antibodies against hVEGF or hVEGF-related protein are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the hVEGF or hVEGF-related protein to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the hVEGF or hVEGF-related protein, which-is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the hVEGF or hVEGF-related protein from the antibody.
  • The following examples are offered by way of illustration only and are not intended to limit the invention in any manner. All patent and literature references cited herein are expressly incorporated.
    • EXAMPLE 1 Preparation of Monoclonal Antibodies
  • To obtain hVEGF conjugated to keyhole limpet hemocyanin (KLH) for immunization, recombinant hVEGF (165 amino acids), Leung, et al., Science 246:1306 (1989), was mixed with KLH at a 4:1 ratio in the presence of 0.05% glutaraldehyde and the mixture was incubated at room temperature for 3 hours with gentle stirring. The mixture then was dialyzed against phosphate buffered saline (PBS) at 4° C. overnight.
  • Balb/c mice were immunized four times every two weeks by intraperitoneal injections with 5 μg of hVEGF conjugated to 20 μg of KLH, and were boosted with the same dose of hVEGF conjugated to KLH four days prior to cell fusion.
  • Spleen cells from the immunized mice were fused with P3X63Ag8U. 1 myeloma cells, Yelton, et al., Curr. Top. Microbiol. Immunol. 81:1 (1978), using 35% polyethylene glycol (PEG) as described. Yarmush, et al., Proc. Nat. Acad. Sci. 77:2899 (1980). Hybridomas were selected in HAT medium,
  • Supernatants from hybridoma cell cultures were screened for anti-hVEGF antibody production by an ELISA assay using hVEGF-coated microtiter plates. Antibody that was bound hVEGF in each of the wells was determined using alkaline phosphatase-conjugated goat anti-mouse IgG immunoglobulin and the chromogenic substrate p-nitrophenyl phosphate. Harlow & Lane, Antibodies: A Laboratory Manual, p. 597 (Cold Spring Harbor Laboratory, 1988). Hybridoma cells thus determined to produce anti-hVEGF antibodies were subcloned by limiting dilution, and two of those clones, designated A4.6.1 and B2.62, were chosen for further studies.
  • The A4.6.1 and B2.6.2 hybridoma cell lines have been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 USA, and are maintained by the ATCC under accession numbers HB 10709 and HB 10710, respectively.
    • EXAMPLE 2 Characterization of Monoclonal Antibodies A. Antigen Specificity
  • The binding specificities of the anti-hVEGF monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas were determined by ELISA. The monoclonal antibodies were added to the wells of microtiter plates that previously had been coated with hVEGF, FGF, HGF, or epidermal growth factor (EGF). Bound antibody was detected with peroxidase conjugated goat anti-mouse IgG immunoglobulins. The results of those assays confirmed that the monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas bind to hVEGF, but not detectably to those other protein growth factors.
    • B. Epitope Mapping
  • A competitive binding ELISA was used to determine whether the monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas bind to the same or different epitopes (sites) within hVEGF. Kim, et al., Infect. Immun. 57:944 (1989). Individual unlabeled anti-hVEGF monoclonal antibodies (A4.6.1 or B2.6.2) or irrelevant anti-HGF antibody (IgG1 isotype) were added to the wells of microtiter plates that previously had been coated with hVEGF. Biotinylated anti-hVEGF monoclonal antibodies (BIO-A4.6.1 or BIO-B2.6.2) were then added. The ratio of biotinylated antibody to unlabeled antibody was 1:1000. Binding of the biotinylated antibodies was visualized by the addition of avidin-conjugated peroxidase, followed by o-phenylenediamine dihydrochloride and hydrogen peroxide. The color reaction, indicating the amount of biotinylated antibody bound, was determined by measuring the optical density (O.D) at 495 nm wavelength.
  • As shown in FIG. 1, in each case, the binding of the biotinylated anti-hVEGF antibody was inhibited by the corresponding unlabeled antibody, but not by the other unlabeled anti-hVEGF antibody or the anti-HGF antibody. These results indicate that the monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas bind to different epitopes within hVEGF.
    • C. Isotyping
  • The isotypes of the anti-hVEGF monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas were determined by ELISA. Samples of culture medium (supernatant) in which each of the hybridomas was growing were added to the wells of microtiter plates that had previously been coated with hVEGF. The captured anti-hVEGF monoclonal antibodies were incubated with different isotype-specific alkaline phosphatase-conjugated goat anti-mouse immunoglobulins, and the binding of the conjugated antibodies to the anti-hVEGF monoclonal antibodies was determined by the addition of p-nitrophenyl phosphate. The color reaction was measured at 405 nm with an ELISA plate reader.
  • By that method, the isotype of the monoclonal antibodies produced by both the A4.6.1 and B2.6.2 hybridomas was determined to be IgG1.
    • D. Binding Affinity
  • The affinities of the anti-hVEGF monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas for hVEGF were determined by a competitive binding assays. A predetermined sub-optimal concentration of monoclonal antibody was added to samples containing 20,000-40,000 cpm 125I-hVEGF (1-2 ng) and various known amounts of unlabeled hVEGF (1-1000 ng). After 1 hour at room temperature, 100 μl of goat anti-mouse Ig antisera (Pel-Freez, Rogers, Ark. USA) were added, and the mixtures were incubated another hour at room temperature. Complexes of antibody and bound protein (immune complexes) were precipitated by the addition of 500 μl of 6% polyethylene glycol (PEG, mol. wt. 8000) at 4° C., followed by centrifugation at 2000×G. for 20 min. at 4° C. The amount of 125I-hVEGF bound to the anti-hVEGF monoclonal antibody in each sample was determined by counting the pelleted material in a gamma counter.
  • Affinity constants were calculated from the data by Scatchard analysis. The affinity of the anti-hVEGF monoclonal antibody produced by the A4.6.1 hybridoma was calculated to 1.2×109 liters/mole. The affinity of the anti-hVEGF monoclonal antibody produced by the B2.6.2 hybridoma was calculated to be 2.5×109 liters/mole.
  • E. Inhibition of hVEGF Mitogenic Activity
  • Bovine adrenal cortex capillary endothelial (ACE) cells, Ferrara et al., Proc. Nat. Acad. Sci. 84:5773 (1987), were seeded at a density of 104 cells/ml in 12 multiwell plates, and 2.5 ng/ml hVEGF was added to each well in the presence or absence of various concentrations of the anti-hVEGF monoclonal antibodies produced by the A4.6.1 or B2.6.2 hybridomas, or an irrelevant anti-HGF monoclonal antibody. After culturing 5 days, the cells in each well were counted in a Coulter counter. As a control, ACE cells were cultured in the absence of added hVEGF.
  • As shown in FIG. 2, both of the anti-hVEGF monoclonal antibodies inhibited the ability of the added hVEGF to support the growth or survival of the bovine ACE cells. The monoclonal antibody produced by the A4.6.1 hybridoma completely inhibited the mitogenic activity of hVEGF, whereas the monoclonal antibody produced by the B2.6.2 hybridoma only partially inhibited the mitogenic activity of hVEGF.
  • F. Inhibition of hVEGF Binding
  • Bovine ACE cells were seeded at a density of 2.5×104 cells/0.5 ml/well in 24 well microtiter plates in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% calf serum, 2 mM glutamine, and 1 ng/ml basic fibroblast growth factor. After culturing overnight, the cells were washed once in binding buffer (equal volumes of DMEM and FI2 medium plus 25 mM HEPES and 1% bovine serum albumin) at 4° C.
  • 12,000 cpm 125I-hVEGF (approx. 5×104 cpm/ng/ml) was preincubated for 30 minutes with 5 μg of the anti-hVEGF monoclonal antibody produced by the A4.6.1, B2.6.2, or A2.6.1 hybridoma (250 μl total volume), and thereafter the mixtures were added to the bovine ACE cells in the microtiter plates. After incubating the cells for 3 hours at 4° C., the cells were washed 3 times with binding buffer at 4° C., solubilized by the addition of 0.5 ml 0.2 N. NaOH, and counted in a gamma counter.
  • As shown in FIG. 3 (upper), the anti-hVEGF monoclonal antibodies produced by the A4.6.1 and B2.6.2 hybridomas inhibited the binding of hVEGF to the bovine ACE cells. In contrast, the anti-hVEGF monoclonal antibody produced by the A2.6.1 hybridoma had no apparent effect on the binding of hVEGF to the bovine ACE cells. Consistent with the results obtained in the cell proliferation assay described above, the monoclonal antibody produced by the A4.6.1 hybridoma inhibited the binding of hVEGF to a greater extent than the monoclonal antibody produced by the B2.6.2 hybridoma.
  • As shown in FIG. 3 (lower), the monoclonal antibody produced by the A4.6.1 hybridoma completely inhibited the binding of hVEGF to the bovine ACE cells at a 1:250 molar ratio of hVEGF to antibody.
  • G. Cross-reactivity with VEGF-related Proteins
  • To determine whether the anti-hVEGF monoclonal antibody produced by the A4.6.1 hybridoma is reactive with hVEGF-related protein, the antibody was assayed for its ability to immunoprecipate hVEGF-related, protein.
  • Human 293 cells were transfected with vectors comprising the nucleotide coding sequence of the 121- and 189-amino acid hVEGF-related proteins, as described. Leung, et al., Science 246:1306 (1989). Two days after transfection, the cells were transferred to medium lacking cysteine and methionine. The cells were incubated 30 minutes in that medium, then 100 μCi/m1 of each 35S-methionine and 35S-cysteine were added to the medium, and the cells were incubated another two hours. The labeling was chased by transferring the cells to serum free medium and incubating three hours. The cell culture media were collected, and the cells were lysed by incubating for 30 minutes in lysis buffer (150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 8.0). Cell debris was removed from the lysates by centrifugation at 200×G. for 30 minutes.
  • 500 μl samples of cell culture media and cell lysates were incubated with 2 μl of A4.6.1 hybridoma antibody (2.4 mg/ml) for 1 hour at 4° C., and then were incubated with 5 μl of rabbit anti-mouse IgG immunoglobulin for 1 hour at 4° C. Immune complexes of 35S-labeled hVEGF-related protein and anti-hVEGF monoclonal antibody were precipitated with protein-A sepharose (Pharmacia), then subjected to SDS—12% polyacrylamide gel electrophoresis under reducing conditions. The gel was exposed to x-ray film for analysis of the immunoprecipitated, radiolabeled proteins by autoradiography.
  • The results of that analysis indicated that the anti-hVEGF monoclonal antibody produced by the A4.6.1 hybridoma was cross-reactive with both the 121- and 189-amino acid hVEGF related proteins.

Claims (6)

1. A method of using a monoclonal antibody to human vascular endothelial growth factor (hVEGF) for treating diseases that are characterized by excessive vascular endothelial cell proliferation, wherein said monoclonal antibody to hVEGF binds to the same hVEGF epitope as monoclonal antibody A4.6.1 (ATCC HB 10709).
2. The method of claim 1, wherein the monoclonal antibody to hVEGF inhibits binding of A4.6.1 (ATCC HB 10709) to hVEGF in a competitive binding ELISA.
3. The method of claim 1, wherein the monoclonal antibody to hVEGF is used as a single agent.
4. The method of claim 1, wherein the monoclonal antibody to hVEGF is used in conjunction with another cytotoxic agent.
5. The method of claim 1, wherein the monoclonal antibody to hVEGF is used in conjunction with another chemotherapeutic agent.
6. The method of claim 1, wherein the monoclonal antibody to hVEGF is administered serially or in combination with a radiological treatment.
US12/546,590 1991-03-29 2009-08-24 Methods of treating diseases with anti-vegf antibodies Abandoned US20100080810A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US12/546,590 US20100080810A1 (en) 1991-03-29 2009-08-24 Methods of treating diseases with anti-vegf antibodies
US12/876,800 US20100330096A1 (en) 1991-03-29 2010-09-07 Methods of treating diseases with anti-vegf antibodies

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
US67721591A 1991-03-29 1991-03-29
US7121493A 1993-06-02 1993-06-02
US18529194A 1994-01-24 1994-01-24
US41654395A 1995-03-29 1995-03-29
US55804295A 1995-11-13 1995-11-13
US71131496A 1996-09-03 1996-09-03
US08/950,863 US6582959B2 (en) 1991-03-29 1997-10-15 Antibodies to vascular endothelial cell growth factor
US10/441,728 US7227004B2 (en) 1991-03-29 2003-05-20 Antibodies to vascular endothelial cell growth factor
US11/682,197 US7482005B2 (en) 1991-03-29 2007-03-05 Methods of treating diseases with anti-VEGF antibodies
US12/329,428 US20090081232A1 (en) 1991-03-29 2008-12-05 Methods of treating diseases with anti-vegf antibodies
US12/546,590 US20100080810A1 (en) 1991-03-29 2009-08-24 Methods of treating diseases with anti-vegf antibodies

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US12/329,428 Continuation US20090081232A1 (en) 1991-03-29 2008-12-05 Methods of treating diseases with anti-vegf antibodies

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/876,800 Continuation US20100330096A1 (en) 1991-03-29 2010-09-07 Methods of treating diseases with anti-vegf antibodies

Publications (1)

Publication Number Publication Date
US20100080810A1 true US20100080810A1 (en) 2010-04-01

Family

ID=38041089

Family Applications (6)

Application Number Title Priority Date Filing Date
US08/950,863 Expired - Fee Related US6582959B2 (en) 1991-03-29 1997-10-15 Antibodies to vascular endothelial cell growth factor
US10/441,728 Expired - Fee Related US7227004B2 (en) 1991-03-29 2003-05-20 Antibodies to vascular endothelial cell growth factor
US11/682,197 Expired - Fee Related US7482005B2 (en) 1991-03-29 2007-03-05 Methods of treating diseases with anti-VEGF antibodies
US12/329,428 Abandoned US20090081232A1 (en) 1991-03-29 2008-12-05 Methods of treating diseases with anti-vegf antibodies
US12/546,590 Abandoned US20100080810A1 (en) 1991-03-29 2009-08-24 Methods of treating diseases with anti-vegf antibodies
US12/876,800 Abandoned US20100330096A1 (en) 1991-03-29 2010-09-07 Methods of treating diseases with anti-vegf antibodies

Family Applications Before (4)

Application Number Title Priority Date Filing Date
US08/950,863 Expired - Fee Related US6582959B2 (en) 1991-03-29 1997-10-15 Antibodies to vascular endothelial cell growth factor
US10/441,728 Expired - Fee Related US7227004B2 (en) 1991-03-29 2003-05-20 Antibodies to vascular endothelial cell growth factor
US11/682,197 Expired - Fee Related US7482005B2 (en) 1991-03-29 2007-03-05 Methods of treating diseases with anti-VEGF antibodies
US12/329,428 Abandoned US20090081232A1 (en) 1991-03-29 2008-12-05 Methods of treating diseases with anti-vegf antibodies

Family Applications After (1)

Application Number Title Priority Date Filing Date
US12/876,800 Abandoned US20100330096A1 (en) 1991-03-29 2010-09-07 Methods of treating diseases with anti-vegf antibodies

Country Status (1)

Country Link
US (6) US6582959B2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9795672B2 (en) 2003-05-30 2017-10-24 Genentech, Inc. Treatment with anti-VEGF antibodies

Families Citing this family (180)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030206899A1 (en) * 1991-03-29 2003-11-06 Genentech, Inc. Vascular endothelial cell growth factor antagonists
US6582959B2 (en) * 1991-03-29 2003-06-24 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
US5965132A (en) * 1992-03-05 1999-10-12 Board Of Regents, The University Of Texas System Methods and compositions for targeting the vasculature of solid tumors
US6749853B1 (en) * 1992-03-05 2004-06-15 Board Of Regents, The University Of Texas System Combined methods and compositions for coagulation and tumor treatment
IL117645A (en) * 1995-03-30 2005-08-31 Genentech Inc Vascular endothelial cell growth factor antagonists for use as medicaments in the treatment of age-related macular degeneration
EP1140173B2 (en) * 1998-12-22 2013-04-03 Genentech, Inc. Vascular endothelial cell growth factor antagonists and uses thereof
US7183387B1 (en) 1999-01-15 2007-02-27 Genentech, Inc. Polypeptide variants with altered effector function
US6703020B1 (en) * 1999-04-28 2004-03-09 Board Of Regents, The University Of Texas System Antibody conjugate methods for selectively inhibiting VEGF
HK1045700B (en) * 1999-04-28 2007-07-27 德克萨斯大学董事会 Compositions an methods for cancer treatment by selectively inhibiting vegf
DE60237282D1 (en) * 2001-06-28 2010-09-23 Domantis Ltd DOUBLE-SPECIFIC LIGAND AND ITS USE
US20050123925A1 (en) 2002-11-15 2005-06-09 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
HUP0600342A3 (en) * 2001-10-25 2011-03-28 Genentech Inc Glycoprotein compositions
EP1600459A3 (en) * 2002-06-28 2005-12-07 Domantis Limited Ligand
US9321832B2 (en) * 2002-06-28 2016-04-26 Domantis Limited Ligand
US7696320B2 (en) 2004-08-24 2010-04-13 Domantis Limited Ligands that have binding specificity for VEGF and/or EGFR and methods of use therefor
CA2511910A1 (en) * 2002-12-27 2004-07-15 Domantis Limited Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand
AU2004253582B2 (en) * 2003-07-02 2011-02-10 Genentech, Inc. TRP-P8 active compounds and therapeutic treatment methods
US7547518B2 (en) 2003-08-19 2009-06-16 Becton, Dickinson And Company Method of screening endothelial cells for angiogenic capability
US8604185B2 (en) 2004-07-20 2013-12-10 Genentech, Inc. Inhibitors of angiopoietin-like 4 protein, combinations, and their use
ZA200701183B (en) * 2004-07-20 2008-05-28 Genentech Inc Inhibitors of angiopoietin-like 4 protein, combinations, an their use
US20060051347A1 (en) 2004-09-09 2006-03-09 Winter Charles M Process for concentration of antibodies and therapeutic products thereof
PT1802334E (en) 2004-10-21 2012-11-28 Genentech Inc Method for treating intraocular neovascular diseases
US8168584B2 (en) 2005-10-08 2012-05-01 Potentia Pharmaceuticals, Inc. Methods of treating age-related macular degeneration by compstatin and analogs thereof
EP3156418A1 (en) 2006-01-05 2017-04-19 Genentech, Inc. Anti-ephb4 antibodies and methods using same
CA2647430A1 (en) * 2006-03-29 2007-10-11 Genentech, Inc. Diagnostics and treatments for tumors
US20070258976A1 (en) * 2006-05-04 2007-11-08 Ward Keith W Combination Therapy for Diseases Involving Angiogenesis
WO2008060705A2 (en) * 2006-06-06 2008-05-22 Genentech, Inc. Anti-dll4 antibodies and methods using same
RU2008152435A (en) * 2006-06-06 2010-07-20 Дженентек, Инк. (Us) COMPOSITIONS AND METHODS OF REGULATING VESSEL DEVELOPMENT
AU2007305443B2 (en) * 2006-09-29 2012-08-23 Oncomed Pharmaceuticals, Inc. Compositions and methods for diagnosing and treating cancer
AU2007323925A1 (en) * 2006-11-10 2008-05-29 Genentech, Inc. Method for treating age-related macular degeneration
WO2008077077A2 (en) 2006-12-19 2008-06-26 Genentech, Inc. Vegf-specific antagonists for adjuvant and neoadjuvant therapy and the treatment of early stage tumors
WO2008091222A1 (en) * 2007-01-26 2008-07-31 Bioinvent International Ab Dll4 signaling inhibitors and uses thereof
PE20090321A1 (en) 2007-06-04 2009-04-20 Genentech Inc ANTI-NOTCH1 NRR ANTIBODIES, METHOD OF PREPARATION AND PHARMACEUTICAL COMPOSITION
JP2010528647A (en) 2007-06-06 2010-08-26 ドマンティス リミテッド Polypeptides, antibody variable domains and antagonists
PE20090983A1 (en) 2007-11-09 2009-08-13 Genentech Inc COMPOSITIONS AND METHODS OF USE OF KINASE-1 SIMILAR TO THE ACTIVIN RECEPTOR
AR069501A1 (en) 2007-11-30 2010-01-27 Genentech Inc ANTI-VEGF ANTIBODIES (VASCULAR ENDOTELIAL GROWTH FACTOR)
MY166446A (en) 2008-03-18 2018-06-27 Genentech Inc Combinations of an anti-her2 antibody-drug conjugate and chemotherapeutic agents,and methods of use
AU2009228158B2 (en) * 2008-03-27 2014-02-27 Zymogenetics, Inc. Compositions and methods for inhibiting PDGFRbeta and VEGF-A
DK2307454T3 (en) 2008-06-25 2017-04-24 Esbatech Alcon Biomed Res Unit Stable and soluble antibodies that inhibit VEGF
US20100029491A1 (en) * 2008-07-11 2010-02-04 Maike Schmidt Methods and compositions for diagnostic use for tumor treatment
ES2644723T3 (en) * 2008-07-23 2017-11-30 F. Hoffmann-La Roche Ag Identification of subjects susceptible to antiangiogenic treatment
EP2324358A1 (en) * 2008-07-23 2011-05-25 F. Hoffmann-La Roche AG Monitoring anti-angiogenesis therapy
AR073231A1 (en) * 2008-08-29 2010-10-20 Genentech Inc DIAGNOSTIC METHODS AND TREATMENTS FOR VEGF INDEPENDENT TUMORS (VASCULAR ENDOTELIAL GROWTH FACTOR)
CN110317272A (en) 2008-10-14 2019-10-11 霍夫曼-拉罗奇有限公司 Immunoglobulin variants and application thereof
EP2752189B1 (en) 2008-11-22 2016-10-26 F. Hoffmann-La Roche AG Use of anti-vegf antibody in combination with chemotherapy for treating breast cancer
CA2746120A1 (en) 2008-12-23 2010-07-01 Genentech, Inc. Methods and compositions for diagnostic use in cancer patients
KR101739598B1 (en) 2009-03-13 2017-06-08 아브락시스 바이오사이언스, 엘엘씨 Combination therapy with thiocolchicine derivatives
SG175289A1 (en) * 2009-04-20 2011-11-28 Genentech Inc Adjuvant cancer therapy
BRPI1007706A2 (en) 2009-05-08 2019-04-02 Genentech, Inc. anti-egfl7 antibody, bispecific antibody, nucleic acid, vector, host cell, composition, method for producing an anti-egfl7 antibody, method of reducing or inhibiting angiogenesis in an individual, method of enhancing the effectiveness of an antiangiogenesis agent in an individual and method of reducing or inhibiting perfusion and permeability of a tumor in an individual
US20110076271A1 (en) 2009-07-13 2011-03-31 Genentech, Inc. Diagnostic methods and compositions for treatment of cancer
WO2011014457A1 (en) 2009-07-27 2011-02-03 Genentech, Inc. Combination treatments
WO2011014750A1 (en) * 2009-07-31 2011-02-03 Genentech, Inc. Inhibition of tumor metastasis using bv8- or g-csf-antagonists
AU2010284446A1 (en) * 2009-08-15 2012-03-08 Genentech, Inc. Anti-angiogenesis therapy for the treatment of previously treated breast cancer
JP2013504595A (en) 2009-09-11 2013-02-07 ジェネンテック， インコーポレイテッド Methods for identifying patients with increased likelihood of response to anticancer drugs
MX2012002909A (en) 2009-09-17 2012-04-19 Hoffmann La Roche METHODS AND COMPOSITIONS FOR USE IN DIAGNOSIS OF PATIENTS WITH CANCER.
EP3072526B1 (en) 2009-10-16 2018-09-26 Oncomed Pharmaceuticals, Inc. Therapeutic combination and use of dll4 antagonist antibodies and anti-hypertensive agents
WO2011056497A1 (en) 2009-10-26 2011-05-12 Genentech, Inc. Activin receptor type iib compositions and methods of use
WO2011056502A1 (en) 2009-10-26 2011-05-12 Genentech, Inc. Bone morphogenetic protein receptor type ii compositions and methods of use
AR078377A1 (en) 2009-12-11 2011-11-02 Genentech Inc ANTI-VEGF-C ANTIBODIES (ISOLATED ANTI-VASCULAR ENDOTELIAL GROWTH FACTOR C) AND ITS METHODS OF USE
KR20190067275A (en) 2009-12-21 2019-06-14 제넨테크, 인크. Antibody formulation
EP2516465B1 (en) 2009-12-23 2016-05-18 F.Hoffmann-La Roche Ag Anti-bv8 antibodies and uses thereof
RU2012140447A (en) * 2010-02-23 2014-03-27 Дженентек, Инк. ANTIANGIOGENIC THERAPY FOR TREATMENT OF OVARIAN CANCER
WO2011153243A2 (en) 2010-06-02 2011-12-08 Genentech, Inc. Anti-angiogenesis therapy for treating gastric cancer
WO2011153224A2 (en) 2010-06-02 2011-12-08 Genentech, Inc. Diagnostic methods and compositions for treatment of cancer
WO2012010549A1 (en) 2010-07-19 2012-01-26 F. Hoffmann-La Roche Ag Method to identify a patient with an increased likelihood of responding to an anti-cancer therapy
CN103180737A (en) 2010-07-19 2013-06-26 霍夫曼-拉罗奇有限公司 Method of identifying patients with increased likelihood of responding to anticancer therapy
AR082693A1 (en) 2010-08-17 2012-12-26 Roche Glycart Ag COMBINATION THERAPY OF AN ANTI-CD20 AFUCOSILATED ANTIBODY WITH AN ANTI-VEGF ANTIBODY
US8551479B2 (en) 2010-09-10 2013-10-08 Oncomed Pharmaceuticals, Inc. Methods for treating melanoma
JP6178240B2 (en) 2010-11-15 2017-08-09 ファイブ プライム セラピューティックス インコーポレイテッド Treatment of cancer with high doses of soluble FGFR1 fusion protein
WO2012092539A2 (en) 2010-12-31 2012-07-05 Takeda Pharmaceutical Company Limited Antibodies to dll4 and uses thereof
KR101614195B1 (en) 2011-03-29 2016-04-20 로슈 글리카트 아게 Antibody fc variants
WO2012135753A1 (en) 2011-04-01 2012-10-04 Genentech, Inc. Combinations of akt inhibitor compounds and erlotinib, and methods of use
US9932386B2 (en) 2011-04-20 2018-04-03 Acceleron Pharma, Inc. Endoglin polypeptides and uses thereof
WO2012151317A1 (en) 2011-05-03 2012-11-08 Genentech, Inc. Vascular disruption agents and uses thereof
EP2744825A1 (en) 2011-08-17 2014-06-25 F.Hoffmann-La Roche Ag Inhibition of angiogenesis in refractory tumors
EP2758073B1 (en) 2011-09-23 2018-10-31 OncoMed Pharmaceuticals, Inc. Vegf/dll4 binding agents and uses thereof
WO2013082511A1 (en) 2011-12-02 2013-06-06 Genentech, Inc. Methods for overcoming tumor resistance to vegf antagonists
SI2825558T1 (en) 2012-03-13 2019-08-30 F. Hoffmann-La Roche Ag Combination therapy for the treatment of ovarian cancer
MX363872B (en) 2012-05-31 2019-04-05 Genentech Inc Methods of treating cancer using pd-l1 axis binding antagonists and vegf antagonists.
KR101915942B1 (en) 2012-06-08 2018-11-06 에프. 호프만-라 로슈 아게 Mutant selectivity and combinations of a phosphoinositide 3 kinase inhibitor compound and chemotherapeutic agents for the treatment of cancer
SMT202100621T1 (en) 2012-07-13 2022-01-10 Roche Glycart Ag Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases
KR20190088571A (en) 2012-08-07 2019-07-26 제넨테크, 인크. Combination therapy for the treatment of glioblastoma
EP2914961A4 (en) 2012-10-31 2016-04-20 Oncomed Pharm Inc METHODS AND MONITORING A TREATMENT BY AN ANTAGONIST OF DLL4
ES2879430T3 (en) 2012-11-15 2021-11-22 Apellis Pharmaceuticals Inc Long-Acting Compstatin Analogs and Related Compositions and Methods
CN103864932B (en) 2012-12-14 2015-11-25 苏州思坦维生物技术有限责任公司 Antagonism suppresses the monoclonal antibody of vascular endothelial growth factor and its receptors bind and encoding sequence thereof and purposes
WO2014100615A1 (en) 2012-12-20 2014-06-26 Purdue Research Foundation Chimeric antigen receptor-expressing t cells as anti-cancer therapeutics
CN110538322A (en) 2013-03-13 2019-12-06 豪夫迈·罗氏有限公司 Antibody formulations
KR102202476B1 (en) 2013-03-15 2021-01-12 제넨테크, 인크. Cell culture media and methods of antibody production
US10308687B2 (en) 2013-03-15 2019-06-04 Apellis Pharmaceuticals, Inc. Cell-penetrating compstatin analogs and uses thereof
BR112015029145A2 (en) 2013-05-23 2017-09-19 Glaxosmithkline Ip No 2 Ltd USES OF A FIBROBLAST GROWTH FACTOR 1 (FGFR1) RECEPTOR EXTRACELLULAR DOMAIN (ECD) AND PATIENT IDENTIFICATION METHODS
US10617755B2 (en) 2013-08-30 2020-04-14 Genentech, Inc. Combination therapy for the treatment of glioblastoma
US10456470B2 (en) 2013-08-30 2019-10-29 Genentech, Inc. Diagnostic methods and compositions for treatment of glioblastoma
EP3060235B1 (en) 2013-10-25 2020-12-02 Acceleron Pharma Inc. Endoglin peptides to treat fibrotic diseases
US10568951B2 (en) 2013-11-18 2020-02-25 Formycon Ag Pharmaceutical composition of an anti-VEGF antibody
JOP20200094A1 (en) 2014-01-24 2017-06-16 Dana Farber Cancer Inst Inc Antibody molecules to pd-1 and uses thereof
JOP20200096A1 (en) 2014-01-31 2017-06-16 Children’S Medical Center Corp TIM-3 Antibody Molecules and Their Uses
LT3116909T (en) 2014-03-14 2020-02-10 Novartis Ag ANTI-BODY MOLECULES AGAINST LAG-3 AND THEIR USE
WO2015148531A1 (en) 2014-03-24 2015-10-01 Genentech, Inc. Cancer treatment with c-met antagonists and correlation of the latter with hgf expression
KR20160146747A (en) 2014-03-31 2016-12-21 제넨테크, 인크. Combination therapy comprising anti-angiogenesis agents and ox40 binding agonists
CN106456757A (en) 2014-05-12 2017-02-22 福尔密孔股份公司 Pre-filled plastic syringe containing VEGF antagonist
WO2016011052A1 (en) 2014-07-14 2016-01-21 Genentech, Inc. Diagnostic methods and compositions for treatment of glioblastoma
US20170224777A1 (en) 2014-08-12 2017-08-10 Massachusetts Institute Of Technology Synergistic tumor treatment with il-2, a therapeutic antibody, and a cancer vaccine
EP3180018B1 (en) 2014-08-12 2019-07-24 Massachusetts Institute Of Technology Synergistic tumor treatment with il-2 and integrin-binding-fc-fusion protein
CA2960824A1 (en) 2014-09-13 2016-03-17 Novartis Ag Combination therapies of alk inhibitors
EP3193932B1 (en) 2014-09-15 2023-04-26 F. Hoffmann-La Roche AG Antibody formulations
ES2774448T3 (en) 2014-10-03 2020-07-21 Novartis Ag Combination therapies
MA41044A (en) 2014-10-08 2017-08-15 Novartis Ag COMPOSITIONS AND METHODS OF USE FOR INCREASED IMMUNE RESPONSE AND CANCER TREATMENT
JP6877339B2 (en) 2014-10-14 2021-05-26 ノバルティス アーゲー Antibody molecule against PD-L1 and its use
CN107530419B (en) 2014-10-31 2021-05-18 昂考梅德药品有限公司 Combination therapy to treat disease
BR112017009728A2 (en) 2014-11-10 2018-02-06 Genentech, Inc. isolated antibodies, isolated nucleic acid, vector, host cell, method for producing antibody, composition, uses of an antibody and methods of treating a disorder
WO2016100882A1 (en) 2014-12-19 2016-06-23 Novartis Ag Combination therapies
SG11201704707PA (en) 2014-12-23 2017-07-28 Genentech Inc Compositions and methods for treating and diagnosing chemotherapy-resistant cancers
US20170000885A1 (en) 2015-06-08 2017-01-05 Genentech, Inc. Methods of treating cancer using anti-ox40 antibodies and pd-1 axis binding antagonists
EP3964528A1 (en) 2015-07-29 2022-03-09 Novartis AG Combination therapies comprising antibody molecules to lag-3
WO2017019896A1 (en) 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to pd-1
EP3878465A1 (en) 2015-07-29 2021-09-15 Novartis AG Combination therapies comprising antibody molecules to tim-3
JP6967003B2 (en) 2015-09-23 2021-11-17 メレオ バイオファーマ ５ インコーポレイテッド Methods and compositions for the treatment of cancer
DK3359555T3 (en) 2015-10-07 2024-03-25 Apellis Pharmaceuticals Inc Dosage plans
EP3368578B1 (en) 2015-10-30 2021-03-17 H. Hoffnabb-La Roche Ag Anti-htra1 antibodies and methods of use thereof
LT3370768T (en) 2015-11-03 2022-05-25 Janssen Biotech, Inc. ANTIBODIES SPECIFICLY BINDING PD-1 AND THEIR USE
RU2734958C2 (en) 2015-11-18 2020-10-26 Формикон Аг Pre-filled pharmaceutical package containing a liquid formulation based on a vegf antagonist
MX2018006171A (en) 2015-11-18 2018-12-19 Formycon Ag Pre-filled plastic syringe containing a vegf antagonist.
CA3005132A1 (en) 2015-11-18 2017-05-26 Sio2 Medical Products, Inc. Coated syringe for ophthalmic formulations containing a vegf antagonist
EP4424322A3 (en) 2015-12-17 2025-04-16 Novartis AG Antibody molecules to pd-1 and uses thereof
EP3407868A1 (en) 2016-01-26 2018-12-05 Formycon AG Liquid formulation of a vegf antagonist
CN109069638B (en) 2016-03-24 2022-03-29 璟尚生物制药公司 Trispecific inhibitors for cancer therapy
EP3439675A4 (en) 2016-04-08 2019-12-18 Purdue Research Foundation METHODS AND COMPOSITIONS FOR T CAR LYMPHOCYTE THERAPY
CN109154613A (en) 2016-04-15 2019-01-04 豪夫迈·罗氏有限公司 For monitoring and the method for the treatment of cancer
EP3443120A2 (en) 2016-04-15 2019-02-20 H. Hoffnabb-La Roche Ag Methods for monitoring and treating cancer
CN109890982B (en) 2016-07-08 2023-07-07 基因泰克公司 Method for diagnosing and treating cancer by expression status and mutation status of NRF2 and target genes downstream thereof
TW202434291A (en) 2016-07-08 2024-09-01 美商建南德克公司 Use of human epididymis protein 4 (he4) for assessing responsiveness of cancer treatment
BR112019002036A2 (en) 2016-08-12 2019-05-14 Genentech Inc methods of treatment of a colorectal cancer subject, colorectal cancer treatment kit in a human subject, and drug combination for colorectal cancer therapy
US10537637B2 (en) 2017-01-05 2020-01-21 Gensun Biopharma Inc. Checkpoint regulator antagonists
EP3568150A4 (en) 2017-01-10 2020-12-02 Xcella Biosciences, Inc. Combination tumor treatment with an integrin-binding-fc fusion protein and immune modulator
US10350266B2 (en) 2017-01-10 2019-07-16 Nodus Therapeutics, Inc. Method of treating cancer with a multiple integrin binding Fc fusion protein
CN110612119B (en) 2017-02-07 2024-10-29 西雅图儿童医院（Dba西雅图儿童研究所） Phosphatidylether (PLE) CAR T cell tumor targeting (CTCT) agents
US11850262B2 (en) 2017-02-28 2023-12-26 Purdue Research Foundation Compositions and methods for CAR T cell therapy
EP3589754B1 (en) 2017-03-01 2023-06-28 F. Hoffmann-La Roche AG Diagnostic and therapeutic methods for cancer
IL314891B1 (en) 2017-04-07 2025-11-01 Apellis Pharmaceuticals Inc Dosage regimens and related compositions and methods
EP3630043A1 (en) 2017-05-24 2020-04-08 Formycon AG Sterilizable pre-filled pharmaceutical packages comprising a liquid formulation of a vegf-antagonist
WO2018215580A1 (en) 2017-05-24 2018-11-29 Formycon Ag Method for sterilizing prefilled plastic syringes containing a vegf antagonist
US20200171244A1 (en) 2017-05-24 2020-06-04 Sio2 Medical Products, Inc. Sterilizable pharmaceutical package for ophthalmic formulations
EP3642240A1 (en) 2017-06-22 2020-04-29 Novartis AG Antibody molecules to cd73 and uses thereof
US11312783B2 (en) 2017-06-22 2022-04-26 Novartis Ag Antibody molecules to CD73 and uses thereof
WO2019020777A1 (en) 2017-07-26 2019-01-31 Formycon Ag Liquid formulation of a vegf antagonist
AU2018386304B2 (en) 2017-12-15 2023-12-07 Apellis Pharmaceuticals, Inc. Dosing regimens and related compositions and methods
US11311576B2 (en) 2018-01-22 2022-04-26 Seattle Children's Hospital Methods of use for CAR T cells
US12398209B2 (en) 2018-01-22 2025-08-26 Janssen Biotech, Inc. Methods of treating cancers with antagonistic anti-PD-1 antibodies
EP3749695A4 (en) 2018-02-06 2021-12-29 Seattle Children's Hospital (DBA Seattle Children's Research Institute) Fluorescein-specific cars exhibiting optimal t cell function against fl-ple labelled tumors
AU2019225174B2 (en) 2018-02-23 2025-11-20 Endocyte, Inc. Sequencing method for CAR T cell therapy
JP7520366B2 (en) 2018-04-16 2024-07-23 オンクオリティー ファーマシューティカルズ チャイナ リミテッド Methods for preventing or treating side effects of oncology therapy
CA3101272A1 (en) 2018-05-24 2019-11-28 Janssen Biotech, Inc. Psma binding agents and uses thereof
TWI869346B (en) 2018-05-30 2025-01-11 瑞士商諾華公司 Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies
WO2019232244A2 (en) 2018-05-31 2019-12-05 Novartis Ag Antibody molecules to cd73 and uses thereof
JP2021529556A (en) 2018-06-29 2021-11-04 ジェンサン バイオファーマ、 インコーポレイテッドＧｅｎｓｕｎ Ｂｉｏｐｈａｒｍａ， Ｉｎｃ． Antitumor antagonist
AU2019361983A1 (en) 2018-10-18 2021-05-20 Genentech, Inc. Diagnostic and therapeutic methods for sarcomatoid kidney cancer
MX2021004774A (en) 2018-10-29 2021-08-24 Hoffmann La Roche Antibody formulation.
EP3956022A1 (en) 2019-04-19 2022-02-23 Janssen Biotech, Inc. Methods of treating prostate cancer with an anti- psma/cd3 antibody
MX2021013222A (en) 2019-05-03 2022-01-06 Genentech Inc Methods of treating cancer with an anti-pd-l1 antibody.
CN114340735B (en) 2019-06-28 2024-11-12 璟尚生物制药公司 Antitumor antagonists composed of mutant TGFβ1-RII extracellular domain and immunoglobulin scaffold
CN114502590A (en) 2019-09-18 2022-05-13 诺华股份有限公司 ENTPD2 antibodies, combination therapies, and methods of using these antibodies and combination therapies
PE20230252A1 (en) 2020-03-13 2023-02-07 Genentech Inc ANTI-INTERLEUKIN-33 ANTIBODIES AND ITS USES FOR THEM
AU2021243864A1 (en) 2020-03-27 2022-11-03 Mendus B.V. Ex vivo use of modified cells of leukemic origin for enhancing the efficacy of adoptive cell therapy
JP2023520515A (en) 2020-04-03 2023-05-17 ジェネンテック， インコーポレイテッド Therapeutic and diagnostic methods for cancer
CA3182607A1 (en) 2020-06-30 2022-01-06 Erik Hans MANTING Use of leukemia-derived cells in ovarian cancer vaccines
TW202535399A (en) 2020-11-13 2025-09-16 美商建南德克公司 Methods and compositions comprising a krasg12c inhibitor and a vegf inhibitor for treating solid tumors
AU2022211682A1 (en) 2021-01-22 2023-08-03 Mendus B.V. Methods of tumor vaccination
KR20230137393A (en) 2021-01-28 2023-10-04 얀센 바이오테크 인코포레이티드 PSMA binding protein and its uses
WO2022190058A1 (en) 2021-03-12 2022-09-15 Dcprime B.V. Methods of vaccination and use of cd47 blockade
EP4329734A4 (en) 2021-04-26 2025-04-02 Celanese EVA Performance Polymers LLC IMPLANTABLE DEVICE FOR THE SUSTAINED RELEASE OF A MACROMOLECULAR DRUG COMPOUND
JP2024516230A (en) 2021-04-30 2024-04-12 ジェネンテック， インコーポレイテッド Therapeutic and diagnostic methods and compositions for cancer
WO2022256820A1 (en) 2021-06-03 2022-12-08 Gensun Biopharma Inc. Multispecific antagonists
TW202309078A (en) 2021-07-02 2023-03-01 美商建南德克公司 Methods and compositions for treating cancer
EP4377351A1 (en) 2021-07-28 2024-06-05 F. Hoffmann-La Roche AG Methods and compositions for treating cancer
WO2023080900A1 (en) 2021-11-05 2023-05-11 Genentech, Inc. Methods and compositions for classifying and treating kidney cancer
WO2023144973A1 (en) 2022-01-27 2023-08-03 中外製薬株式会社 Pharmaceutical composition containing anti-pd-l1 antibody to be used in combination with anti-vegf antibody and paclitaxel
KR20240144944A (en) 2022-01-28 2024-10-04 온퀄리티 파마슈티컬스 차이나 리미티드 Methods for preventing or treating diseases or symptoms associated with antineoplastic agents
EP4484429A1 (en) 2022-02-21 2025-01-01 OnQuality Pharmaceuticals China Ltd. Compound and use thereof
WO2024263904A1 (en) 2023-06-23 2024-12-26 Genentech, Inc. Methods for treatment of liver cancer
WO2024263195A1 (en) 2023-06-23 2024-12-26 Genentech, Inc. Methods for treatment of liver cancer

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4456550A (en) * 1982-11-22 1984-06-26 President And Fellows Of Harvard College Vascular permeability factor
US4816567A (en) * 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5008196A (en) * 1987-08-21 1991-04-16 Monsanto Company Stimulation of endothelial cell growth
US5036003A (en) * 1987-08-21 1991-07-30 Monsanto Company Antibodies to VPF
US5116964A (en) * 1989-02-23 1992-05-26 Genentech, Inc. Hybrid immunoglobulins
US5219739A (en) * 1989-07-27 1993-06-15 Scios Nova Inc. DNA sequences encoding bVEGF120 and hVEGF121 and methods for the production of bovine and human vascular endothelial cell growth factors, bVEGF120 and hVEGF121
US5283354A (en) * 1991-04-02 1994-02-01 The Trustees Of Princeton University Nucleic acids encoding hematopoietic stem cells receptors flk-1
US5725856A (en) * 1988-01-12 1998-03-10 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5840301A (en) * 1994-02-10 1998-11-24 Imclone Systems Incorporated Methods of use of chimerized, humanized, and single chain antibodies specific to VEGF receptors
US5861499A (en) * 1994-02-10 1999-01-19 Imclone Systems Incorporated Nucleic acid molecules encoding the variable or hypervariable region of a monoclonal antibody that binds to an extracellular domain
US5955311A (en) * 1994-02-10 1999-09-21 Imclone Systems Incorporated Monoclonal antibodies specific to VEGF receptors and uses thereof
US6582959B2 (en) * 1991-03-29 2003-06-24 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
US6811779B2 (en) * 1994-02-10 2004-11-02 Imclone Systems Incorporated Methods for reducing tumor growth with VEGF receptor antibody combined with radiation and chemotherapy

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5240848A (en) 1988-11-21 1993-08-31 Monsanto Company Dna sequences encoding human vascular permeability factor having 189 amino acids
US5332671A (en) 1989-05-12 1994-07-26 Genetech, Inc. Production of vascular endothelial cell growth factor and DNA encoding same
WO1991000916A2 (en) 1989-07-06 1991-01-24 The Regents Of The University Of California Receptors for fibroblast growth factors
US5194596A (en) 1989-07-27 1993-03-16 California Biotechnology Inc. Production of vascular endothelial cell growth factor
ES2180535T3 (en) 1991-02-22 2003-02-16 American Cyanamid Co IDENTIFICATION OF A NEW GENE OF HUMAN RECEPTORA TIROSINA QUINASA.
CA2119580A1 (en) 1991-10-01 1993-04-15 George Cachianes Production of gpa neurotrophic factor
DE69233803D1 (en) 1992-10-28 2011-03-31 Genentech Inc Use of vascular endothelial growth factor antagonists
TW406127B (en) 1992-12-21 2000-09-21 Ciba Sc Holding Ag Novel liquid phenolic antioxidants
US5629327A (en) 1993-03-01 1997-05-13 Childrens Hospital Medical Center Corp. Methods and compositions for inhibition of angiogenesis
US5731294A (en) 1993-07-27 1998-03-24 Hybridon, Inc. Inhibition of neovasularization using VEGF-specific oligonucleotides
ATE501170T1 (en) 1997-04-07 2011-03-15 Genentech Inc METHOD FOR PRODUCING HUMANIZED ANTIBODIES BY RANDOMIZED MUtagenesis
NZ500078A (en) 1997-04-07 2001-10-26 Genentech Inc Humanized anti-VEGF antibodies and their use in inhibiting VEGF-induced angiogenesis in mammals
US20050186208A1 (en) * 2003-05-30 2005-08-25 Genentech, Inc. Treatment with anti-VEGF antibodies
WO2005044853A2 (en) 2003-11-01 2005-05-19 Genentech, Inc. Anti-vegf antibodies
ZA200701183B (en) * 2004-07-20 2008-05-28 Genentech Inc Inhibitors of angiopoietin-like 4 protein, combinations, an their use

Patent Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4456550A (en) * 1982-11-22 1984-06-26 President And Fellows Of Harvard College Vascular permeability factor
US4816567A (en) * 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5008196A (en) * 1987-08-21 1991-04-16 Monsanto Company Stimulation of endothelial cell growth
US5036003A (en) * 1987-08-21 1991-07-30 Monsanto Company Antibodies to VPF
US5725856A (en) * 1988-01-12 1998-03-10 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5116964A (en) * 1989-02-23 1992-05-26 Genentech, Inc. Hybrid immunoglobulins
US5219739A (en) * 1989-07-27 1993-06-15 Scios Nova Inc. DNA sequences encoding bVEGF120 and hVEGF121 and methods for the production of bovine and human vascular endothelial cell growth factors, bVEGF120 and hVEGF121
US6582959B2 (en) * 1991-03-29 2003-06-24 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
US7482005B2 (en) * 1991-03-29 2009-01-27 Genentech, Inc. Methods of treating diseases with anti-VEGF antibodies
US7227004B2 (en) * 1991-03-29 2007-06-05 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
US5283354A (en) * 1991-04-02 1994-02-01 The Trustees Of Princeton University Nucleic acids encoding hematopoietic stem cells receptors flk-1
US5840301A (en) * 1994-02-10 1998-11-24 Imclone Systems Incorporated Methods of use of chimerized, humanized, and single chain antibodies specific to VEGF receptors
US6365157B2 (en) * 1994-02-10 2002-04-02 Imclone Systems, Inc. Monoclonal antibodies specific to VEGF receptors and uses thereof
US6448077B1 (en) * 1994-02-10 2002-09-10 Imclone Systems, Inc. Chimeric and humanized monoclonal antibodies specific to VEGF receptors
US5955311A (en) * 1994-02-10 1999-09-21 Imclone Systems Incorporated Monoclonal antibodies specific to VEGF receptors and uses thereof
US6811779B2 (en) * 1994-02-10 2004-11-02 Imclone Systems Incorporated Methods for reducing tumor growth with VEGF receptor antibody combined with radiation and chemotherapy
US5874542A (en) * 1994-02-10 1999-02-23 Imclone Systems Incorporated Single chain antibodies specific to VEGF receptors
US5861499A (en) * 1994-02-10 1999-01-19 Imclone Systems Incorporated Nucleic acid molecules encoding the variable or hypervariable region of a monoclonal antibody that binds to an extracellular domain

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9795672B2 (en) 2003-05-30 2017-10-24 Genentech, Inc. Treatment with anti-VEGF antibodies

Also Published As

Publication number Publication date
US7482005B2 (en) 2009-01-27
US7227004B2 (en) 2007-06-05
US6582959B2 (en) 2003-06-24
US20090081232A1 (en) 2009-03-26
US20030203409A1 (en) 2003-10-30
US20070154391A1 (en) 2007-07-05
US20100330096A1 (en) 2010-12-30
US20020122797A1 (en) 2002-09-05

Similar Documents

Publication Publication Date Title
US7482005B2 (en) Methods of treating diseases with anti-VEGF antibodies
EP1238986B1 (en) Use of Vascular endothelial cell growth factor antagonists
US5668003A (en) Nucleic acid encoding monoclonal antibodies to leukemia inhibitory factor
AU696487B2 (en) Vascular endothelial cell growth factor antagonists
US20030206899A1 (en) Vascular endothelial cell growth factor antagonists
US20020032313A1 (en) Vascular endothelial cell growth factor antagonists
US20030023046A1 (en) Vascular endothelial cell growth factor antagonists
US20010021382A1 (en) Vascular endothelial cell growth factor antagonists
US20060193862A1 (en) Vascular endothelial cell growth factor antagonists
HK1117549A1 (en) Use of vascular endothelial cell growth factor antagonists
HK1117549B (en) Use of vascular endothelial cell growth factor antagonists

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION